Universal red blood cells—enzymatic conversion of blood group A and B antigens

Transfusion Clinique et Biologique 11 (2004) 33–39 www.elsevier.com/locate/tracli

EUROSAT 2003

Universal red blood cells—enzymatic conversion of blood group A and B antigens Globules rouges universels—conversion enzymatique des antigens de groupes sanguins A et B Martin L. Olsson a,*,b,c, Cheryl A. Hill c, Humberto de la Vega c, Qiyong P. Liu c, Mark R. Stroud c, Jean Valdinocci c, Steven Moon c, Henrik Clausen b,c,d, Margot S. Kruskall b,c a

Department of Transfusion Medicine, Institution of Laboratory Medicine, Lund University and Blood Center, University Hospital, 221 85 Lund, Sweden b Division of Laboratory and Transfusion Medicine, Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, 330 Brookline Avenue, Boston, MA 02215, USA c ZymeQuest, Inc., 100 Cummings Center, Beverly, MA 01915-6122, USA d Department of Oral Diagnostics, School of Dentistry, University of Copenhagen, Nørre Allé 20, DK-2200 N, Copenhagen, Denmark Received 25 September 2003; accepted 2 December 2003

Abstract Accidental transfusion of ABO-incompatible red blood cells (RBCs) is a leading cause of fatal transfusion reactions. To prevent this and to create a universal blood supply, the idea of converting blood group A and B antigens to H using specific exo-glycosidases capable of removing the immunodominant sugar residues was pioneered by Goldstein and colleagues at the New York Blood Center in the early 1980s. Conversion of group B RBCs to O was initially carried out with a-galactosidase extracted from coffee beans. These enzyme-converted O (ECO) RBCs appeared to survive normally in all recipients independent of blood group. The clinical trials moved from small infusions to single RBC units and finally multiple and repeated transfusions. A successful phase II trial utilizing recombinant enzyme was reported by Kruskall and colleagues in 2000. Enzymatic conversion of group A RBCs has lagged behind due to lack of appropriate glycosidases and the more complex nature of A antigens. Identification of novel bacterial glycosidases with improved kinetic properties and specificities for the A and B antigens has greatly advanced the field. Conversion of group A RBCs can be achieved with improved glycosidases and the conversion conditions for both A and B antigens optimized to use more cost-efficient quantities of enzymes and gentler conditions including neutral pH and short incubation times at room temperature. Of the different strategies envisioned to create a universal blood supply, the ECO concept is the only one, for which human clinical trials have been performed. This paper discusses some biochemical and clinical aspects of this developing technology. © 2003 Elsevier SAS. All rights reserved. Résumé La transfusion accidentelle de CGR incompatibles dans le système ABO est une des principales causes de réaction transfusionnelle graves mortelles. Pour prévenir cela et créer un approvisionnement en sang universel, l’idée de convertir des antigènes de groupe A ou B en groupe H, en utilisant des exo-glycosidases spécifiques à même d’enlever/les résidus sucrés immunodominants, fut mise au point par Goldstein et coll au centre de transfusion sanguine de New York au début des années 80. La conversion des globules rouges de groupe B en O avait été réalisée initialement avec de l’exo-galactosidase extraite de grains de café. Ces globules rouges de groupe O convertis par réaction enzymatique ont montré une survie normale chez tous les receveurs quel que soit leur groupe sanguin. Ces essais cliniques sont passés du stade des petites injections d’une seule unité de globules rouges à celui de transfusions multiples et répétées. Kruskall et coll. ont rapporté en 2000 le succès d’un essai clinique de phase II utilisant une enzyme recombinante. La conversion enzymatique de globules rouges de groupe A était en retard en raison du manque des glycosidases appropriées et de la nature plus complexe des antigènes A. La découverte de nouvelles glycosidases

* Corresponding author. Tel.:+ 46-46-173207; fax: +46-46-173226. E-mail address: [email protected] (M.L. Olsson). © 2003 Elsevier SAS. All rights reserved. doi:10.1016/j.tracli.2003.12.002

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bactériennes ayant de meilleures propriétés kinétiques et plus spécifiques pour les antigènes A et B a beaucoup fait avancer ce champ de recherche. La conversion de globules rouges de groupe A peut être réalisée avec des glycosidases améliorées, et des conditions de conversion valables pour les antigènes A et B de manière à utiliser des quantités d’enzymes dont le rapport coût/efficacité est meilleur, et des conditions de réalisation plus faciles. Parmi les différentes stratégies envisagées pour développer une forme de sang universel, le concept ECO est le seul pour lequel des essais cliniques ont été effectués. Cet article rapporte certains aspects biochimiques et cliniques de la technologie mise au point. © 2003 Elsevier SAS. All rights reserved. Keywords: Red blood cells; Transfusion; Blood group ABO; Glycosidase; Clinical trial Mots clés : Globules rouges ; Transfusion ; Groupage sanguine ABO ; Glycosidase ; Essai clinique

1. Background There are now 29 established blood group systems [1], the clinically most important of which is still the ABO system discovered in 1900 by Landsteiner [2]. This was the first major break-through in research relating to transfusion safety and was rightfully awarded the Nobel Prize in 1930. The system is based on the presence or absence of the blood group antigens A and/or B. These antigens are found on the surface of erythrocytes and platelets as well as on the surface of endothelial and most epithelial cells [3]. The ABH blood group structures are found on glycoproteins and glycolipids, and considerable work has been done to identify the specific structures making up the A and B determinants or antigens. The ABH blood group specificity is determined by the nature and linkage of monosaccharides at the ends of the carbohydrate chains. The carbohydrate chains are attached to peptide (glycoprotein) or lipid (glycosphingolipid) backbones, which are attached to the cell membrane of the cells. The immunodominant monosaccharide determining type A specificity is a terminal a1-3 linked N-acetylgalactosamine (GalNAc), while the corresponding monosaccharide of B type specificity is an a1-3 linked galactose (Gal). Group O cells lack either of these monosaccharides at the termini of oligosaccharide chains, which instead are terminated with a1-2 linked fucose (Fuc) residues and called the H antigen (for a review, see Ref. [4]). A great diversity of blood group ABH carbohydrate structures is found due to structural variations in the oligosaccharide chains that carry ABH immunodominant saccharides (for a review, see Ref. [5]). Red cells contain ABH antigens on N-linked glycoproteins and glycosphingolipids, while it is generally believed that O-linked glycans on erythrocyte glycoproteins, mainly glycophorins, are terminated by sialic acid and not with ABH antigens. Type 1 chain glycosphingolipids are not endogenous products of red cells, but rather adsorbed from plasma. The genetic basis of ABO variation was largely elucidated in the 1990s. Following isolation of A transferase, the enzyme capable of adding the terminal sugar required for A antigen expression, transcripts from the ABO locus were cloned and the amino acid sequence of the primary gene product characterized [6,7]. In another seminal paper, poly-

morphisms accounting for the common blood group phenotypes A, B, AB and O were determined [8]. A schematic representation of the relationship between the ABO genes, the A and B glycosyltransferases and the immunodominant ABH carbohydrate epitopes is shown in Fig. 1. Blood groups A and B exist in several inherited variants. Blood group A subtypes are the most frequent, and there are three recognized major subtypes of blood type A expressing high levels of A antigen. These subtypes are known as A1, A intermediate (Aint) and A2. There are both quantitative and qualitative differences that distinguish these three subtypes. Quantitatively, A1 erythrocytes have more antigenic A sites, i.e. terminal N-acetylgalactosamine residues, than Aint erythrocytes which in turn have more antigenic A sites than A2 erythrocytes [9]. Qualitatively, A1 erythrocytes have a dual repeated A structure on a subset of glycosphingolipids, while A2 cells have an H structure on an internal A structure on a similar subset of glycolipids [10,11]. These differences between A1 and weaker A subtypes are thought to relate to differences in the kinetic properties of blood group A isoenzyme variants responsible for the formation of A antigens [12]. The genetic basis of the A1 and A2 phenotypes has been described [13] whilst the genetic basis of Aint is still unclear. In addition to A1, Aint and A2, numerous unusual A subgroups showing decreasing A antigen site densities on red blood cells (RBCs) exist. As is the case with the rare blood group B subtypes, differences in A/B antigen expression are believed to be mainly of quantitative nature. Several research groups have explored the genetic diversity at the ABO locus in detail, including the molecular basis of weak A and B subgroups (for a review of the genetic diversity underlying common and rare ABO phenotypes, see Ref. [14]). The plasma of blood group A individuals contains naturally occurring antibodies to B antigen. Conversely, plasma of blood group B contains antibodies to A antigen. Blood of group AB has neither antibody, and blood group O has both. This is generally referred to as Landsteiner’s rule. Antibodies to these and other carbohydrate-defined blood group antigens are believed to be elicited by continuous exposure to microbial organisms carrying related carbohydrate structures. An individual whose blood contains either (or both) of the anti-A or anti-B antibodies cannot receive a transfusion of blood containing the corresponding incompatible antigen(s). If an

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Fig. 1. Schematic representation of the relationship between common ABO genes, the A and B glycosyltransferases and the immunodominant ABH carbohydrate epitopes. In the top section, the consensus open reading frames of three common ABO alleles are shown as grey bars. The frame-shift and truncation caused by the 261delG mutation in the common O alleles are represented by white and black bars, respectively. Missense mutations in the B allele compared to the A1 allele are shown as vertical white lines. In the middle section, predicted topology models of the primary gene products (A and B transferases and the enzymatically inactive hypothetical O protein) are shown. In the B transferase, the four amino acid substitutions that differ between A and B are given. The amino acid corresponding to the start of the 261delG frameshift is noted in the O protein. The Golgi membrane encompassing the transmembrane domains of the glycosyltransferases is represented by two bold horizontal lines. The relative locations of amino- and carboxyterminals of the transferases are also indicated. In the bottom section, the resulting secondary gene products (A, B and H antigens) are depicted using the following symbols for carbohydrate structures: filled square, N-acetylglucosamine; open square, N-acetylgalactosamine; filled circle, galactose; open triangle, fucose. Bindings between monosaccharide residues are characterized using alpha and beta symbols and the carbon atom (in addition to C-1) involved in the glycosidic linkage. The scissors symbols indicate the linkages in A and B oligosaccharides affected by alpha-N-acetylgalactosidase and alpha-galactosidase treatment, respectively.

individual receives a transfusion of ABO-incompatible blood, the transfusion recipient’s ABO antibodies will coat these RBCs and destroy them, not primarily due to agglutination as seen in vitro but due to complement activation and induction of membrane attack complexes (MAC) with the capacity to lyse cells. The resulting clinical syndrome will often be an acute intravasal hemolytic transfusion reaction with multiple alarming symptoms including hemoglobinuria, back pain, chest pressure and hypotonia. According to all current hemovigilance systems used to monitor the transfusion safety in different countries around the world, this reaction type remains the most common fatal adverse event following transfusion, only matched by septic

reactions due to bacterial contamination of blood components and transfusion-related acute lung injury (TRALI). Even if far from all ABO-incompatible transfusions end with the death of the recipient, every possible effort to decrease this risk should be considered a cost-efficient way of improving transfusion safety. In order to avoid ABO-related transfusion reactions, and hemolysis, all blood donations are checked to confirm the blood group of the donor. In addition, either a classical cross-match procedure (RBCs from the donor are mixed with plasma from the recipient to ensure that they are compatible, i.e. no agglutination or lysis occurs in vitro) or an electronic cross-match following a type-and-screen procedure (the

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blood group of the recipient is confirmed by RBC typing with anti-A and anti-B whilst the recipient plasma is mixed with test RBCs from three to four representative group O donors to exclude the presence of non-ABO-related irregular antibodies to RBCs) is performed. In general, a blood group A recipient will safely be transfused with group A blood, which contains compatible antigens. Because group O blood contains no A or B antigens, it can be transfused into recipients with any blood group, i.e. recipients with group A, B, AB or O. Thus, type O blood is considered “universal”, and may be used for all RBC transfusions. Hence, it is desirable for blood banks to maintain large quantities of type O blood. However, due to many reasons, there is a paucity of blood group O donors. Therefore, it is desirable and useful to remove the immunodominant A and B antigens on types A, B and AB blood in order to maintain large quantities of universal blood products and at the same time avoid one of the most dangerous, and indeed the most common fatal, transfusion reactions. In an attempt to increase the supply of type O blood, efforts have been made to develop methods for converting group A, B and AB blood to group O blood. Conversion of group B cells to group O has been accomplished in the past. However, conversion of the more abundant group A cells has only been achieved with the less abundant A subgroups. The major obstacle for development and utilization of enzyme converted universal O cells in the past has been the failure to enzymatically convert the strongly expressing A1 cells. This obstacle has remained. As will be explained below in detail, the enzymes and methods used in the prior work are inefficient, impractical, and/or too costly to be used in a commercial process to supply universal group O cells.

2. Conversion of group B RBCs Enzymatic conversion of group B blood using purified or recombinant coffee bean (Coffea canephora) a-galactosidase has been achieved using 100–200 U/ml (US Pat. No. 4,427,777 [15,16]). The specific activity of coffee bean a-galactosidase was reported to be 32 U/mg using p-nitrophenyl (pNP) a-D-Gal as the substrate, with one unit (U) defined as 1 µmol substrate hydrolyzed per min [15]. Enzymatic conversions were done at pH 5.5 with approximately 6 mg/ml enzyme at 80–90% hematocrit. The resulting converted O cells functioned normally in transfusion experiments and no significant adverse clinical parameters were observed [16]. These data along with earlier publications clearly demonstrate that enzymatic conversion of RBCs is feasible and that such group B enzyme-converted O (B-ECO) cells can function as well as blood-group-matched untreated cells in clinical transfusion medicine. Nevertheless, the quantities of enzymes used in these studies, even with the effective recombinant expression technology used today, constitute an economic obstacle for use in transfusion medicine.

An apparently improved protocol for conversion of B cells using recombinant soy bean (Glycine max) a-galactosidase with a specific activity of approximately 200 U/mg has been reported using 5–10 U/ml with 16% hematocrit (US Pat. Nos. 5,606,042; 5,633,130; 5,731,426; 6,184,017). This conversion protocol has, however, not been fully evaluated by routine blood typing procedures. The conversion buffer used was Na citrate and glycine at pH 5.8. Approximately, 0.5 mg of enzyme was required per ml packed group B cells for conversion, and the protocol included additional protein in the form of bovine serum albumin (BSA) for stabilization. More recently, Bakunina et al. [17] has isolated and used a novel a-galactosidase from the marine bacterium Pseudoalteromonas spp. for conversion of group B cells to O. This enzyme had a neutral pH optimum and converted B cells after 24 h incubation, although conversion efficiency evaluated by standard typing procedures remains to be tested. Recombinant forms of the enzyme are apparently not available yet and the quantity of protein required for conversion is unknown.

3. Conversion of group A RBCs Levy and Aminoff [18] used purified Clostridium perfringens a-N-acetylgalactosaminidase to convert A cells, and found reduction in antigen expression but considerable blood group A activity remained. Further studies of this enzyme have lead to its purification to apparent homogeneity with a specific activity using the aGalNAc pNP substrate of 43.92 U/mg (PCT Application No. WO 99/23210 and [19]). The purified enzyme was reported to have neutral pH optimum with the aGalNAc pNP substrate. The purified enzyme removed A antigens from A2 cells as measured by an ELISA assay. Calcutt et al. [20] later cloned and expressed a C. perfringens a-N-acetylgalactosaminidase and similarly showed that the enzyme was able to degrade the A2 activity of erythrocyte membranes as measured by ELISA, but a protocol for conversion of intact cells has not been reported [21]. Goldstein [22] used a chicken liver a-Nacetylgalactosaminidase ([23] and US Pat. No. 4,609,627) to achieve similar conversion of weak A subgroups, Aint and A2. The specific activity of the chicken liver a-Nacetylgalactosaminidase was reported to be approximately 50 U/mg using pNP aGalNAc as substrate and the pH optimum 3.65 [24]. The described conversion conditions for Aint and A2 cells included 180 U/ml cells at acidic pH 5.7. The protocol requires more than 3 mg/ml enzyme protein to convert A2 cells. Hata et al. [25] also reported conversion of A2 cells using chicken liver a-N-acetylgalactosaminidase at acidic pH (US Pat. No. 5,606,042) and found similar results. Falk et al. [26] identified and purified an a-Nacetylgalactosaminidase from Ruminococcus torques strain IX-70 that could destroy Dolichus biflorus agglutinability indicating that the A antigenic strength of A1 cells was reduced to the level of A2 cells. This enzyme was reported to

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have relatively poor kinetic properties [27], but the enzyme has not been tested in homogeneous or recombinant form. Based on a recent report, it is likely that the purified enzyme preparation contained contaminating sialidase activity [28]. Izumi et al. [29] tested purified Acremonium sp. a-Nacetylgalactosaminidase on type A1 cells. Although some reduction in agglutination titer was observed conversion was not complete. Finally, a recent attempt to degrade blood group A antigens has been reported by Bakunina et al. [30]. They identified and purified a novel a-N-acetylgalactosaminidase enzyme with neutral pH optimum. They were able to show conversion of both A1 and A2 cells but conversion was not evaluated by routine typing procedures. The reported conversion procedure involved incubation for 24 h and functionality of the cells post-conversion were not studied.

4. Selection of enzymes for RBC conversion Previous methods for searching, identification and characterization of exo-glycosidases have generally relied on the use of simple monosaccharide derivatives as substrates to identify saccharide and potential linkage specificity. Derivatized monosaccharide, or rarely oligosaccharide, substrates include e.g. pNP, benzyl (Bz), 4-methyl-umbrelliferyl (Umb), and 7-amino-4-methyl-coumarin (AMC). The use of such substrates provides easy, fast, and inexpensive tools to identify glycosidase activities, and makes large scale screening of diverse sources of enzymes practically applicable. However, the kinetic properties and fine substrate specificities of glycosidase enzymes may not necessarily be reflected in assays with such simple structures. It is also possible that novel enzymes with high degree of specificity and/or selective efficiency for complex oligosaccharide and unique glycoconjugate structures exist, but that these may have been overlooked and remain unrecognized due to methods of analysis. Thus, in order to identify and select the optimal exo-glycosidase for a particular complex oligosaccharide or glycoconjugate structure, it may be preferable to use such complex structures in assays used for screening sources of enzymes. Furthermore, assays used for screening may include selection for preferable kinetic properties such as pH requirement and performance on substrates, e.g. attached to the membrane of cells. In the past, a-galactosidases (EC 3.2.1.22) and a-Nacetylgalactosaminidases (EC 3.2.1.49) used for destroying blood group B and A antigens, respectively, have mainly been identified and characterized using primarily pNP monosaccharide derivatives. Interestingly, most a-galactosidase and a-N-acetylgalactosaminidase enzymes used in past studies to attempt removal of A and B antigens on cells are evolutionarily homologous as indicated by significant DNA and amino acid sequence similarities. Thus, the human a-galactosidase and a-N-acetylgalactosaminidase are close homologues [31], and other enzymes previously used in

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blood cell conversion including the chicken liver a-Nacetylgalactosaminidase, fungal Acremonium a-N-acetylgalactosaminidase, and bacterial a-galactosidases and a-Nacetylgalactosaminidases all exhibit significant sequence similarities. All known O-glycoside hydrolases have been grouped in 91 distinct families based on sequence analysis, and the above mentioned a-galactosidases and a-Nacetylgalactosaminidases are grouped in families 27 and 36, respectively (see http://afmb.cnrs-mrs.fr/~pedro/CAZY). These enzymes are characterized by having a retaining mechanism of catalysis and use aspartic acid as the catalytic nucleophile [32]. We have screened for novel blood-group-degrading enzymes with the aim to make the conversion procedure work on intact RBCs in a clinical setting. New and efficient glycoside hydrolases of both specificities, i.e. both B-degrading a-galactosidases and A-degrading a-N-acetylgalactosaminidases have been cloned, characterized and used for conversion of native RBCs up to full RBC units. The main goal has been to use short (1 h) incubations at neutral pH and room temperature in order to facilitate the transfer of this technology to blood component laboratories eventually. An a-N-acetylgalactosaminidase from Chryseobacterium spp. available as a recombinant enzyme has proven effective in conversion of both A1 and A2 RBCs as evaluated by standard monoclonal or oligoclonal anti-A, anti-B and antiA,B typing reagents used in blood bank practice. The enzyme quantities required for conversion of A or B cells have been greatly reduced such that 20 mg or less can convert a full unit of RBCs, which holds promise for use of the technology in transfusion medicine.

5. Clinical trials with enzymatically converted O (ECO) RBCs After successful in vitro studies and transfusion of enzymatically converted group B cells to gibbons [33], a series of pioneering studies in humans were reported, the first of which appeared in 1982 [34]. Clinical phase I trials were conducted using B-ECO RBCs given to healthy group A and O volunteers. The trials moved from small infusions to full single RBC units [35] and finally multiple and repeated full-unit transfusions [36,37]. The main conclusion from these studies was that the transfused B-ECO RBCs appeared to survive normally in the circulation of the recipients irrespective of blood group. The conversions were initially carried out with a-galactosidase extracted from coffee beans but the later studies involved the production of recombinant enzyme. At the end of their phase I trials in 1995 the investigators concluded that B-ECO RBCs prepared with recombinant or native enzyme are equivalent in vivo [37]. The next step was a successful phase II clinical trial reported in 2000 [16]. Twenty-one patients underwent transfusion with full unit B-ECO RBCs converted in a modified blood cell processor. Eighteen of them were also given control transfusions

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with blood-group-identical untreated RBCs. The hemoglobin increments were comparable between B-ECO and control transfusions. An increase in anti-B titre was noted in five of the patients but only in one case did this result in a (non-hemolytic) positive direct anti-globulin test (DAT). Whilst serum samples from random group A, B and AB individuals were non-reactive with B-ECO RBCs in all cross-match methods tested including highly sensitive environments like albumin, low-ionic strength solution (LISS) and polyethylene glycol (PEG), a certain proportion of sera from O donors (5–40%) agglutinated the B-ECO cells, especially if the IgG anti-globulin test was used. Despite this, however, the RBC recovery and half-life (T50) of ECO cells were comparable to ABO-matched units based on 51Crlabelling studies [16]. In analogy with previous studies, no signs of antibody formation against the enzyme used for conversion were detected by enzyme-linked immunosorbent assay (ELISA) when comparing serum samples drawn before exposure and 4 weeks after B-ECO transfusion. So far clinical studies for A-ECO RBCs have not been reported. Currently, phase I clinical trials are under way at ZymeQuest Inc, MA, using recombinant a-Nacetylgalactosaminidase at neutral pH.

[2]

6. The future

[11]

Of the different strategies envisioned to create a universal blood component supply, the ECO concept is the only one where human clinical trials have been performed. Until recently no enzyme capable of converting blood group A RBCs was available but through systematic screening and optimization of enzymes and conversion conditions, this is about to change. Future work includes clinical phase II trials for A-ECO RBCs and eventually phase III studies for both A-ECO and B-ECO RBCs. Development of innovative devices for large-scale conversion of the whole blood inventory as well as optimization of ECO RBC storage conditions are performed in parallel with the early clinical trials. Effective and safe enzyme conversion processes for the clinical component preparation laboratory would have an enormous impact on the field of transfusion medicine at large. Most importantly it would be possible to prevent the most common reason to die from blood transfusion today, thus saving numerous lives around the world every year. Furthermore, the logistic advantages of ECO RBCs could not be exaggerated for blood centers and transfusion services that are constantly struggling with blood group O unit shortages while trying to maintain viable blood inventories.

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