Method for preparing cellular universal blood products for blood group independent transfusion

Abstract

A first aspect of the invention is directed to a process for the preparation of cellular blood components with reduced expression of antigen(s) (blood groups) compared to the native state, comprising: (a) Providing a solid carrier carrying at least one enzyme; (b) Providing a liquid phase LP1 comprising at least one cellular blood component having antigen(s) with a native expression of antigen(s) LA(1); and (c) Contacting the solid carrier carrying at least one enzyme provided according to step (a) with the liquid phase provided according to step (b) under conditions allowing a removal of at least a part of the antigen(s) from the at least one cellular blood component; thereby obtaining a liquid phase LP2 comprising the at least one cellular blood component having antigen(s) with a reduced expression of antigen(s) LA(2), wherein LA(2) is < LA(1), and the solid carrier carrying the at least one enzyme at a concentration CSC(1). In a second aspect, the invention relates to a system for removing antigen(s) from cellular blood components, and a third aspect is directed to specific α-1,3-galactosidases. A fourth aspect of the invention is related to a cellular blood component having a reduced expression of antigen(s) LA(2) compared to a native expression of antigen(s) LA(1) of said cellular blood component with LA(2) < LA(1), obtained or obtainable by a process according to the first aspect of the invention, wherein a fifth aspect of the invention is directed to the cellular blood component with LA(2) < LA(1) per se. In a sixth aspect, the invention relates to the cellular blood component having a reduced expression of antigen(s) LA(2) compared to a native expression of antigen(s) LA(1) of said cellular blood component with LA(2) < LA(1) of the fourth or the fifth aspect of the invention for use in transfusion.

Description

[0001] Universitatsmedizin Greifswald PVA21246PC

[0002] Universitat Greifswald

[0003] -1-

[0004] Method for preparing cellular universal blood products for blood group independent transfusion

[0005] A first aspect of the invention is directed to a process for the preparation of cellular blood components with reduced expression of antigen(s) (blood groups) compared to the native state, comprising: (a) Providing a solid carrier carrying at least one enzyme; (b) Providing a liquid phase LP1 comprising at least one cellular blood component having antigen(s) with a native expression of antigen(s) LA(1); and (c) Contacting the solid carrier carrying at least one enzyme provided according to step (a) with the liquid phase provided according to step (b) under conditions allowing a removal of at least a part of the antigen(s) from the at least one cellular blood component; thereby obtaining a liquid phase LP2 comprising the at least one cellular blood component having antigen(s) with a reduced expression of antigen(s) LA(2), wherein LA(2) is < LA(1), and the solid carrier carrying the at least one, optionally modified, enzyme at a concentration Csc(l).

[0006] In a second aspect, the invention relates to a system for removing antigen(s) from cellular blood components, and a third aspect is directed to specific a-l,3-galactosidases. A fourth aspect of the invention is related to a cellular blood component having a reduced expression of antigen(s) LA(2) compared to a native expression of antigen(s) LA(1) of said cellular blood component with LA(2) < LA(1), obtained or obtainable by a process according to the first aspect of the invention, wherein a fifth aspect of the invention is directed to the cellular blood component with LA(2) < LA(1) per se. IN a sixth aspect, the invention relates to the cellular blood component having a reduced expression of antigen(s) LA(2) compared to a native expression of antigen(s) LA(1) of said cellular blood component with LA(2) < LA(1) of the fourth or the fifth aspect of the invention for use in transfusion.

[0007] Blood transfusion has evolved from transfusing whole blood to the transfusion of blood components such as red blood cell concentrates, and platelet concentrates. For example, the transfusion of platelet concentrates can prevent bleeding or stop ongoing bleeding in patients with platelet disorders or low platelet counts since platelets are the most important elements of primary hemostasis.[1]Platelet concentrates are manufactured from a single donor by apheresis or by pooling blood group identical buffy coats that contain the platelets (and leukocytes) from 4-5 whole blood donations. The supply of platelet concentrates for transfusion poses a challenge due to increasing demand, decreasing donor numbers, and the short shelf-life (4-7 days).[2]

[0008] Generally, transfusion of blood products requires compatibility within respective blood group system, for example, the ABO blood group system. This system is based on the presence of the two antigens A and B on blood cell surfaces. All blood cells contain the fucosyl galactose H- antigen on their surface, determining blood type O. In blood types A and B, an additional N- Universitatsmedizin Greifswald PVA21246PC

[0009] Universitat Greifswald

[0010] -2-

[0011] Acetylgalactosamine (GalNAc) or galactose (Gal) are attached to the fucosyl galactose H-antigen, respectively (Fig. 1).

[0012] Human platelets also carry detectable quantities of A and B antigens on their surface. However, the expression of these antigens is approximately 100-fold weaker than on red blood cells (RBC). Expression density also varies between individuals.13 51Human anti- A and anti-B antibodies in blood plasma (isoagglutinins) also target A and B antigens on platelets.[6]This causes a reduction in platelet survival.[7]

[0013] The concept of enzymatic removal of ABO-antigens was first demonstrated for RBC.[8]For B- antigen conversion, an a-galactosidase from green coffee beans was used. However, due to the low pH optimum (pH 3.5) a large amount of enzyme was needed (3-7 mg per mL RBC) for effective blood group removal. Fully converted blood from B-type to O-type blood was then transfused in a first clinical trial with three patients of blood type A, B, and 0. The transfused RBC exhibited normal circulation times and were well-tolerated by the recipients. A large phase 2 clinical trial concluded that enzymatic conversion could indeed be used to create universal donor blood, but shows that other enzymes with higher activity and a pH optimum close to the blood pH of 7.35-7.45 are required.19 1 1 1

[0014] Pooled platelet concentrate can be produced from buffy coats. However, the selection of AB0- identical buffy coats for the production of pooled platelet concentrate causes logistical challenges.

[0015] For example, with a view to platelet concentrates, it was aimed to produce pooled platelet concentrates from a plurality of buffy coats from whole blood donations of non-identical blood groups. This would allow for the use of all buffy coats of the daily donation and buffy coats whose blood group is unknown at the time of platelet concentrate production.

[0016] Generally, a need exists for the provision of cellular blood components, irrespective whether this means platelets, red blood cells or other cellular blood components, said cellular blood components having a reduced expression of antigen(s) (blood groups) compared to the native state.

[0017] An object of the present invention was thus the provision of a process for the preparation of cellular blood components with reduced expression of antigen(s) (blood groups) compared to the native state, as well as the provision of the respective cellular blood components with reduced expression of antigen(s) (blood groups) compared to the native state themselves.

[0018] In a first aspect, the invention is directed to a process for the preparation of cellular blood components with reduced expression of antigen(s) (blood groups) compared to the native state, comprising: Universitatsmedizin Greifswald PVA21246PC

[0019] Universitat Greifswald

[0020] -3-

[0021] (a) Providing a solid carrier carrying at least one enzyme;

[0022] (b) Providing a liquid phase LP1 comprising at least one cellular blood component having antigen(s) with a native expression of antigen(s) LA(1);

[0023] (c) Contacting the solid carrier carrying at least one enzyme provided according to step (a) with the liquid phase provided according to step (b) under conditions allowing a removal of at least a part of the antigen(s) from the at least one cellular blood component; thereby obtaining a liquid phase LP2 comprising the at least one cellular blood component having antigen(s) with a reduced expression of antigen(s) LA(2), wherein LA(2) is < LA(1), and the solid carrier carrying the at least one enzyme at a concentration Csc(l).

[0024] A “expression of antigen(s)” means the concentration of the respective antigen(s), with which these antigen(s) are presented on the surface of respective the cellular blood component. A “native expression of antigen(s)” means the concentration of the respective antigen(s), with which these antigen(s) are originally presented on the surface of the respective cellular blood component, before any treatment is done to remove or alter these antigen(s). “A removal of at least a part of the antigen(s) from the at least one cellular blood component” means a removal of a part of an individual antigen as well as removal of the complete individual antigen. For example, as described in more detail below, in cases where a cellular blood component carries an A antigen of the ABO blood group system, the removal of at least a part of the antigen(s) means that at least the terminal acetyl glucosamine residue representing the A antigen is removed, leaving only the remaining (fucosyl galactose) H-antigen, determining blood type 0, on the surface of the cellular blood component.

[0025] Preferably, the reduced expression of antigen(s) LA(2) is in the range of from 0.01 x LA(1) to 0.8 x LA(1), more preferably in the range of from 0.01 x LA(1) to 0.3 X LA(1).

[0026] Solid carrier (a)

[0027] Preferably, the at least one solid carrier provided in step (a) is selected from the group consisting of microparticle, membrane, foil and mixed forms of two or more thereof.

[0028] Microparticle as solid carrier (a)

[0029] Preferably, the at least one solid carrier provided in step (a) is a microparticle, said microparticle comprising a core (C) and optionally one or more shell(s) (S).

[0030] The core (C) preferably comprises or consists of a component selected from the group consisting of polymer, lipid, metal, metal carbide, metal nitride, metal sulfide, metal phosphide, metal oxide, Universitatsmedizin Greifswald PVA21246PC

[0031] Universitat Greifswald

[0032] -4- metal chelate, glass, silica, earth alkali metal phosphate, carbon and mixed forms of two or more thereof.

[0033] In some preferred embodiments, the core (C) comprises or consists of a (supra)magnetic material, preferably selected from the group consisting of metal carbide, metal nitride, metal sulfide, metal phosphide, metal oxide, metal chelate, and mixtures of two or more thereof, preferably at least an iron oxide, in particular an iron oxide selected from the group consisting of FesCU, a-Fe2Os, y- Fe2C>3, MnFepOq, CoFepOq, NiFepOq, CuFepOq, ZnFepOq, CdFepOq, BaFepO and SrFepO, wherein p and q vary depending on the method of synthesis, and wherein p is preferably an integer of from 1 to 3, more preferably 2, and wherein q is preferably 3 or 4, most preferably y-Fe2O3 (maghemite) and / or FesCU (magnetite).

[0034] In some alternatively preferred embodiments, the core (C) comprises or consists of a polymer, preferably selected from the group consisting of poly(meth)acrylate, polymethyl(meth)acrylate, polystyrene, melamin resin, polylactic acid, methylcellulose, polycaprolacton, chitosan, gelatine, copolymers of two or more of these polymers and mixtures of two or more of these polymers and / or copolymers.

[0035] In some alternatively preferred embodiments, the core (C) comprises or consists of a lipid, preferably selected from the group consisting of triglyceride, partial glyceride, fatty acid, steroid, wax and mixtures of two or more of these lipids.

[0036] Preferably, the optional one or more shell(s) (S) comprises or consists of a polymeric material, more preferably selected from the group consisting of poly(meth)acrylate, polymethyl(meth)acrylate, polystyrene, polyvinylbenzene, melamin resin, polylactic acid, methylcellulose, polycaprolacton, chitosan, gelatine, dextrane, hydroxyl ethyl starch, copolymers of two or more of these polymers and mixtures of two or more of these polymers and / or copolymers.

[0037] Preferably, the optional one or more shell(s) (S) comprises on its surface at least one functional group for protein binding, wherein the at least one functional group for protein binding is preferably selected from the group consisting of halogenated Cl-C3-alkyl group, halogen atom, epoxy group, carboxy group, activated carboxy group, (preferably -acid halide or acid anhydride or succinimide), amino group, hydroxyl group, streptavidin, avidin, biotin, and two or more thereof.

[0038] In some preferred embodiments, the microparticle has an average diameter in the range of from 10 to 1000 pm, preferably in the range of from 250 to 1000 pm, more preferably in the range of from 250 to 500 pm. Universitatsmedizin Greifswald PVA21246PC

[0039] Universitat Greifswald

[0040] -5-

[0041] The microparticle may, in principle, display any geometrical form, however, preferably, the microparticle is substantially spherical. As used herein, the term “substantially spherical” refers to particles with rounded shapes that are preferably non-faceted or substantially free of sharp corners. In certain embodiments, the substantially spherical microparticle typically has an average aspect ratio of less than 3: 1 or 2: 1, for example, an aspect ratio less than 1.5: 1, or less than 1.2: 1. In a certain embodiment, the substantially spherical microparticle may have an aspect ratio of about 1 : 1. The aspect ratio (AR) is defined as being a function of the largest diameter (dmax) and the smallest diameter (dmin) orthogonal to it (AR = d min / dmax ). The diameters are determined or determinable via a method selected from the group consisting of scanning electron microscopy (SEM), dynamic light scattering (DLS) and light microscope measurements.

[0042] Preferably, the microparticle has only pores with a pore size of < 1mm.

[0043] The microparticle is preferably biocompatible and non-toxic in the human system, especially nontoxic for cellular blood components. The microparticle does not induce directly or indirectly an activation of cellular blood components and / or of coagulation factors. The microparticle is preferably stable, especially non-dissolving, at a pH value of a surrounding medium in the range of from 3 to 10, preferably in the range of from 4.0 to 9.5, more preferably in the range of from 5.0 to 9.0, more preferably in the range of from 6.0 to 8.5, more preferably in the range of from 6.5 to 8.2, more preferably in the range of from 7.0 to 8.0, more preferably in the range of from 7.2 to 7.8.

[0044] In some preferred embodiments, the microparticle has an average diameter in the range of from 250 to 500 pm and / or, preferably and, comprises poly(meth)acrylate, or comprises at least a shell (S) comprising poly(meth)acrylate, which carries on its surface at least an amino group and / or an epoxy group. membrane, foil as solid carrier (a)

[0045] In some preferred embodiments, the at least one solid carrier provided in step (a) is a membrane and / or a foil, said membrane and / or foil comprising a material selected from the group consisting of polymer, hydroxyapatite, silica, titanium dioxide, zirconium oxide, lipid, hydrogel (preferably polyvinyl alcohol, agarose, agar) and mixed forms of two or more thereof.

[0046] Preferably, the membrane and / or foil comprises oris a polymer, more preferably selected from the group consisting of polylactide, polylactide-co-glycolide, polycaprolactone, polyurethane, polyethylene oxide, polyethylene glycol, chitosan, gelatine, collagen, hyaluronic acid, alginate, Universitatsmedizin Greifswald PVA21246PC

[0047] Universitat Greifswald

[0048] -6- rubber, cellulose, modified cellulose, copolymers of two or more of these polymers, and mixtures of two or more of these polymers and / or copolymers.

[0049] A “membrane” is preferably a membrane of a blood bag, and is preferably porous. A “foil” is preferably a foil of a blood bag and is preferably non-porous.

[0050] The membrane and / or foil is preferably biocompatible and non-toxic in the human system, especially non-toxic for cellular blood components. The membrane and / or foil does not trigger directly or indirectly activation of cellular blood components and / or of coagulation factors. The membrane and / or foil is preferably stable, especially non-dissolving, at a pH value of a surrounding medium in the range of from 3 to 10, preferably in the range of from 4.0 to 9.5, more preferably in the range of from 5.0 to 9.0, more preferably in the range of from 6.0 to 8.5, more preferably in the range of from 6.5 to 8.2, more preferably in the range of from 7.0 to 8.0, more preferably in the range of from 7.2 to 7.8.

[0051] Cellular blood components

[0052] Preferably, the cellular blood components are selected from the group consisting of platelets (thrombocytes), red blood cells (erythrocytes), leukocytes, stem cells and mixtures of two or more thereof.

[0053] In some preferred embodiments, the liquid phase LP1 comprises at least erythrocytes, and is more preferably an erythrocyte concentrate.

[0054] An erythrocyte concentrate (blood product) is obtained or obtainable from whole blood. Preferably, an erythrocyte concentrate is enriched during separation from whole blood, i.e. the erythrocyte concentrate has a higher concentration of erythrocytes than the underlying whole blood, the specification of the erythrocyte concentrate according to the German hemotherapy guideline means a hematocrit in the range of 0.5-0.7 liters / liter (whole blood 0.3-0.5 liters / liter), as well as hemoglobin > 40 g / unit (unit: approx. 250 mL) (whole blood approx. 35 g / 250 mL). The separation of the erythrocytes from the whole blood is usually done gravimetrically, preferably by centrifugation, whereby the residue containing the erythrocytes is reabsorbed into additive solution, whereby the erythrocyte concentrate is obtained. An erythrocyte concentrate is preferably selected from the group consisting of apheresis erythrocyte concentrate, irradiated erythrocyte concentrate, filtered erythrocyte concentrate, leukocyte-depleted erythrocyte concentrate, pathogen-reduced, preferably pathogen-inactivated erythrocyte concentrate, washed erythrocyte concentrate and mixtures of these erythrocyte concentrates. Mixed forms of these erythrocyte concentrates are known to the person skilled in the art, for example leukocyte-depleted irradiated erythrocyte concentrate, as is known under the approval / reg. no. (AMG76): PEI.H.02806.01.1. In Universitatsmedizin Greifswald PVA21246PC

[0055] Universitat Greifswald

[0056] -7- some embodiments, an erythrocyte concentrate comprises erythrocyte concentrates from more than one donor, especially from at least two donors having different blood groups.

[0057] In some alternatively preferred embodiments, the liquid phase LP1 comprises at least platelets, and is more preferably a platelet concentrate.

[0058] A platelet concentrate is obtained or obtainable from human blood (blood product). Platelet concentrates contain blood platelets, thrombocytes, which are suspended in stabilized blood plasma from the donor or in an additive solution or composition of both in different ratios. Platelet concentrates are either platelet apheresis concentrates from a single donor or pooled platelet concentrates from several (suitable) donors. A platelet concentrate is preferably selected from the group consisting of leukocyte-depleted pool platelet concentrate, leukocyte-depleted apheresis platelet concentrate and irradiated platelet concentrate. In some embodiments, a platelet concentrate comprises platelets from more than one donor, especially from at least two donors having different blood groups.]

[0059] In some alternatively preferred embodiments, the liquid phase LP1 comprises at least human stem cells (e.g. hematopoietic and mesenchymal stem cells), and is more preferably a human hematopoietic stem cell preparation.

[0060] A human hematopoietic stem cell preparation is obtained from human peripheral blood or bone marrow. Human hematopoietic stem cell preparations obtained from peripheral blood by apheresis contain human hematopoietic stem cells, suspended in an additive solution. Human hematopoietic stem cell preparations from bone marrow are obtained from bone marrow by direct harvesting, suspended in additive solution.

[0061] Antigen(s)

[0062] Preferably, the one or more antigen(s) are covalently bound to the cellular blood components.

[0063] In some preferred embodiments, the one or more antigen(s) are blood group specific antigen(s).

[0064] Preferably, if the cellular blood components comprise leukocytes, the one or more antigen(s) are blood group specific antigen(s) and / or human leukocyte antigen(s) (HLAs).

[0065] Preferably, if the cellular blood components comprise granulocytes, the one or more antigen(s) comprise human neutrophil antigen(s) (HNAs) and / or human leukocyte antigen(s) (HLAs). Universitatsmedizin Greifswald PVA21246PC

[0066] Universitat Greifswald

[0067] -8-

[0068] Preferably, if the cellular blood components comprise platelets, the one or more antigen(s) comprise blood group specific antigen(s) and / or human leukocyte antigen(s) (HLAs) of class I and / or human platelet antigens (HP As).

[0069] Preferably, if the cellular blood components comprise erythrocytes, the one or more antigen(s) comprise blood group specific antigen(s).

[0070] Blood group specific antigen(s) are preferably selected from the group consisting of antigen of the ABO blood group system, MNS blood group system, P1PK blood group system, Rh blood group system, Lutheran blood group system, Kell blood group system, Lewis blood group system, DARC (Duffy) blood group system, Kidd blood group system, Diego blood group system, Yt blood group system, Xg blood group system, Scianna blood group system, Dombrock blood group system, Colton blood group system, Landsteiner-Wiener blood group system, Chido / Rodgers blood group system, H blood group system, Kx blood group system, Gerbich blood group system, Cromer blood group system, Knops blood group system, Indian blood group system, Ok blood group system, Raph blood group system, JohnMiltonHagen blood group system, I blood group system, Globoside blood group system, Gill blood group system, Rh-associated glycoprotein blood group system, FORS blood group system, JR blood group system, LAN blood group system, Vel blood group system, CD59 blood group system, Augustine blood group system, Kanno blood group system, SID blood group system, CTL2 blood group system, PEL blood group system, MAM blood group system, EMM blood group system, ABCC1 blood group system, Er blood group system, CD36 blood group system, and mixtures of antigens of two or more of these blood group systems.

[0071] More preferably, blood group specific antigen(s) are selected from the group consisting of antigen of the ABO blood group system, Rh blood group system, DARC (Duffy) blood group system and mixtures of antigens of two or more of these blood group systems.

[0072] Enzyme

[0073] Preferably, the at least one enzyme is selected from the group of enzymes capable of removing at least a part of an antigen from a cellular blood component.

[0074] In some preferred embodiments, if the blood group specific antigen(s) are A antigen(s) of the ABO blood group system, at least one enzyme is selected from the group consisting of hydrolases, preferably deacetylase, carbohydrate active enzymes such as galactosaminidase, enzymes of the Glycoside Hydrolase Family 109 (GH109) and mixtures of two or more thereof. Universitatsmedizin Greifswald PVA21246PC

[0075] Universitat Greifswald

[0076] -9-

[0077] Preferably, the at least one enzyme is selected from the group consisting of enzymes having at least 95%, more preferably at least 98%, more preferably at least 99%, more preferably 100%, sequence identity with FpGalNAcDeAc (7V-acetyl-a-D-galactosamine deacetylase from Flavonifractor plautii , FpGalNase (a-D-galactosamine galactosaminidase from Flavonifractor plautii), AmGH109B (a-N-acetylgalactosaminidase from Akkermansia muciniphila ATCC BAA-835), AmGH109A (a-N-acetylgalactosaminidase from Akkermansia muciniphila ATCC BAA-835), NAGA (a-N-acetylgalactosaminidase from Elizabethkingia meningoseptica ATCC 13253), nagA (a-N-acetylgalactosaminidase from Elizabethkingia meningoseptica ATCC 33958), Nag68 (a-N-acetylgalactosaminidase from Alkalimonas sp), NAg69 (a-N-acetylgalactosaminidase from Pararheinheimera sp), Nag71 (a-N- acetylgalactosaminidase from Amycolatopsis sp) and mixtures of two or more thereof.

[0078] More preferably, the at least one enzyme is a combination of an enzyme having at least 95%, more preferably at least 98%, more preferably at least 99%, sequence identity with FpGalNAcDeAc (7V- acetyl-a-D-galactosamine deacetylase from Flavonifractor plautii) and an enzyme having at least 95%, preferably at least 98%, more preferably at least 99%, sequence identity with FpGalNase (a- D-galactosamine galactosaminidase from Flavonifractor plautii .

[0079] In some preferred embodiments, the at least one enzyme is a combination of FpGalNAcDeAc (7V- acetyl-a-D-galactosamine deacetylase from Flavonifractor plautii and FpGalNase (a-D- galactosamine galactosaminidase from Flavonifractor plautii .

[0080] Preferably, if the blood group specific antigen(s) are B antigen(s) of the ABO blood group system, the at least one enzyme is selected from the group of a-galactosidases of the Glycoside Hydrolase Family 110 (GH110) and mixtures of two or more thereof.

[0081] More preferably, the at least one enzyme is selected from the group consisting of enzymes having at least 95%, more preferably at least 98%, more preferably at least 99%, more preferably 100%, sequence identity with BfGalB (a-l,3-galactosidase from Bacteroides fragilis), PmGal (a-1,3- galactosidase from Phocaeicola massiliensis). BpGal (a- 1,3 -galactosidase from Bacteroides pyogenes), PjGal (a- 1,3 -galactosidase from Parabacteroides johnsonii), PpaGal (a- 1,3- galactosidase from Pedobacter panaciterrae), CcGal (a- 1,3 -galactosidase from Capnocytophaga canimorsus, ), AkMuGalB (a- 1,3 -galactosidase from Akkermansia muciniphila), AkMuGalA (a- 1,3-galactosidase from Akkermansia muciniphila , BbGal (a- 1,3 -galactosidase from Bifidobacterium bifidum), Gall lOA (a-l,3-galactosidase from Bacteroides thetaiotaomicron VPI- 5482), Gall lOB (a-l,3-galactosidase from Bacteroides thetaiotaomicron VPI-5482), SgGall lOA (a- 1,3 -galactosidase Fom Pelerkaempfera griseoplana 2357), Gall lOA (a- 1,3 -galactosidase from Streptomyces avermitilis MA-4680) and mixtures of two or more thereof. Universitatsmedizin Greifswald PVA21246PC

[0082] Universitat Greifswald

[0083] -10-

[0084] More preferably, the at least one enzyme is selected from the group consisting of enzymes having at least 95%, more preferably at least 98%, more preferably at least 99%, more preferably 100%, sequence identity with BfGalB (a-l,3-galactosidase from Bacteroides fragilis), PmGal (a-1,3- galactosidase from Phocaeicola massiliensis . BpGal (a- 1,3 -galactosidase from Bacteroides pyogenes), PjGal (a- 1,3 -galactosidase from Parabacteroides johnsonii), PpaGal (a- 1,3- galactosidase from Pedobacter panacilerrae), CcGal (a- 1,3 -galactosidase from Capnocytophaga canimorsus, ), and mixtures of two or more thereof.

[0085] In some preferred embodiments, the at least one enzyme is selected from the group consisting of PpaGal (a- 1,3 -galactosidase from Pedobacter panacilerrae), PpaGal_W260Y of SeqID No. 1, PpaGal_K238Q of SeqID No. 2, PpaGal_P182M of SeqID No. 3 and mixtures of two or more thereof.

[0086] Bonding enzyme / carrier

[0087] The at least one enzyme is preferably bound to the solid carrier by a bonding type selected from the group consisting of covalent bonding, physical bonding, affinity bonding, bonding via antibodies, covalent cross-linking and mixed forms of two or more of these bonding types.

[0088] “Covalent bonding” means the formation of a covalent bond between the at least one enzyme and the solid carrier, “physical bonding” means preferably a bonding formed due Van der Waals forces, hydrophobic interactions and / or electrostatic attraction between the at least on enzyme and the solid carrier, “affinity bonding” preferably means a biotin-(strept)avidin bonding and / or the formation of His-tag-nickel complexes between the at least one enzyme and the solid carrier, “bonding via antibodies” means that antibodies and their respective antigens are connected to enzyme and solid carrier respectively and form the bond, “covalent cross-linking” preferably means cross-linking by covalent bonds with cross-linking reagents such as glutaraldehyde.

[0089] Preferably, the at least one enzyme is bound to the solid carrier by at least covalent bonding, more preferably via an amine and / or thiol group of the enzyme, more preferably by at least covalent bonding between an amine group of the amine and a carboxyl group and / or epoxy group of the solid carrier and / or by at least covalent bonding between a thiol group of the enzyme and a maleimide group and / or a sulfhydryl group of the solid carrier.

[0090] When the liquid phase LP1 comprises at least erythrocytes and / or platelets carrying blood group specific antigen(s) of the ABO blood group system, preferably platelets carrying blood group specific antigen(s) of the ABO blood group system, wherein for platelets carrying at least A antigen(s) of the ABO blood group system, the reduced expression of A antigen(s) LA-A(2) is in the range of from 0.01 x LA-A(1) to 0.8 x LA-A(1), preferably in the range of from 0.01 x LA-A(1) to 0.5 Universitatsmedizin Greifswald PVA21246PC

[0091] Universitat Greifswald

[0092] -11- x LA-A(1), more preferably in the range of from 0.01 x LA-A(1) to 0.2 x LA-A(1), more preferably in the range of from 0.01 x LA-A(1) to 0.1 x LA-A(1), more preferably in the range of from 0.01 x LA- A(1) to 0.05 x LA-A(1); and / or wherein for platelets carrying at least B antigen(s) of the ABO blood group system, the reduced expression of B antigen(s) LA-B(2) is in the range of from 0.01 x LA-B (1) to 0.8 x LA-B (1), preferably in the range of from 0.01 x LA-B (1) to 0.5 X LA-B (1), more preferably in the range of from 0.01 x LA-B(1) to 0.3 x LA-B(1).

[0093] Contacting in step (c)

[0094] Contacting the solid carrier carrying at least one enzyme with the liquid phase is done in step (c) preferably at a pH value in the range of from 3 to 10, preferably in the range of from 4.0 to 9.5, more preferably in the range of from 5.0 to 9.0, more preferably in the range of from 6.0 to 8.5, more preferably in the range of from 6.5 to 8.2, more preferably in the range of from 7.0 to 8.0, more preferably in the range of from 7.2 to 7.8.

[0095] Preferably, contacting the solid carrier carrying at least one enzyme with the liquid phase is done in step (c) at a temperature in the range of from 0 °C to 40 °C.

[0096] If the cellular blood components comprise platelets, contacting the solid carrier carrying at least one enzyme with the liquid phase is done in step (c) preferably at a temperature in the range of from 20-24 °C.

[0097] If the cellular blood components comprise erythrocytes, contacting the solid carrier carrying at least one enzyme with the liquid phase is done in step (c) preferably at a temperature in the range of from 2-6 °C.

[0098] Preferably, contacting the solid carrier carrying at least one enzyme with the liquid phase is done in step (c) for a period of time of at least 30 minutes, more preferably for a period of time in the range of from 30 minutes to 1 month.

[0099] Liquid phases

[0100] The liquid phase LP1 is preferably an aqueous liquid phase and / or, preferably and, the liquid phase LP2 is preferably an aqueous liquid phase.

[0101] “Mixed” antigens Universitatsmedizin Greifswald PVA21246PC

[0102] Universitat Greifswald

[0103] -12-

[0104] In some preferred embodiments, the process comprises

[0105] (a.1) Providing at least one solid carrier carrying at least a first enzyme and a second enzyme, or (a.2) Providing at least a first solid carrier carrying at least a first enzyme and at least a second solid carrier carrying a second enzyme, wherein the first enzyme is different from the second enzyme;

[0106] (b.l) Providing a liquid phase LP1 comprising at least a first cellular blood component having first antigen(s) with a native expression of antigen(s) L1A(1) and at least a second cellular blood component having second antigen(s) with a native expression of antigen(s) L2A(1), with the first antigen(s) being different from the second antigen(s);

[0107] (c.1) Contacting the solid carrier carrying at least a first enzyme and a second enzyme according to step (a. l) or the at least a first solid carrier carrying at least a first enzyme and at least a second solid carrier carrying a second enzyme according to step (a.2), with the liquid phase provided according to step (b.l) under conditions allowing a removal of at least a part of the antigen(s) from the cellular blood components; thereby obtaining a liquid phase LP2 comprising at least the first cellular blood component having first antigen(s) with a reduced antigen(s) expression L1A(2) compared to the native expression of antigen(s) L1A(1), wherein L1A(2) < L1A(1), at least the second cellular blood component having second antigen(s) with a reduced antigen(s) expression L2A(2) compared to the native expression of antigen(s) L2A(1), wherein L2A(2) < L2A(1), and the solid carrier(s) carrying the enzyme(s) in a concentration Clsc(l) and C2sc(l).

[0108] Separation step

[0109] In some preferred embodiments, the process further comprises

[0110] (d) separating the liquid phase LP2 obtained in (c) or (c.1), thereby obtaining the solid carrier(s) in separated form and a liquid phase LP3 comprising the cellular blood component(s) having antigen(s) with expressions of antigen(s) LA(2) or LI A(2) and L2A(2) respectively, said liquid phase LP3 having (a) reduced concentration(s) of the solid carrier(s) carrying the enzyme(s) Csc(2) or Clsc(2) and C2sc(2) respectively, with Csc(2) < Csc(l) or Clsc(2) < Clsc(l) and C2sc(2) < C2sc(l) respectively.

[0111] Separation in step (d) is preferably done by a mechanical separation method, more preferably by filtration, more preferably by filtration via a filter having a pore size in the range of from 10 pm to 250 pm, more preferably via a filter integrated in a transfusion set.

[0112] In some preferred embodiments, separation in step (d) is done by magnetic forces, more preferably via a magnet. Universitatsmedizin Greifswald PVA21246PC

[0113] Universitat Greifswald

[0114] -13-

[0115] The process is preferably an in vitro or ex vivo process, more preferably an in vitro process.

[0116] 2ndaspect - System

[0117] A second aspect of the invention is directed to a system for removing antigen(s) from cellular blood components comprising:

[0118] (a) at least one solid carrier;

[0119] (b) at least one enzyme; wherein the at least one enzyme is bound to the at least one solid carrier.

[0120] All details, embodiments, preferred and alternatively preferred embodiments described in the section above related to the process, i.e. the first aspect of the invention, also apply for the system of the second aspect of the invention.

[0121] 3rdaspect - PpaGal mutations

[0122] In a third aspect, the invention is directed to PpaGal mutations, preferably an a-l,3-galactosidase of SeqID No. 1 (PpaGal_W260Y), and / or an a- 1,3 -galactosidase of SeqID No. 2 (PpaGal_K238Q) and / or an a-l,3-galactosidase of SeqID No. 3 (PpaGal_P182M).

[0123] Compared to the wildtype of PpaGal (PpaGal wt), these mutations resulted in an increased or comparable activity with respect to B antigen removal. Relative activity and specific activity of PpaGal_W260Y, PpaGal_K238Q and PpaGal_P182M are listed in Table 1, as well as turnover rates (kcat) the values of the Michaelis Menten constant Km and the quotient of Kcat / Km. In addition, melting points are indicated in Table 1. Model substrate was a simple galactose p- nitrophenyl ( / ?NP) substrate ( / ?-nitrophenyl a-D-galactopyranoside).

[0124] Table 1

[0125] Characteristics of PpaGal mutations compared to the wildtype (investigation with model substrate Universitatsmedizin Greifswald PVA21246PC

[0126] Universitat Greifswald

[0127] -14-

[0128] Furthermore, activity tests with erythrocytes as a substrate were performed and the agglutination rates of these mutations in comparison to the wildtype of PpaGal (PPaGal wt) were evaluation at different concentrations. The results are summarized in Table 2.

[0129] Table 2

[0130] Agglutination rates of enzyme mutants and wildtype at different concentrations

[0131] The amino acid sequences of these PpaGal mutations are as follows:

[0132] SeqID No. 1 (PpaGal_W260Y)

[0133] MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSEFNVKIYKLSAYGIKPNSGKNTTPLLTSLL KEIKSKTSDLDKVIIQFEKGRYDFYPEGAIKREYYISNHDQDNPKTVGIGIEKFNNITLIGKGTDLM FHGRMLPLALIESSNVKIKDLNIDFEKPQITQVKIISNDTTAGNIVFETAPWVKYKLKDSTFYNTGE GWEMQPTSGIAFENGTKHIIFNSGDIGVGTKSVSEVSPGKIMAHHWKNKKLVPGTVIAMRSYQR PAPGIFVHKGKNISFENVKVHYAEGMGLLAQLTENIYMDGFGVCLRGKNDPRYFTTQADATHFS GCKGEIVSKNGLYEGMMDDAINIHGTYLKITKKLDDHTVIANYMHEQSYGFDWGNIRDTVQFIQ SKTMELWDAKNTIASIKPILRNSTDPIKEFRIEFTKALDPVIDPSKQDIGIENLSWTPSVVFTGNTIR

[0134] NNRARGALFSTPKPTLVANNLFDHTSGCAILLCGDSNGWYETGSCRDITIRDNKFVNALTSMYQF TSAIISIYPEIPDLTNQKKYFHSGIRILNNQFDTFDQPILYAKSVDGLVFTGNKIQTNKEYPAFHSNK KRFLFERVIGVDFSDNKVDGKPIEML

[0135] SeqID No. 2 (PpaGal_K238Q)

[0136] MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSEFNVKIYKLSAYGIKPNSGKNTTPLLTSLL KEIKSKTSDLDKVIIQFEKGRYDFYPEGAIKREYYISNHDQDNPKTVGIGIEKFNNITLIGKGTDLM FHGRMLPLALIESSNVKIKDLNIDFEKPQITQVKIISNDTTAGNIVFETAPWVKYKLKDSTFYNTGE GWEMQPTSGIAFENGTKHIIFNSGDIGVGTKSVSEVSPGQIMAHHWKNKKLVPGTVIAMRSWQR PAPGIFVHKGKNISFENVKVHYAEGMGLLAQLTENIYMDGFGVCLRGKNDPRYFTTQADATHFS GCKGEIVSKNGLYEGMMDDAINIHGTYLKITKKLDDHTVIANYMHEQSYGFDWGNIRDTVQFIQ SKTMELWDAKNTIASIKPILRNSTDPIKEFRIEFTKALDPVIDPSKQDIGIENLSWTPSVVFTGNTIR

[0137] NNRARGALFSTPKPTLVANNLFDHTSGCAILLCGDSNGWYETGSCRDITIRDNKFVNALTSMYQF TSAIISIYPEIPDLTNQKKYFHSGIRILNNQFDTFDQPILYAKSVDGLVFTGNKIQTNKEYPAFHSNK KRFLFERVIGVDFSDNKVDGKPIEML

[0138] SeqID No. 3 (PpaGal_P182M)

[0139] MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSEFNVKIYKLSAYGIKPNSGKNTTPLLTSLL

[0140] KEIKSKTSDLDKVIIQFEKGRYDFYPEGAIKREYYISNHDQDNPKTVGIGIEKFNNITLIGKGTDLM FHGRMLPLALIESSNVKIKDLNIDFEKPQITQVKIISNDTTAGNIVFETAMWVKYKLKDSTFYNTG EGWEMQPTSGIAFENGTKHIIFNSGDIGVGTKSVSEVSPGKIMAHHWKNKKLVPGTVIAMRSWQ RPAPGIFVHKGKNISFENVKVHYAEGMGLLAQLTENIYMDGFGVCLRGKNDPRYFTTQADATHF SGCKGEIVSKNGLYEGMMDDAINIHGTYLKITKKLDDHTVIANYMHEQSYGFDWGNIRDTVQFI QSKTMELWDAKNTIASIKPILRNSTDPIKEFRIEFTKALDPVIDPSKQDIGIENLSWTPSVVFTGNTI RNNRARGALFSTPKPTLVANNLFDHTSGCAILLCGDSNGWYETGSCRDITIRDNKFVNALTSMY Universitatsmedizin Greifswald PVA21246PC

[0141] Universitat Greifswald

[0142] -15-

[0143] QFTSAIISIYPEIPDLTNQKKYFHSGIRILNNQFDTFDQPILYAKSVDGLVFTGNKIQTNKEYPAFHS NKKRFLFERVIGVDFSDNKVDGKPIEML

[0144] All details, embodiments, preferred and alternatively preferred embodiments described in the section above related to the first aspect of the invention, also apply for the third aspect of the invention.

[0145] Other enzymes disclosed herein are to be found in the Sequence Listing as indicated below in Table 3.

[0146] Table 3

[0147] Enzymes, data base, identifier, SeqID No. Universitatsmedizin Greifswald PVA21246PC

[0148] Universitat Greifswald

[0149] -16-

[0150] 4thaspect - Product-by-process

[0151] A fourth aspect of the invention is directed to a cellular blood component having a reduced expression of antigen(s) LA(2) compared to a native expression of antigen(s) LA(1) of said cellular blood component with LA(2) < LA(1), obtained or obtainable by a process according to any one of embodiments 1 to 45.

[0152] All details, embodiments, preferred and alternatively preferred embodiments described in the section above related to the first aspect of the invention and in the section related to the third aspect of the invention, also apply for the fourth aspect of the invention.

[0153] 5thaspect - Product

[0154] In a fifth aspect, the invention is directed to a cellular blood component having a reduced expression of antigen(s) LA(2) compared to a native expression of antigen(s) LA(1) of said cellular blood component with LA(2) < LA(1).

[0155] All details, embodiments, preferred and alternatively preferred embodiments described in the section above related to the first aspect of the invention, in the section related to the third aspect of the invention, and in the e section related to the fourth aspect of the invention also apply for the fifth aspect of the invention.

[0156] 6thaspect - Medical Use

[0157] A sixth aspect of the invention is directed to a cellular blood component having a reduced expression of antigen(s) LA(2) compared to a native expression of antigen(s) LA(1) of said cellular blood component with LA(2) < LA(1) as described in detail herein above for use in transfusion.

[0158] All details, embodiments, preferred and alternatively preferred embodiments described in the section above related to the first aspect of the invention, in the section related to the third aspect of the invention, in the section related to the fourth aspect of the invention, and in the section related to the fifth aspect of the invention also apply for the sixth aspect of the invention. Universitatsmedizin Greifswald PVA21246PC

[0159] Universitat Greifswald

[0160] -17-

[0161] Cellular blood component for transfusion

[0162] The present invention further relates to a cellular blood component having a reduced expression of antigen(s) LA(2) compared to a native expression of antigen(s) LA(1) of said cellular blood component with LA(2) < LA(1) as described in detail herein above for use in medicine, preferably in transfusion.

[0163] The present invention also relates to a method for treating a subject in need of transfusion, said method comprising

[0164] (I) administering a cellular blood component having a reduced expression of antigen(s) LA(2) compared to a native expression of antigen(s) LA(1) of said cellular blood component with LA(2) < LA(1) as described in detail herein above to said subject, and

[0165] (II) thereby treating and / or preventing said need for transfusion in said subject.

[0166] The method for treating is an in vivo method comprising administration of a cellular blood component having a reduced expression of antigen(s) LA(2) compared to a native expression of antigen(s) LA(1) of said cellular blood component with LA(2) < LA(1) to a subject, as specified herein above. The method may, however, comprise further steps such as evaluating or having evaluated the need for transfusions from said subject.

[0167] Thus, the method may in particular be method for treating a need for transfusion in a subject, said method comprising

[0168] (0) evaluating or having evaluated the need for transfusion of said subject;

[0169] (I) based on the result of the evaluation in step (0), administering a cellular blood component having a reduced expression of antigen(s) LA(2) compared to a native expression of antigen(s) LA(1) of said cellular blood component with LA(2) < LA(1) to said subject, and

[0170] (II) thereby treating said need for transfusion in said subject.

[0171] The term "subject", as referred to herein, relates to a vertebrate animal, preferably a mammal, for example, a livestock, companion, or laboratory animal. Most preferably, the subject is a human. Preferably, the subject has been diagnosed to suffer from a need for transfusion or is suspected to suffer from a need for transfusion. Preferably, the need for transfusion of said subject has been identified to be susceptible to treatment with a cellular blood component having a reduced expression of antigen(s) LA(2) compared to a native expression of antigen(s) LA(1) of said cellular blood component with LA(2) < LA(1), preferably as specified herein below, and / or the need for transfusion of said subject has been stratified as described herein elsewhere. Universitatsmedizin Greifswald PVA21246PC

[0172] Universitat Greifswald

[0173] -18-

[0174] The terms "treating" and “treatment” refer to an amelioration of the diseases or disorders, especially the need for transfusion, referred to herein or the symptoms accompanied therewith to a significant extent. Said treating as used herein also includes an entire restoration of health with respect to the diseases or disorders referred to herein. It is to be understood that treating, as the term is used herein, may not be effective in all subjects to be treated. However, the term shall require that, preferably, a statistically significant portion of subjects suffering from a disease or disorder referred to herein can be successfully treated. Whether a portion is statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools, e.g., determination of confidence intervals, p-value determination, Student's t-test, Mann- Whitney test etc. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99%. The p-values are, preferably, 0.1, 0.05, 0.01, 0.005, or 0.0001. Preferably, the treatment shall be effective for at least 10%, at least 20% at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the subjects of a given cohort or population. Preferably, treating a need for transfusion is increasing the load of the respective cellular blood component in a subject. As will be understood by the skilled person, methods and effectiveness of treatment of e.g. a need for transfusion is dependent on a variety of factors including, e.g. stage and severity.

[0175] As used herein, the term "susceptible to treatment" relates to the property of a subject and / or a need of transfusion thereof, to be improved by said treatment. Thus, in a subject susceptible to treatment as described herein, upon administration of an effective dose of a cellular blood component having a reduced expression of antigen(s) LA(2) compared to a native expression of antigen(s) LA(1) of said cellular blood component with LA(2) < LA(1), said need of transfusion is preferably decreased. As the skilled person understands from the description herein above, the subject may be found to be susceptible to treatment with a single therapy, e.g. single treatment with a cellular blood component having a reduced expression of antigen(s) LA(2) compared to a native expression of antigen(s) LA(1) of said cellular blood component with LA(2) < LA(1)„ but may also be found to be susceptible to a multi-phase treatment, e.g. by repeated treatment with a cellular blood component having a reduced expression of antigen(s) LA(2) compared to a native expression of antigen(s) LA(1) of said cellular blood component with LA(2) < LA(1). Using the method of, optionally repeatedly, treatment steps can be optimized in accordance with the physiological state of the need of transfusion.

[0176] The invention is disclosed herein below in more details with respect to platelet concentrates:

[0177] Regarding platelet concentrates, there were two aspects to consider in this approach: First, plasma isoagglutinins in the platelet concentrate transfused in a recipient of a different blood group can cause red cell hemolysis and platelet aggregation due to antibody-cell binding (minor incompatibility). Said problem of the described ABO minor incompatibility due to isoagglutinins in the plasma was solved Universitatsmedizin Greifswald PVA21246PC

[0178] Universitat Greifswald

[0179] -19- without the need to interact by adsorbing anti-A and anti-B antibodies onto residual red blood cells in buffy coats during their manufacturing. Red-cell-isoagglutinin complexes are removed by centrifugation and separation during platelet concentrate manufacturing.

[0180] Second, platelets from platelet concentrate of a different blood group can bind recipient isoagglutinins leading to decreased platelet survival as a result of immune complex formation followed by degradation and removal of platelets by liver and spleen (major incompatibility). The A and B antigens on the platelet surface causing major incompatibility were eliminated by enzymatic removal of the A and B antigens using enzymes coated on microparticles. A-antigen were jointly removed by the A-acetyl-a- D-galactosamine deacetylase (FpGalNAcDeAc) and a-D-galactosaminidase from Flavonifractor plautii (FpGalNase) as described (Fig. la). For B-antigen removal (Fig. lb), several previously unstudied a-l,3-galactosidases of the GH110 family were investigated. The a- 1,3- galactosidase from Pedobacter panaciterrae (PpaGal) is more efficient than previously known a- 1,3-galactosidases, which reduces the enzyme needed for B-antigen removal. This method preserves the quality parameters required for platelet transfusions, and the enzyme-coated microparticles can be easily removed by filtration with standard transfusion equipment. Additionally, the risk of leakage of covalently linked enzymes from the microparticles is minimal, making this an effective and safe technique for producing universal platelet concentrates.

[0181] Results and Discussion

[0182] Production of Pooled Platelet Concentrates from Buffy Coats of Non-Identical ABO Blood Groups

[0183] Pooled platelet concentrates from four ABO-non-identical buffy coats were produced (see also Table 4). The A- and B-RBC in the buffy coats adsorb the corresponding anti-A and anti-B antibodies in the remaining plasma and the isoagglutinin-red-cell complexes are then removed by centrifugation within the production process; the centrifugation parameters were set that red cells sediment while platelets remain in the supernatant (Fig. 2a). Herewith the first issue of producing universal platelets was solved, i.e. removing the anti-A / anti-B antibodies from the platelet concentrates without a loss in product quality.

[0184] Table 4

[0185] Isoagglutinin titer and buffy coat blood groups of pooled platelet concentrates from buffy coats of non-identical blood groups. Universitatsmedizin Greifswald PVA21246PC

[0186] Universitat Greifswald

[0187] -20-

[0188] The regulatory quality parameters of platelet concentrates, like platelet count, pH, and preserved thrombin receptor-activating peptide-6 (TRAP6) platelet activation, determined by CD62P expression, are comparable between pooled platelet concentrates of blood group identical and nonidentical buffy coats for up to day 4 (Fig. 2b-d). Adsorption of anti-A and anti- B IgM on RBC did not cause an increase in free hemoglobin (Fig. 2e, 2f). Increased anti-A and / or anti-B IgG binding on platelet surface proteins expressing A and B blood groups were also excluded, i.e. glycoprotein (GP) Illblla, GP IbIX and PEC AM- 1 (CD31) by the gold standard monoclonal antibody immobilization of platelet antigens (MAIPA) assay (Fig. 2g).1'51

[0189] Enzymatic Removal of A- and B-Antigens

[0190] Next, platelet concentrates incompatibility due to the A and B antigens on the platelet surface was addressed by enzymatic deglycosylation, as it was previously demonstrated for RBCs. In this context, the prokaryotic family of a-galactosidases (CAZy GH110), which selectively cleaves terminal a-l,3-linked galactose residues of B-antigens at neutral pH optimum, attracted attention (Fig. 1).

[0016] Bacteroides fragilis expresses two a- galactosidases (=B-zymes) of the GH110 family (BfGalA and BfGalB), which show similar behaviour in the removal of a-1,3- linked galactose residues from B-antigens. For the enzymatic conversion of the A antigen to the H antigen, an enzyme pair was selected that efficiently works in concert (=A-zymes). First, a deacetylase generates a galactosamine intermediate (GalN) and second, a GH36 a-galactosaminidase cleaves the remaining galactosamine (Fig. la). These two enzymes were highly efficient and required 15-30-fold less enzyme for the removal of the A antigen compared to previously applied enzymes like the a-N-

[0191]

[0017] acetylgalactosaminidases from Elizabethkingia meningoseptica.

[0192] First, the activity of the A-zymes by RBC (Fig. 3a) was assessed. For this, the A-zymes were recombinantly expressed in E. coli and the enzymes were purified afterward (see Table 5 below). Four different enzyme concentrations were incubated with A-type RBC, followed by an agglutination test with an anti-A-antibody. No agglutination could be seen even with the second lowest enzyme concentration (0.15 mg mL‘1 FpGalNAcDeAc and 0.3 mg mL‘l FpGalNase). For B-zymes, several unstudied a- 1,3 -galactosidases were investigated using a galactosidase from Bacteroides fragilis Universitatsmedizin Greifswald PVA21246PC

[0193] Universitat Greifswald

[0194] -21-

[0195] (BfGalB) as a benchmark / 16’181Five homologs of BfGalB with a sequence similarity between 52% and 79% were identified and recombinantly expressed in E. coli followed by purification (see Table 6 below). The enzymes were biochemically characterized regarding their kinetic parameters for a simple galactose / ?-nitrophenyl ( / ?NP) substrate ( / ?-nitrophenyl a-D-galactopyranoside), pH optima, residual activities, and thermostabilities (Table 6, Table 7, Fig. 4). The benchmark enzyme BfGalB showed the highest activity towards the NP model substrate (Table 6, Table 7). However, using RBC, the new galactosidase PpaGal from Pedobacter panaciterrae is most efficient (Fig. 3b). Even the lowest used enzyme concentration of PpaGal (0.05 mg mL-1) led to complete B antigen removal. Moreover, the lower activity of PpaGal for pNP indicates that it also has a higher specificity for the branched B-antigen compared to BfGalB.

[0196] Next, A- and B-zymes were incubated with platelets from pooled platelet concentrates of the corresponding blood type. The lowest A-zyme concentrations completely removed the A-antigen within 24 hours (Fig. 3c). Also, the lowest concentration of PpaGal reduced the B-antigen completely (Fig. 3d).

[0197] Table 5

[0198] Expression yields of the A-zymes FpGalNAcDeAc and FpGalNase for the enzymatic removal of A-antigens expressed in E. coli BL21 Gold (DE3) for one liter of culture volume.

[0199] Table 6

[0200] List of the original organisms of the investigated B-zymes (a-l,3-galactosidases) used for the enzymatic removal of B-antigens, their sequence similarities towards the a- 1,3 -galactosidase of Bacteroides fragilis, protein yields for the recombinant expression in E. coli BL21 Gold (DE3) for one liter of culture volume after by IMAC purification, melting points (Tm) in 50 mM sodium phosphate buffer pH 7.5 (storage buffer) and SSP+ buffer measured with nanoDSF, residual activities after 81 days storage at 4°C, further kinetic parameters as the catalytic constant kcat, the Universitatsmedizin Greifswald PVA21246PC

[0201] Universitat Greifswald

[0202] -22-

[0203] Michaelis-Menten constant Kmand the catalytic efficiency kcat / Kmvalues with p-nitrophenyl a-D- galactopyranoside as quantified at 405 nm.

[0204] Table 7 Original organisms of the investigated B-zymes (a-l,3-galactosidases) used for the enzymatic removal of B-antigens and their specific activities a. n=3, mean±standard deviation.

[0205] Hereby, the issue of removing A and B antigens from platelets was solved. However, before the transfusion of universal platelets to patients, the enzymes had to be removed from the platelet concentrates to avoid potential immune reactions.

[0206] Removal of Blood Group Antigen by Immobilized Enzymes Universitatsmedizin Greifswald PVA21246PC

[0207] Universitat Greifswald

[0208] -23-

[0209] As an alternative to tedious washing and centrifugation steps to remove the enzymes from the platelet concentrates, which would also compromise the platelet concentrate quality, a method to covalently bind A-zymes and B-zymes to polymethacrylate microparticles was developed (Fig. 5a and 5b). The enzymes, immobilized on 200-500 pm polymethacrylate ReliZyme™ HFA403-M microparticles were removed -after having removed the blood group antigen from the platelets - by 200 pm filters already integrated today mandatorily in the transfusion set to remove small blood clots. The platelet activation ability was not affected by the enzymes or the microparticles (Fig. 6) and the immobilized enzymes were still able to remove the antigen A and B structures from platelet surfaces (Fig. 5c and 5d). The percentage of A positive platelets was decreased to 3.8%±1.1% after 4 hours of incubation with enzyme-linked microparticles, while B positive platelets were decreased to 25.7%±10.0% after 24 hours.

[0210] In the next step, it was aimed to investigate the possible leakage of enzymes from the microparticles since this could cause an immune response when given to a patient. The leakage of enzymes from microparticles into the platelet concentrates was negligible. By mass spectroscopy only traces of FpGalNAcDeAc of 0.6 pg mL‘l ±0.1 pg mL‘l (0.2% of the bead- bound enzyme), translating into a single digit nM concentration (see Table 8) and no leakage of FpGalNase and PpaGal were detected after 24 h incubation. Furthermore, the addition of A- and B-zymes on microparticles to pooled platelet concentrates fully preserved platelet function.

[0211] Universitatsmedizin Greifswald PVA21246PC

[0212] Universitat Greifswald

[0213] -24-

[0214] Table 8

[0215] Target protein characteristics and mass spectrometry results from complex platelet lysate / blood serum background (477 proteins, 192,440 PSMs*)

[0216] PSM = Peptide spectra match (unique fragment pattern identifying the peptide), number correlates positively with the protein concentration in the analyte, number of PSMs found in control and test samples is given

[0217] $DDA =Data dependent analysis approach; PD = ProteomeDiscoverer, data represent number of peptides / number of PSMs detected from test samples and attributed to the target protein

[0218] &DIA = data independent analysis approach; column specifies if, and in which estimated amount the target proteins could be detected in test sample; n.d. - no precursor or fragment ions detected for the target protein

[0219] Removal of Blood Group Antigen by Immobilized Enzymes from red blood cells

[0220] In order to assess the usability also for red blood cells, A-zymes and B-zymes covalently bound to polymethacrylate microparticles as described above were used. The enzymes, immobilized on 200- 500 pm polymethacrylate ReliZyme™ HFA403-M microparticles were removed - after having removed the blood group antigen from the red blood cells - by 200 pm filters already integrated today mandatorily in the transfusion set to remove small blood clots. The percentage of B positive platelets was decreased to less than 5% after 48 hours of incubation with enzyme-linked microparticles at 20-24 °C and also at 2-6 °C (Figures 8, 9).

[0221] Conclusion Universitatsmedizin Greifswald PVA21246PC

[0222] Universitat Greifswald

[0223] -25-

[0224] A method was developed for producing universal pooled platelet concentrates by adsorbing anti-A and anti-B antibodies from residual plasma onto red cells in buffy coats, obtained by pooling buffy coats from donors with non-identical blood groups.

[0225] To overcome the issue of ABO blood group incompatibilities in patients and reduce supply shortage, blood group antigens A and B were removed from the platelet surface through enzymatic degradation. This process involved the addition of enzymes covalently bound to microparticles to platelet concentrates of blood group A or B. The galactosidase sourced from Pedobacter panaciterrae proves to be more efficient at removing B-antigens compared to previously described galactosidases, resulting in a reduced enzyme requirement. The quality parameters of the platelet concentrates remained fully compliant with approval standards. Additionally, the enzyme-linked microparticles could be easily removed using standard transfusion sets, and any leakage of free enzymes from the microparticles was negligible making the applied method easy, safe, and promising for the production of universal blood products on a larger scale. Essentially the same result was also found for red blood cell concentrates.

[0226] The present invention is further illustrated by the following set of embodiments and combinations of embodiments resulting from the dependencies and back-references as indicated. In particular, it is noted that in each instance where a range of embodiments is mentioned, for example in the context of a term such as "The process of any one of embodiments 1 to 4", every embodiment in this range is meant to be explicitly disclosed for the skilled person, i.e. the wording of this term is to be understood by the skilled person as being synonymous to "The process of any one of embodiments 1, 2, 3 and 4". Further, it is explicitly noted that the following set of embodiments represents a suitably structured part of the general description directed to preferred aspects of the present invention, and, thus, suitably supports, but does not represent the claims of the present invention.

[0227] 1. Process for the preparation of cellular blood components with reduced expression of antigen(s) (blood groups) compared to the native state, comprising:

[0228] (a) Providing a solid carrier carrying at least one enzyme;

[0229] (b) Providing a liquid phase LP1 comprising at least one cellular blood component having antigen(s) with a native expression of antigen(s) LA(1);

[0230] (c) Contacting the solid carrier carrying at least one enzyme provided according to step (a) with the liquid phase provided according to step (b) under conditions allowing a removal of at least a part of the antigen(s) from the at least one cellular blood component; thereby obtaining a liquid phase LP2 comprising the at least one cellular blood component having antigen(s) with a reduced expression of antigen(s) LA(2), Universitatsmedizin Greifswald PVA21246PC

[0231] Universitat Greifswald

[0232] -26- wherein LA(2) is < LA(1), and the solid carrier carrying the at least one, enzyme at a concentration Csc(l).

[0233] 2. The process of embodiment 1, wherein the reduced expression of antigen(s) LA(2) is in the range of from 0.01 x LA(1) to 0.8 x LA(1), preferably in the range of from 0.01 x LA(1) to 0.3 X LA(1).

[0234] 3. The process of embodiment 1 or 2, wherein the at least one solid carrier provided in step (a) is selected from the group consisting of microparticle, membrane, foil and mixed forms of two or more thereof.

[0235] 4. The process of embodiment 3, wherein the at least one solid carrier provided in step (a) is a microparticle, said microparticle comprising a core (C) and optionally one or more shell(s) (S).

[0236] 5. The process of embodiment 4, wherein the core (C) comprises or consists of a component selected from the group consisting of polymer, lipid, metal, metal carbide, metal nitride, metal sulfide, metal phosphide, metal oxide, metal chelate, glass, silica, earth alkali metal phosphate, carbon and mixed forms of two or more thereof.

[0237] 6. The process of embodiment 4, wherein the core (C) comprises or consists of a (supra)magnetic material, preferably selected from the group consisting of metal carbide, metal nitride, metal sulfide, metal phosphide, metal oxide, metal chelate, and mixtures of two or more thereof, preferably at least an iron oxide, in particular an iron oxide selected from the group consisting of FesCU, a-Fe2Os, y-Fe2O3, MnFepOq, CoFepOq, NiFepOq, CuFepOq, ZnFepOq, CdFepOq, BaFepO and SrFepO, wherein p and q vary depending on the method of synthesis, and wherein p is preferably an integer of from 1 to 3, more preferably 2, and wherein q is preferably 3 or 4, most preferably y-Fe2O3 (maghemite) and / or FesCU (magnetite).

[0238] 7. The process of embodiment 4, wherein the core (C) comprises or consists of a polymer, preferably selected from the group consisting of poly(meth)acrylate, polymethyl(meth)acrylate, polystyrene, melamin resin, polylactic acid, methylcellulose, polycaprolacton, chitosan, gelatine, copolymers of two or more of these polymers and mixtures of two or more of these polymers and / or copolymers.

[0239] 8. The process of embodiment 4, wherein the core (C) comprises or consists of a lipid, preferably selected from the group consisting of triglyceride, partial glyceride, fatty acid, steroid, wax and mixtures of two or more of these lipids. Universitatsmedizin Greifswald PVA21246PC

[0240] Universitat Greifswald

[0241] -27-

[0242] 9. The process of any one of embodiments 4 to 8, wherein the optional one or more shell(s) (S) comprises or consists of a polymeric material, preferably selected from the group consisting of poly(meth)acrylate, polymethyl(meth)acrylate, polystyrene, polyvinylbenzene, melamin resin, polylactic acid, methylcellulose, polycaprolacton, chitosan, gelatine, dextrane, hydroxyl ethyl starch, copolymers of two or more of these polymers and mixtures of two or more of these polymers and / or copolymers.

[0243] 10. The process of any one of embodiments 4 to 9, wherein the optional one or more shell(s) (S) comprises on its surface at least one functional group for protein binding, wherein the at least one functional group for protein binding is preferably selected from the group consisting of halogenated C1-C3 -alkyl group, halogen atom, epoxy group, carboxy group, activated carboxy group, (preferably -acid halide or acid anhydride or succinimide), amino group, hydroxyl group, streptavidin, avidin, biotin, and two or more thereof.

[0244] 11. The process of any one of embodiments 4 to 10, wherein the microparticle has an average diameter in the range of from 10 to 1000 pm, preferably in the range of from 250 to 1000 pm, more preferably in the range of from 250 to 500 pm.

[0245] 12. The process of any one of embodiments 4 to 11, wherein the microparticle has only pores with a pore size of < 1mm.

[0246] 13. The process of any one of embodiments 4 to 12, wherein the microparticle has an average diameter in the range of from 250 to 500 pm and / or, preferably and, comprises poly(meth)acrylate, or comprises at least a shell (S) comprising poly(meth)acrylate, which carries on its surface at least an amino group and / or an epoxy group.

[0247] 14. The process of embodiment 3, wherein the at least one solid carrier provided in step (a) is a membrane and / or a foil, said membrane and / or foil comprising a material selected from the group consisting of polymer, hydroxyapatite, silica, titanium dioxide, zirconium oxide, lipid, hydrogel (preferably polyvinyl alcohol, agarose, agar) and mixed forms of two or more thereof.

[0248] 15. The process of embodiment 14, wherein the membrane and / or foil comprises or is a polymer, preferably selected from the group consisting of polylactide, polylactide-co- glycolide, polycaprolactone, polyurethane, polyethylene oxide, polyethylene glycol, chitosan, gelatine, collagen, hyaluronic acid, alginate, rubber, cellulose, modified cellulose, copolymers of two or more of these polymers, and mixtures of two or more of these polymers and / or copolymers. Universitatsmedizin Greifswald PVA21246PC

[0249] Universitat Greifswald

[0250] -28-

[0251] 16. The process of any one of embodiments 1 to 15, wherein the cellular blood components are selected from the group consisting of platelets (thrombocytes), red blood cells (erythrocytes), leukocytes, stem cells and mixtures of two or more thereof.

[0252] 17. The process of embodiment 16, wherein the liquid phase LP1 comprises at least erythrocytes, and is preferably an erythrocyte concentrate.

[0253] 18. The process of embodiment 16, wherein the liquid phase LP1 comprises at least platelets, and is preferably a platelet concentrate.

[0254] 19. The process of embodiment 16, wherein the liquid phase LP1 comprises at least human stem cells ( e.g. hematopoietic and mesenchymal stem cells), and is preferably a human hematopoietic stem cell preparation.

[0255] 20. The process of any one of embodiments 1 to 19, wherein the one or more antigen(s) are covalently bound to the cellular blood components.

[0256] 21. The process of any one of embodiments 1 to 20, wherein the one or more antigen(s) are blood group specific antigen(s).

[0257] 22. The process of any one of embodiments 1 to 20, wherein, if the cellular blood components comprise leukocytes, the one or more antigen(s) are blood group specific antigen(s) and / or human leukocyte antigen(s) (HLAs).

[0258] 23. The process of embodiment 22, wherein, if the cellular blood components comprise granulocytes, the one or more antigen(s) comprise human neutrophil antigen(s) (HNAs) and / or human leukocyte antigen(s) (HLAs).

[0259] 24. The process of any one of embodiments 1 to 20, wherein, if the cellular blood components comprise platelets, the one or more antigen(s) comprise blood group specific antigen(s) and / or human leukocyte antigen(s) (HLAs) of class I and / or human platelet antigens (HPAs).

[0260] 25. The process of any one of embodiments 1 to 20, wherein, if the cellular blood components comprise erythrocytes, the one or more antigen(s) comprise blood group specific antigen(s). Universitatsmedizin Greifswald PVA21246PC

[0261] Universitat Greifswald

[0262] -29-

[0263] 26. The process of any one of embodiments 21 to 25, wherein blood group specific antigen(s) are selected from the group consisting of antigen of the ABO blood group system, MNS blood group system, P1PK blood group system, Rh blood group system, Lutheran blood group system, Kell blood group system, Lewis blood group system, DARC (Duffy) blood group system, Kidd blood group system, Diego blood group system, Yt blood group system, Xg blood group system, Scianna blood group system, Dombrock blood group system, Colton blood group system, Landsteiner-Wiener blood group system, Chido / Rodgers blood group system, H blood group system, Kx blood group system, Gerbich blood group system, Cromer blood group system, Knops blood group system, Indian blood group system, Ok blood group system, Raph blood group system, JohnMiltonHagen blood group system, I blood group system, Globoside blood group system, Gill blood group system, Rh-associated glycoprotein blood group system, FORS blood group system, JR blood group system, LAN blood group system, Vel blood group system, CD59 blood group system, Augustine blood group system, Kanno blood group system, SID blood group system, CTL2 blood group system, PEL blood group system, MAM blood group system, EMM blood group system, ABCC1 blood group system, Er blood group system, CD36 blood group system, and mixtures of antigens of two or more of these blood group systems.

[0264] 27. The process of embodiment 26, wherein the blood group specific antigen(s) are selected from the group consisting of antigen of the ABO blood group system, Rh blood group system, DARC (Duffy) blood group system and mixtures of antigens of two or more of these blood group systems.

[0265] 28. The process of any one of embodiments 1 to 26, wherein the at least one enzyme is selected from the group of enzymes capable of removing at least a part of an antigen from a cellular blood component.

[0266] 29. The process of any one of embodiments 1 to 28, wherein, if the blood group specific antigen(s) are A antigen(s) of the ABO blood group system, at least one enzyme is selected from the group consisting of hydrolases, preferably deacetylase, carbohydrate active enzymes such as galactosaminidase, enzymes of the Glycoside Hydrolase Family 109 (GH109) and mixtures of two or more thereof.

[0267] 30. The process of embodiment 29, wherein the at least one enzyme is selected from the group consisting of enzymes having at least 95%, preferably at least 98%, more preferably at least 99%, more preferably 100%, sequence identity with FpGalNAcDeAc (Macetyl-a-D- galactosamine deacetylase from Flavonifractor plautii), FpGalNase (a-D- galactosamine galactosaminidase from Flavonifractor plautii), AmGH109B (a-N- Universitatsmedizin Greifswald PVA21246PC

[0268] Universitat Greifswald

[0269] -30- acetylgalactosaminidase from Akkermansia muciniphila ATCC BAA-835), AmGH109A (a-N-acetylgalactosaminidase from Akkermansia muciniphila ATCC BAA-835), NAGA (a-N-acetylgalactosaminidase from Elizabethkingia meningoseptica ATCC 13253), nagA (a-N-acetylgalactosaminidase from Elizabethkingia meningoseptica ATCC 33958), Nag68 (a-N-acetylgalactosaminidase from Alkalimonas sp), NAg69 (a-N- acetylgalactosaminidase from Pararheinheimera sp), Nag71 (a-N- acetylgalactosaminidase from Amycolatopsis sp) and mixtures of two or more thereof.

[0270] 31. The process of embodiment 29 or 30, wherein the at least one enzyme is a combination of an enzyme having at least 95%, preferably at least 98%, more preferably at least 99%„ sequence identity with FpGalNAcDeAc ( -acetyl-a-D-galactosamine deacetylase from Flavonifractor plautii) and an enzyme having at least 95%, preferably at least 98%, more preferably at least 99%, sequence identity with FpGalNase (a-D- galactosamine galactosaminidase from Flavonifractor plautii).

[0271] 32. The process of any one of embodiments 29 to 31, wherein the at least one enzyme is a combination of FpGalNAcDeAc (7V-acetyl-a-D-galactosamine deacetylase from Flavonifractor plautii) and FpGalNase (a-D-galactosamine galactosaminidase from Flavonifractor plautii).

[0272] 33. The process of any one of embodiments 1 to 28, wherein, if the blood group specific antigen(s) are B antigen(s) of the ABO blood group system, the at least one enzyme is selected from the group of a-galactosidases of the Glycoside Hydrolase Family 110 (GH110) and mixtures of two or more thereof.

[0273] 34. The process of embodiment 33, wherein the at least one enzyme is selected from the group consisting of enzymes having at least 95%, preferably at least 98%, more preferably at least 99%, more preferably 100%, sequence identity with BfGalB (a- 1,3 -galactosidase from Bacteroides fragilis). PmGal (a- 1,3 -galactosidase from Phocaeicola massiliensis). BpGal (a-l,3-galactosidase from Bacteroides pyogenes), PjGal (a-l,3-galactosidase from Parabacteroides johnsonii), PpaGal (a-l,3-galactosidase from Pedobacter panacilerrae), CcGal (a- 1,3 -galactosidase from Capnocytophaga canimorsus,), AkMuGalB (a- 1,3- galactosidase from Akkermansia muciniphila), AkMuGalA (a-l,3-galactosidase from Akkermansia muciniphila), BbGal (a-l,3-galactosidase from Bifidobacterium bifidum), Gall 10A (a- 1,3 -galactosidase from Bacteroides thetaiotaomicron VPI-5482), Gall 10B (a- 1,3-galactosidase from Bacteroides thetaiotaomicron VPI-5482), SgGall lOA (a-1,3- galactosidase from Peterkaempfera griseoplana 2357), Gall lOA (a- 1,3 -galactosidase from Streptomyces avermitilis MA-4680) and mixtures of two or more thereof. Universitatsmedizin Greifswald PVA21246PC

[0274] Universitat Greifswald

[0275] -31-

[0276] 35. The process of embodiment 33 or 34, wherein the at least one enzyme is selected from the group consisting of enzymes having at least 95%, preferably at least 98%, more preferably at least 99%, more preferably 100%, sequence identity with BfGalB (a- 1,3 -galactosidase from Bacteroides fragilis), PmGal (a- 1,3 -galactosidase from Phocaeicola massiliensis . BpGal (a-l,3-galactosidase from Bacteroides pyogenes'), PjGal (a- 1,3 -galactosidase from Parabacteroides johnsonii), PpaGal (a-l,3-galactosidase from Pedobacter panacilerrae), CcGal (a-l,3-galactosidase from Capnocytophaga canimorsus, ), and mixtures of two or more thereof.

[0277] 36. The process of any one of embodiment 33 to 35, wherein the at least one enzyme is selected from the group consisting of PpaGal (a-l,3-galactosidase from Pedobacter panacilerrae), PpaGal_W260Y of SeqID No. 1, PpaGal_K238Q of SeqID No. 2, PpaGal_P182M of SeqID No. 3 and mixtures of two or more thereof.

[0278] 37. The process of any one of embodiments 1 to 36, wherein the at least one enzyme is bound to the solid carrier by a bonding type selected from the group consisting of covalent bonding, physical bonding, affinity bonding, bonding via antibodies, covalent cross-linking and mixed forms of two or more of these bonding types.

[0279] 38. The process of any embodiment 37, wherein the at least one enzyme is bound to the solid carrier by at least covalent bonding, preferably via an amine and / or thio group of the enzyme, more preferably by at least covalent bonding between an amine group of the amine and a carboxyl group and / or epoxy group of the solid carrier and / or by at least covalent bonding between a thio group of the enzyme and a maleimide group and / or a sulfhydryl group of the solid carrier.

[0280] 39. The process of any one of embodiments 1 to 38, wherein contacting the solid carrier carrying at least one enzyme with the liquid phase is done in step (c) at a pH value in the range of from 3 to 10, preferably in the range of from 4.0 to 9.5, more preferably in the range of from 5.0 to 9.0, more preferably in the range of from 6.0 to 8.5, more preferably in the range of from 6.5 to 8.2, more preferably in the range of from 7.0 to 8.0, more preferably in the range of from 7.2 to 7.8.

[0281] 40. The process of any one of embodiments 1 to 39, wherein contacting the solid carrier carrying at least one enzyme with the liquid phase is done in step (c) at a temperature in the range of from 0 °C to 40 °C. Universitatsmedizin Greifswald PVA21246PC

[0282] Universitat Greifswald

[0283] -32-

[0284] 41. The process of embodiment 40, wherein, if the cellular blood components comprise platelets, contacting the solid carrier carrying at least one enzyme with the liquid phase is done in step (c) at a temperature in the range of from 20-24 °C.

[0285] 42. The process of embodiment 40, wherein, if the cellular blood components comprise erythrocytes, contacting the solid carrier carrying at least one enzyme with the liquid phase is done in step (c) at a temperature in the range of from 2-6 °C.

[0286] 43. The process of any one of embodiments 1 to 42, wherein contacting the solid carrier carrying at least one enzyme with the liquid phase is done in step (c) for a period of time of at least 30 minutes, preferably for a period of time in the range of from 30 minutes to 1 month.

[0287] 44. The process of any one of embodiments 1 to 43, wherein the liquid phase LP1 is an aqueous liquid phase and / or, preferably and, wherein the liquid phase LP2 is an aqueous liquid phase.

[0288] 45. The process of any one of embodiments 1 to 44, comprising

[0289] (a. l) Providing at least one solid carrier carrying at least a first enzyme and a second enzyme, or

[0290] (a.2) Providing at least a first solid carrier carrying at least a first enzyme and at least a second solid carrier carrying a second enzyme, wherein the first enzyme is different from the second enzyme;

[0291] (b.l) Providing a liquid phase LP1 comprising at least a first cellular blood component having first antigen(s) with a native expression of antigen(s) L1A(1) and at least a second cellular blood component having second antigen(s) with a native expression of antigen(s) L2A(1), with the first antigen(s) being different from the second antigen(s);

[0292] (c. l) Contacting the solid carrier carrying at least a first enzyme and a second enzyme according to step (a.l) or the at least a first solid carrier carrying at least a first enzyme and at least a second solid carrier carrying a second enzyme according to step (a.2), with the liquid phase provided according to step (b.l) under conditions allowing a removal of at least a part of the antigen(s) from the cellular blood components; thereby obtaining a liquid phase LP2 comprising at least the first cellular blood component having first antigen(s) with a reduced antigen(s) expression L1A(2) compared to the native expression of antigen(s) L1A(1), wherein L1A(2) < L1A(1), at least the second cellular blood component having second antigen(s) with a reduced antigen(s) expression L2A(2) compared to the native expression of antigen(s) L2A(1), wherein L2A(2) < L2A(1), and Universitatsmedizin Greifswald PVA21246PC

[0293] Universitat Greifswald

[0294] -33- the solid carrier(s) carrying the enzyme(s) in a concentration Clsc(l) and C2sc(l).

[0295] 46. The process of any one of embodiments 1 to 45 further comprising

[0296] (d) separating the liquid phase LP2 obtained in (c) or (c.1), thereby obtaining the solid carrier(s) in separated form and a liquid phase LP3 comprising the cellular blood component(s) having antigen(s) with expressions of antigen(s) LA(2) or L1A(2) and L2A(2) respectively, said liquid phase LP3 having (a) reduced concentration s) of the solid carrier(s) carrying the enzyme(s) Csc(2) or Clsc(2) and C2sc(2) respectively, with Csc(2) < Csc(l) or Clsc(2) < Clsc(l) and C2sc(2) < C2sc(l) respectively.

[0297] 47. The process of embodiment 46, wherein separation in step (d) is done by a mechanical separation method, preferably by filtration, more preferably by filtration via a filter having a pore size in the range of from 10 pm to 250 pm, more preferably via a filter integrated in a transfusion set.

[0298] 48. The process of embodiment 47, wherein separation in step (d) is done by magnetic forces, preferably via a magnet.

[0299] 49. The process of any one of embodiments 1 to 48 being an in vitro or ex vivo, preferably an in vitro process.

[0300] 50. A system for removing antigen(s) from cellular blood components comprising:

[0301] (c) at least one solid carrier;

[0302] (d) at least one enzyme; wherein the at least one enzyme is bound to the at least one solid carrier.

[0303] 51. An a- 1,3 -galactosidase of SeqID No. 1 (PpaGal_W260Y).

[0304] 52. An a- 1,3 -galactosidase of SeqID No. 2 (PpaGal_K238Q).

[0305] 53. An a- 1,3 -galactosidase of SeqID No. 3 (PpaGal_P182M).

[0306] 54. A cellular blood component having a reduced expression of antigen(s) LA(2) compared to a native expression of antigen(s) LA(1) of said cellular blood component with LA(2) < LA(1), obtained or obtainable by a process according to any one of embodiments 1 to 45. Universitatsmedizin Greifswald PVA21246PC

[0307] Universitat Greifswald

[0308] -34-

[0309] 55. A cellular blood component having a reduced expression of antigen(s) LA(2) compared to a native expression of antigen(s) LA(1) of said cellular blood component with LA(2) < LA(1).

[0310] 56. A cellular blood component having a reduced expression of antigen(s) LA(2) compared to a native expression of antigen(s) LA(1) of said cellular blood component with LA(2) < LA(1) of embodiment 54 or 55 for use in transfusion.

[0311] Experimental part

[0312] 1. Methods

[0313] 1.1 Manufacturing Pooled Platelet Concentrates

[0314] Whole blood was collected from healthy donors according to the German guidelines for hemotherapy with written informed consent. Pooled platelet concentrates (PC) from whole blood buffy coats were produced according to the standard method for producing therapeutic PC. In brief, after centrifugation (4000 g, 10 min) whole blood in citrate phosphate dextrose solution (CPD, Macopharma, France) was separated into red cell concentrate, plasma, and buffy coat. Buffy coats of 4 donations, which were ABO-non-identical, were pooled (total approximately 280 mL) and 250 mL additive solution PAS-E (SSP+, Macopharma) was added. By centrifugation (720 g, 15 min) platelets were separated from residual red blood cells and leukocyte depleted (LEUCOFLEX® LXT Filter, Macopharma). PC bags were stored under agitation at room temperature (RT) for 4 days.

[0315] 1.2 Manufacturing Red Cell Concentrates

[0316] Whole blood was collected from healthy donors in accordance with German hemotherapy guidelines, with written informed consent. Whole blood in citrate phosphate dextrose solution (CPD, Macopharma, Tourcouring, France) was centrifuged at 4000 x g for 10 minutes at a temperature in the range of from 20 to 24 °C, and separated into erythrocyte concentrate (= red cell concentrate, RCC), plasma, and buffy coat. The RCC was leukocyte-depleted (LCRD2-Filter, Macopharma) and stored in phosphate-adenine-glucose-guanosine-saline-mannitol (PAGGS-M) solution at 2-6 °C for 49 days.

[0317] 1.3 Quality Control of Pooled Platelet Concentrates

[0318] Platelet count. Platelet count was measured in a 1 :8 dilution by cell counter Sysmex XP-300 (Sysmex, Germany).

[0319] Platelet function. Platelet activation was determined by CD62P expression before and after the addition of thrombin receptor activating peptide 6 (TRAP-6). 3xl08mL'1platelets were incubated for 10 min at 37°C with 20 pM TRAP-6 (Hart Biologicals, UK) or phosphate buffered saline (PBS buffer; w / o Ca2+, Mg2+; pH 7.2) as the negative control, followed by addition of 5 pL / 100 pL PE- Universitatsmedizin Greifswald PVA21246PC

[0320] Universitat Greifswald

[0321] -35-

[0322] Cy 5 Mouse Anti-Human CD62P (BD Bioscience, USA) for additional 10 min. Samples were fixed 20 min with 0.5% paraformaldehyde (PF A) and washed twice (2 mL PBS; 650 g, 7 min, RT). The pellet was resuspended in 500 pL PBS and analyzed by flow cytometry (Cytoflex S, Beckman Coulter, USA). The increase of CD62P expression on platelets was determined using Pe-Cy5 mean fluorescence intensity (MFI) of the platelet-population given as fold increase in comparison to the respective buffer controls.

[0323] Residual red blood cell count. Residual red cells of PC units were counted using a Nageotte chamber (Assistent, Germany) under a microscope.

[0324] Free hemoglobin. Free hemoglobin, as a marker for hemolysis of red cells, was measured as cyanmethemoglobin spectroscopically using potassium hexacyanoferrate III and potassium cyanide in cell free supernatant. A multi wavelength absorption analysis was performed at 540 nm (Al) and 680 nm (A2) in a spectrophotometer (UV-1700, Shimadzu, Japan).

[0325] Antibody binding on platelets. Antibody binding to glycoprotein (GP) Ilbllla, GP IbIX and Platelet Endothelial Cell Adhesion Molecule (PECAM-1; CD31) on platelet surfaces was determined by the monoclonal antibody immobilization of platelet antigens (MAIPA) assay.15

[0326] Isoagglutinin titer. Titers of anti-A and anti-B were determined by a microcolumn gel card system. For IgG titers cards containing 6 microcolumns with an anti-human globulin phase (ID-Card LISS / Coombs, Bio-Rad Laboratories Inc., USA), for IgM titers saline cards (ID-Card for sodium chloride, enzyme test and cold agglutinins, Bio-Rad Laboratories Inc., USA) were used according to the manufacturer’s instructions. PC supernatant samples in 1 :2 dilution series (dilution media: 0.9% (w / v) NaCl-solution) were incubated (IgG: 37°C, 15 min; IgM: RT, 15 min) with group B red cells for anti-A and group A red cells for anti-B. Group O red cells were used as negative control. Agglutination strengths were evaluated by two independent individuals. Antibody titer was defined as the last sample dilution inducing agglutination.

[0327] 1.4 Protein Expression and Purification

[0328] Synthetic genes for the different six galactosidases to investigate B-Antigen cleavage as well as two synthetic genes for the enzymatic removal of A-antigens were ordered in a pET28(a) vector (BioCat GmbH, Heidelberg, Germany). All genes contained an N- or C-terminal His-tag sequence for affinity chromatography. The sequences and NCBI accession codes are listed in Table S5.

[0329] For protein expression, the pET28(a) vectors harboring the synthetic genes were transformed into E. coli BL21 Gold (DE3) by the heat-shock method. In the case of each gene expression, a single colony of the cells with the desired amino acid sequence was picked and used to inoculate 4 mL LB media supplemented with 50 pg mL'1kanamycin, which was grown overnight at 37°C at 140 rpm. These starter cultures were used to inoculate the 50 mL main cultures (TB medium) in which the cells were grown until the optical density at 600 nm (OD600) reached approximately 0.6 - 0.8. Universitatsmedizin Greifswald PVA21246PC

[0330] Universitat Greifswald

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[0332] The gene expressions of the cultures were induced with a final concentration of 0.5 mM isopropyl- P-D-thiogalactopyranoside (IPTG) and lasted around 20 hours at 20°C at 160 rpm. The main cultures were harvested by centrifugation (10 min, 2000 g, 4°C) and washed once with sodium phosphate buffer (50 mM, pH 7.5). The harvested bacteria pellets were resuspended in 4 mL washing buffer (50 mM sodium phosphate, 300 mM NaCl, 20 mM imidazole, 2 mM MgCh, pH 8.0) for each gram of cell pellet. The cells were disrupted via ultrasonication with 30% power and 50% cycle on ice. Thereby, the sonication procedure consisted of 5 min sonication followed by a 2 min break and another 5 min of sonication. For separation of the cell debris from the supernatant, the samples were centrifuged at 10.000 g for 30 min at 4°C. The clarified lysates containing the desired proteins were transferred onto Ni-IDA columns (ROTI®Garose-His / Ni Beads, Carl Roth GmbH + Co. KG, Germany) for affinity chromatography, washed ten times with washing buffer and eluted in fractions with elution buffer (50 mM sodium phosphate, 300 mM NaCl, 2 mM MgCh, 250 mM imidazole, pH 8.0). The fractions with the highest protein content were pooled and rebuffered in 50 mM sodium phosphate buffer pH 7.5 using Ami con Ultra-15 centrifugal filter units (MWCO 10 kD; Merck KGaA, Germany). The protein solutions were stored at 4°C until further use.

[0333] 1.5 Blood Group Antigen Determination on Blood Cells

[0334] Red blood cells. RBC of blood group A or B were used to test the antigen removal by FpGalNAcDeAc and FpGalNase for the A-antigen and PpaGal for B-antigen. For B-antigen removal, 200 pL RBC of diluted whole blood samples were incubated with 200 pL of a 0.4, 0.2, and 0.1 mg mL'1PpaGal solution for 1 h (final enzyme concentration of 0.2, 0.1, and 0.05 mg mL'x). For A-antigen removal, 200 pl of an enzyme mixture of FpGalNAcDeAc / FpGalNase with 0.6 / 1.2, 0.3 / 0.6, 0.06 / 0.12, 0.03 / 0.06 mg mL'1(final enzyme concentrations of 0.3 / 0.6, 0.15 / 0.3, 0.03 / 0.06, 0.015 / 0.03) were used. Samples were washed three times with 2 mL PBS pH 7.4 and agglutination tests with anti- A or anti-B antibodies (Optima Testseren, Germany) were performed on size exclusion chromatography dextran gel cards (LISS / Coombs, Bio-Rad, Germany). Cards were centrifuged for 15 min at 140 g at RT using ID centrifuge 24 S (Bio-Rad) and the extent of antigen removal from the RBC was evaluated by the location of the RBC in the gel of the cards. Due to complete antigen removal RBC migrate to the bottom (Score 0, no agglutination). In the presence of the antigen, the antibody addition results in a complex formation that is therefore present on top of the column (Score 4, full agglutination). RBC with partially removed antigens migrate in between (Score 1 to 3).

[0335] Platelets. The effect of enzyme addition to platelet blood group antigens was determined by measuring the percentage of antigen A- and B-positive platelets by flow cytometry. 50 pL of platelets incubated with the enzyme solutions were fixated with 50 pL of 4% paraformaldehyde (PF A, Morphisto Laborchemikalien, Germany) and incubated for 15 min at RT and washed 2 times with 200 pL PBS (w / o Ca2+, Mg2+; PAN-Biotech, Germany) by centrifugation (7 min at 650 g) and the pellet was finally resuspended in 50 pL PBS. 20 pL of the fixed platelets were incubated Universitatsmedizin Greifswald PVA21246PC

[0336] Universitat Greifswald

[0337] -37- with 20 pL primary antibody Anti-A or Anti-B Monoclonal Immunoglobuline M (Optima Testseren, Germany) for 20 min at RT. The sample was then washed twice and the pellet was incubated with 50 pL of 1 : 10 diluted secondary antibody Polyclonal Rabbit Anti-Mouse Ig / FITC (Dako Deutschland GmbH, Germany) for 30 min at RT. Samples were washed once and resuspended in 500 pL PBS. Incubation of blood group A platelets with Anti-B Immunoglobuline and vice versa served as negative controls for background stain determination (Figure 7 a-e). All samples were analysed by flow cytometry (Cytoflex S, Beckman Coulter, USA).

[0338] 1.6 Enzyme Immobilization on Microparticles

[0339] Enzymes were immobilized on polymethacrylate ReliZyme 200 - 500 pm HFA 403 microparticles (MP, Resindion S.r.l., Italy). Covalent binding of the enzymes to the oxirane group on the particle shell was performed with 10 mg MP / 2 mL sodium phosphate buffer (50 mM, pH 7.5) after two washing steps (650 g; 3 min). MP were incubated at RT with either FpGalNAcDeAc and FpGalNase (final concentrations: FpGalNAcDeAc / FpGalNase: 4.7 / 9.4 pg mL'1, 9.4 / 18.8 pg mL' 18.8 / 37.5 pg mL'1) or PpaGal (final concentrations: 0.41, 4.1, 41 pg mL'1) overnight. Samples were washed once with sodium phosphate buffer and stored at 4°C.

[0340] Enzyme amount immobilized on microparticles. Samples were taken before and after 1, 2, and 4 hours of incubation to determine the protein concentration decrease in the supernatant (after centrifugation at 650 g for 3 min) by UV / VIS spectrometry at 280 nm (Nanodrop 2000 Spectrophotometer, Thermo Fisher Scientific, Germany). We calculated the immobilization rate from the total enzyme amount applied to the sample before MP-addition minus the supernatant enzyme amount after the MP-addition.

[0341] 1.7 Impact of Enzyme Addition on Platelet Function

[0342] The impact of the enzymes or microparticles addition to pooled PC was determined by the CD62P expression by flow cytometry as described above (Figure 6, Figure 7 f-g).

[0343] 1.8 Statistics and Reproducibility

[0344] The data are shown as mean with standard error of mean. All replicates are biological replicates from distinct samples. GraphPad Prism 8.0.1 software (GraphPad Software, La Jolla, CA) was used for statistical analysis. Samples were tested for lognormal distribution by Shapiro-Wilk-test. When normal distributed ordinary one way ANOVA followed by Fishers LSD Test was used (Figures 2b, 2c, 2d, 2g, 3a, 3c, 3d). Non normal distributed data were analyzed by Kruskal-Wallis Test followed by uncorrected Dunn’s test (Fig. 3b). Data with only two comparable data sets were analyzed by unpaired (Figures 2e, 2f) or paired (Figures 5c, 5d) t-test. P-values <0.05 were considered to be statistically significant: *P < 0.05, **P<0.01, ***P<0.001, and ****P<0.0001.

[0345] 1.9 Protein Characterization

[0346] Protein yields. The protein concentrations were measured via NanoDrop 1000 (Thermo Scientific, Wilmington, DE, USA), and protein yields were calculated based on the molecular weight and the Universitatsmedizin Greifswald PVA21246PC

[0347] Universitat Greifswald

[0348] -38- extinction coefficients from the Expasy tool ProtParam (https: / / web.expasy.org / protparam / ), Tables S2-S3.

[0349] Activity tests. First, activity measurements were performed with a common / / ra-nitrophenol-assay ( NP-assay). For this, -nitrophenyl a-D-galactopyranoside was used as a chromogenic substrate. The product, -nitrophenol, is colorless when protonated and becomes yellow in aqueous alkaline solutions. The production of the yellow-colored / ?-nitrophenolate can be measured by absorption at 405 nm. 400 pg mL'1enzyme solutions of each enzyme were prepared. 80 pL of 50 mM sodium phosphate buffer pH 7.5 and 20 pL of a 400 pg mL'1enzyme solution was pipetted into a 96-well plate. Reactions were started by the addition of 100 pL of a 5 mM substrate solution dissolved in 50 mM sodium phosphate buffer pH 7.5 to a final reaction volume of 200 pL (final concentration of 2.5 mM). Absorption at 405 nm was measured every 30 seconds over 10 min. pH Optima. Activity tests were performed similarly as described above. However, instead of 80 pL of 50 mM sodium phosphate buffer pH 7.5, 80 pl of 50 mM sodium phosphate buffer with different pH values were added to the 96-well plate (pH 5.0, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 or 9.0) to adjust the desired pH value. In contrast to the previous activity tests, where the substrate was dissolved in 50 mM sodium phosphate buffer pH 7.5, the substrate solutions were prepared in MilliQ water to not influence the pH value.

[0350] Determination of Km values. Km values were determined by measuring initial rates with varying substrate concentrations. First, the substrate concentrations 0 mM, 0.75 mM, 1.5 mM, 2.5 mM, 4 mM, 6 mM, 9 mM and 12 mM for CcGal and PpaGal, and 0 mM, 1.25 mM, 2.5 mM, 3.75 mM, 5.0 mM, 7.5 mM, 11 mM and 15 mM for BpGal, PmGal, PjGal and BfGalB were pipetted in a reaction plate with the same volume of 100 pl in each well. The reactions were started by the addition of 100 pl of the enzyme solutions with a concentration of 40 pg mL'1of CcGal, BpGal, PmGal, PjGal, BfGalB (final concentration 20 pg mL'1) and 20 pg mL'1of PpaGal (final concentration 10 pg mL'1).

[0351] Calculation of Km values occurred with a nonlinear regression for the fit function for the Michaelis-Menten kinetics of the software GraphPad Prism7 (GraphPad Software, USA).

[0352] Thermostability. The stability of the proteins was investigated by measuring melting points that serve as an indicator of the overall stability of proteins. After protein purification, the melting points of the protein samples stored in 50 mM sodium phosphate buffer pH 7.5 were investigated by using Nano differential scanning fluorimetry (Prometheus NT.48 nanoDSF, NanoTemper Technologies GmbH, Munich, Germany). Moreover, the proteins were diluted 1 : 10 in 70% PAS- E buffer and melting points were measured.

[0353] Long-term stability. Besides the thermostability, the long-term stability of a protein can be determined which indicates the stability of a protein over time. For this approach, the activity of Universitatsmedizin Greifswald PVA21246PC

[0354] Universitat Greifswald

[0355] -39- enzymes can be used. An activity test was performed as described above. A second activity test was performed after 81 days of storage at 4°C and residual activities were calculated.

[0356] For the PpaGal mutations PpaGal_W260Y, PpaGal_K238Q and PpaGal_P182M, activity tests, determination of Km values and thermostability were determined in the same manner as described above.

[0357] 1.10 Determination of Enzyme Residues in Platelet Concentrate Supernatant by Mass Spectroscopy

[0358] Potential leakage of FpGalNAcDeAc (NCBI WP 009260926.1), FpGalNase (WP_044942952.1), and PpaGal (NQX53349.1) from beads into PC supernatant was determined using nano-Liquid Chromatography / Tandem Mass Spectrometry (LC-MS / MS). Sample preparation: A fraction of the supernatant containing 250 pg of proteins was diluted 1 :5 in PBS w / o Ca2+ / Mg2+, and precipitated in 80% acetone at -20°C overnight. The precipitate was spun down for 5 min at 5000 g, washed once with 80% acetone, air-dried for 5 min and dissolved in 50 mM ammonium bicarbonate buffer (pH 8). Reduction was achieved by adding a final concentration of 10 mM dithiothreitol and incubation at 60 °C for 30 min, followed by alkylation by iodoacetamide (10 mM final concentration, 20 min at RT). For digestion, sequencing-grade trypsin (Promega, V5111) was used in a ratio of 1 :40 at 37°C overnight. The reaction was stopped by reducing the pH to 1 by adding HC1. For desalting and clean-up, sample was subjected to Cl 8 solid phase extraction (Luna C18 3um, Phenomenex, Aschaffenburg). Peptides were diluted by 0.1% formic acid in 80% of acetonitrile in water (uHPLC-MS quality, Thermo Fisher). The eluate was dried in a SpeedVac at room temperature and reconstituted in 10 pl 0.1% formic acid in water (Buffer A). For mass spectrometry analysis, 500 ng peptide was loaded onto a PepMap C18 trap column (5 pm particles, 20 x 0.1 mm) and separated on a PepMap Cl 8 analytical column (3 pm particles, 150 x 0.075 mm; Dionex UltiMate 3000 RSLCnano) using buffer A and 0.1 % formic acid in 95:5 ACN:MS grade water (buffer B) at a flow rate of 300 nL min'1at 40°C. A linear gradient of 2 to 35 % buffer B over 75 min was used. Analytes were ionized by electrospray in positive mode (+2.4 kV) using a metal emitter and transferred into an Exploris 480 orbitrap mass analyser (Thermo Fisher). The mass spectrometer was operated in data dependent acquisition mode (DDA). MSI full scan parameters were set as follows: 350-1200 m z'1, R = 120,000 at 200 m z'1, target of 5 * 103ions, followed by up to 15 data-dependent MS / MS scans with higher energy collision dissociation (HCD, maximum injection time (IT) 50 ms, isolation width 1.0 m z'1, NCE 30%, R = 15,000 at 200 m z'1). Dynamic exclusion was enabled and set to 30 s. For quantification, the instrument was used in data independent acquisition mode (DI A), a full MSI spectrum was recorded for 390-1010 m z'1, R = 15,000 at 200 m z'1, target of 5 x 103ions, followed by 60 MS2 scans with each 10 m z'1isolation window, spanning from 399.5 m z'1to 1000.5 m z'1with a fixed injection time of 22 ms and a resolution of R = 15,000 at 200 m z'1. DDA raw data analysis was achieved by ProteomeDiscoverer 2.5 (Thermo Fisher, Dreieich, Germany), using a hybrid fasta file containing Universitatsmedizin Greifswald PVA21246PC

[0359] Universitat Greifswald

[0360] -40- the sequences of FpGalNAcDeAc FpGalNase, and PpaGal and the human proteome (uniprot.org, Sep 2023, 26255 sequences), yielding to the unambiguous detection of FpGalNAcDeAc, FpGalNase or PpaGal in control samples with 32, 38, and 38 unique peptides, respectively. Information of identified peptides was subsequently used for relative quantification of FpGalNAcDeAc, FpGalNase and PpaGal in DIA raw data by Skyline 21.2.0.565 (MacCoss Lab, University of Washington, USA) within the human proteome as the background. For each protein, three unique peptides showing best performance regarding quality and numbers of detection in control samples were selected. To visualize and summarize the results the areas of the precursor ions M, M+l, M+2 (natural13C isotope containing variants) and the related fragment ions (mainly y-ions) of the tryptic peptides DLVASGSDWALDAK (597 - 610, FpGalNAcDeAc), TDEAGAYAELTFR (865 - 877, FpGalNase), and NTTPLLTSLLK (55 - 65, PpaGal) were shown. Unless otherwise stated, all consumables were purchased from Thermo Fisher, Dreieich, Germany.

[0361] Description of Figures

[0362] Fig. 1. shows a schematic overview of enzymatic antigen removal of A- antigen by N-acetyl-a-D- galactosamine deacetylase (FpGalNAcDeAc) and a-D-galactosaminidase from Flavonifractor plautii (FpGalNase), and B-antigen by a newly characterized a- 1,3- galactosidase from Pedobacter panaciterrae (PpaGal). Sugars are shown using the

[0013] Consortium for Functional Glycomics notation.

[0363] Fig. 2 shows removal of anti-A and anti-B antibodies with red blood cells (RBCs). a) During the pooling process of buffy coats of non-identical blood types isoagglutinins anti-A and anti- B are adsorbed on red cells and are removed. Comparison of pooled platelet concentrates from buffy coats of non-identical and identical blood groups regarding b) platelet count, c) pH, d) platelet function determined as fold increase of CD62P expression after TRAP6 addition (20 pM), e) red blood cell count, f) free hemoglobin, g) antibody binding on glycoprotein Ilbllla, glycoprotein IbIX, CD31. n=12, mean±standard deviation, all n.s. between identical and non- identical blood groups.

[0364] Fig- 3 shows enzymatic removal of blood group antigens. Red blood cell agglutination after A- or B-antibody addition is reduced a) for A-antigen by the addition of N- Acetyl-a-D- galactosamine deacetylase (FpGalNAcDeAc) and a-D-galactosaminidase from Flavonifractor plautii (FpGalNase) with increasing concentration and b) for B-antigen by galactosidases of different origins (Table 3) with increasing concentrations. Blood group antigens are removed from platelets after the addition of c) FpGalNAcDeAc / FpGalNase to blood group A platelet concentrates and d) a-l,3-Galactosidase from Pedobacter panaciterrae (PpaGal) to blood group B platelet concentrates. n=3-4, mean±standard deviation. Universitatsmedizin Greifswald PVA21246PC

[0365] Universitat Greifswald

[0366] -41-

[0367] Fig- 4 shows the determination of the pH optima of the six investigated a-galactosidases. Activity tests were performed at pH values between 5.0 and 9.0 in 50 mM sodium phosphate buffer and relative activities were calculated. n=3, mean±standard deviation.

[0368] Fig. 5 shows enzymes immobilized on polymethacrylate microparticles reduce A- and B-antigens on platelets. Schematic illustration of enzyme-linked microparticles added to platelet concentrates a) blood group A and b) blood group B. Enzymes linked to ReliZyme HF403 microparticles are able to remove blood group antigens on platelets of c) blood group A platelet concentrates and d) blood group B platelet concentrates. n=3, mean±standard deviation.

[0369] Fig. 6 shows the impact of the addition of a) Flavonifractor plautii (FpGalNAcDeAc) and a-d- galactosaminidase from Flavonifractor plautii (FpGalNase), b) a- 1,3 -galactosidase from Pedobacter panaciterrae (PpaGal), and c) ReliZyme HF403 microparticles on platelet activation ability determined as fold increase of the CD62P expression after 20 pM thrombin receptor-activating peptide-6 (TRAP6) addition; each n=3, mean±standard deviation; d) Immobilization rate of FpGalNAcDeAc / FpGalNase and PpaGal on ReliZyme HF403 microparticles during 4 hours of incubation, n=2, mean; all n.s. between samples without and with enzymes or microparticles.

[0370] Fig- 7 shows flow cytometry gating and analysis of platelets (PLT) blood groups (B-E) or CD62P activation status (F-H). a) PLT population was determined by size in the forward / sideward scatter blot (FSC / SSC) and analyzed for binding of primary mouse anti-blood-group- antibodies followed by secondary FITC-conjugated anti-mouse-IgG (B-E) or direct binding of PeCy5 -conjugated CD62P activation marker (F-H).b) representative blood group-A platelet concentrate (PC-A) at time point tO (before addition of enzymes) incubated with primary anti-B-antibody as negative control to set the background stain, c) PC-A at tO incubated with primary anti-A-antibody to set the base-value of group-A positive platelets (this value was normalized to reflect 100% A-positive PLT), and d) PC- A at t3 (4h after enzyme addition), e) blood-group stain histogram overlay of negative control, base-value and all 4 time points after enzyme incubation (tl=lh, t2=2h, t3=4h, t4=24h).f-g) representative CD62P histograms of untreated PC before (F) and after (G) addition of 40 pM TRAP-6 at time point tO. h: CD62P-activation histogram overlay of untreated PC and after 4h of enzyme incubation, each with buffer (PBS) or 40 pM TRAP.

[0371] Fig- 8 shows enzymatic B-antigen removal from RBCs by galactosidases from Pedobacter panaciterae wild type (PpaGal WT) and engineered (PpaGal_W260Y) and galactosidases from Akkermansia muciniphila A (AmGHl 10 A) and B (AmGHl 10B) each 1.18 pM linked to ReliZymeTM HFA403 microparticles at 20-24 °C. n=3, mean±standard deviation. *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001 (Fishers LSD Test). Differences indicated compared to incubation without enzymes.

[0372] Fig. 9 shows enzymatic B-antigen removal from RBCs by galactosidases from Pedobacter panaciterae wild type (PpaGal WT) and engineered (PpaGal_W260Y) and Universitatsmedizin Greifswald PVA21246PC

[0373] Universitat Greifswald

[0374] -42- galactosidases from Akkermansia muciniphila A (AmGHl 10 A) and B (AmGHl 10B) each 1.18 pM linked to ReliZymeTM HFA403 microparticles at 2-6 °C. n=3, mean±standard deviation. *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001 (Fishers LSD Test). Differences indicated compared to incubation without enzymes.

[0375] Cited Literature

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[0382] [7] R. M. Kaufman, B. Djulbegovic, T. Gemsheimer, S. Kleinman, A. T. Tinmouth, K. E. Capocelli, M. D. Cipolle, C. S. Cohn, M. K. Fung, B. J. Grossman, P. D. Mintz, B. A. O’Malley, D. A. Sesok-Pizzini, A. Shander, G. E. Stack, K. E. Webert, R. Weinstein, B. G. Welch, G. J. Whitman, E. C. Wong, A. A. R. Tobian, AABB, Ann. Intern. Med. 2015, 162, 205-213.

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[0388] Kizhakkedathu, S. G. Withers, Nat. Microbiol. 2019, 4, 1475-1485.

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[0013] A. Varki, R. D. Cummings, M. Aebi, N. H. Packer, P. H. Seeberger, J. D. Esko, P. Stanley, G. Hart, A. Darvill, T. Kinoshita, J. J. Prestegard, R. L. Schnaar, H. H. Freeze, J. D. Marth, C. R. Bertozzi, M. E. Etzler, M. Frank, J. F. Vliegenthart, T. Liitteke, S. Perez, E. Bolton, P. Rudd, J. Paulson, M. Kanehisa, P. Toukach, K. F. Aoki-Kinoshita, A. Dell, H. Narimatsu, W. York, N. Taniguchi, S. Komfeld, Glycobiology 2015, 25, 1323-1324.

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Claims

Universitatsmedizin Greifswald PVA21246PCUniversitat Greifswald-43-Claims1. Process for the preparation of cellular blood components with reduced expression of antigen(s) (blood groups) compared to the native state, comprising:(a) Providing a solid carrier carrying at least one enzyme;(b) Providing a liquid phase LP1 comprising at least one cellular blood component having antigen(s) with a native expression of antigen(s) LA(1);(c) Contacting the solid carrier carrying at least one enzyme provided according to step (a) with the liquid phase provided according to step (b) under conditions allowing a removal of at least a part of the antigen(s) from the at least one cellular blood component; thereby obtaining a liquid phase LP2 comprising the at least one cellular blood component having antigen(s) with a reduced expression of antigen(s) LA(2), wherein LA(2) is < LA(1), and the solid carrier carrying the at least one enzyme at a concentration Csc(l).

2. The process of claim 1, wherein the at least one solid carrier provided in step (a) is selected from the group consisting of microparticle, membrane, foil and mixed forms of two or more thereof.

3. The process of claim 1 or 2, wherein the cellular blood components are selected from the group consisting of platelets (thrombocytes), red blood cells (erythrocytes), leukocytes, stem cells and mixtures of two or more thereof; and / or, preferably and, wherein the one or more antigen(s) are blood group specific antigen(s).

4. The process of any one of claims 1 to 3, wherein the liquid phase LP1 comprises at least erythrocytes, and is preferably an erythrocyte concentrate; or wherein the liquid phase LP1 comprises at least platelets, and is preferably a platelet concentrate; or wherein the liquid phase LP1 comprises at least human stem cells (e.g. hematopoietic and mesenchymal stem cells), and is preferably a human hematopoietic stem cell preparation.

5. The process of any one of claims 1 to 4, wherein, if the cellular blood components comprise leukocytes, the one or more antigen(s) are blood group specific antigen(s) and / or human leukocyte antigen(s) (HLAs); or, if the cellular blood components comprise granulocytes, the one or more antigen(s) comprise human neutrophil antigen(s) (HNAs) and / or human leukocyte antigen(s) (HLAs); or, if the cellular blood components comprise platelets, the one or more antigen(s) comprise blood group specific antigen(s) and / or human leukocyte antigen(s) (HLAs) of class I and / or human platelet antigens (HP As); or, if the cellular bloodUniversitatsmedizin Greifswald PVA21246PCUniversitat Greifswald-44- components comprise erythrocytes, the one or more antigen(s) comprise blood group specific antigen(s).

6. The process of any one of claims 1 to 5, wherein the at least one enzyme is selected from the group of enzymes capable of removing at least a part of an antigen from a cellular blood component; wherein preferably, if the blood group specific antigen(s) are A antigen(s) of the ABO blood group system, at least one enzyme is selected from the group consisting of hydrolases, preferably deacetylase, carbohydrate active enzymes such as galactosaminidase, enzymes of the Glycoside Hydrolase Family 109 (GH109) and mixtures of two or more thereof; and / or wherein preferably, if the blood group specific antigen(s) are B antigen(s) of the ABO blood group system, the at least one enzyme is selected from the group of a-galactosidases of the Glycoside Hydrolase Family 110 (GH110) and mixtures of two or more thereof.

7. The process of any one of claims 1 to 6, comprising(a. l) Providing at least one solid carrier carrying at least a first enzyme and a second enzyme, or(a.2) Providing at least a first solid carrier carrying at least a first enzyme and at least a second solid carrier carrying a second enzyme, wherein the first enzyme is different from the second enzyme;(b.l) Providing a liquid phase LP1 comprising at least a first cellular blood component having first antigen(s) with a native expression of antigen(s) L1A(1) and at least a second cellular blood component having second antigen(s) with a native expression of antigen(s) L2A(1), with the first antigen(s) being different from the second antigen(s);(c. l) Contacting the solid carrier carrying at least a first enzyme and a second enzyme according to step (a.l) or the at least a first solid carrier carrying at least a first enzyme and at least a second solid carrier carrying a second enzyme according to step (a.2), with the liquid phase provided according to step (b.l) under conditions allowing a removal of at least a part of the antigen(s) from the cellular blood components; thereby obtaining a liquid phase LP2 comprising- at least the first cellular blood component having first antigen(s) with a reduced antigen(s) expression L1A(2) compared to the native expression of antigen(s) L1A(1), wherein L1A(2) < L1A(1),- at least the second cellular blood component having second antigen(s) with a reduced antigen(s) expression L2A(2) compared to the native expression of antigen(s) L2A(1), wherein L2A(2) < L2A(1), andUniversitatsmedizin Greifswald PVA21246PCUniversitat Greifswald-45-- the solid carrier(s) carrying the enzyme(s) in a concentration Clsc(l) and C2sc(l).

8. The process of any one of claims 1 to 7 further comprising(d) separating the liquid phase LP2 obtained in (c) or (c.1), thereby obtaining the solid carrier(s) in separated form and a liquid phase LP3 comprising the cellular blood component(s) having antigen(s) with expressions of antigen(s) LA(2) or L1A(2) and L2A(2) respectively, said liquid phase LP3 having (a) reduced concentration(s) of the solid carrier(s) carrying the enzyme(s) Csc(2) or Clsc(2) and C2sc(2) respectively, with Csc(2) < Csc(l) or Clsc(2) < Clsc(l) and C2sc(2) < C2sc(l) respectively; wherein separation in step (d) is preferably done by a mechanical separation method, more preferably by filtration, more preferably by filtration via a filter having a pore size in the range of from 10 pm to 250 pm, more preferably via a filter integrated in a transfusion set; or wherein separation in step (d) is preferably done by magnetic forces, more preferably via a magnet.

9. The process of any one of claims 1 to 8 being an in vitro or ex vivo, preferably an in vitro process.

10. A system for removing antigen(s) from cellular blood components comprising:(a) at least one solid carrier;(b) at least one enzyme; wherein the at least one enzyme is bound to the at least one solid carrier.

11. An a- 1,3 -galactosidase of SeqID No. 1 (PpaGal_W260Y); or an a-l,3-galactosidase of SeqID No. 2 (PpaGal_K238Q); or an a-l,3-galactosidase of SeqID No. 3 (PpaGal_P182M).

12. A cellular blood component having a reduced expression of antigen(s) LA(2) compared to a native expression of antigen(s) LA(1) of said cellular blood component with LA(2) < LA(1), preferably obtained or obtainable by a process according to any one of claims 1 to 9.

13. The cellular blood component having a reduced expression of antigen(s) LA(2) compared to a native expression of antigen(s) LA(1) of said cellular blood component with LA(2) < LA(1) of claim 12 for use in transfusion.

Images