Abstract:
Methods of neutralising circulating antibody and mitigating the risk of clinically significant adverse responses to incompatible transfusions and transplantations are described. The methods comprise the administration to the subject of dispersible antigen-lipid constructs.

Description:
TECHNICAL FIELD 
       [0001]    The invention relates to a method of modifying the immune response in a mammal. In particular, the invention relates to a method of neutralizing circulating antibody. 
       BACKGROUND ART 
       [0002]    The circulating antibodies of the ABO blood groups may trigger a hyperacute reaction where a recipient receives a transfusion of blood from an incompatible donor. Circulating antibodies against antigenic determinants (epitopes), including those of the ABO blood groups (glycotopes), are also a major impediment to achieving successful transplantation. Removing anti-A/B antibodies by plasma exchange or plasmapheresis, splenectomy, and use of anti-B cell immunosuppressants are widely adopted strategies to avoid antibody dependant rejection of ABO-incompatible allografts. 
         [0003]    Both the hyperacute reactions and delayed vascular rejections arising from ABO-incompatible transfusions and transplantations are triggered by the binding of anti-A/B antibodies to the A/B glycotopes expressed on the transfused red blood cells (RBCs) or vascular endothelial cells of the transplanted allografts. The binding of anti-A/B antibodies activates complement, platelet aggregation, and inflammation, resulting in intravascular haemolysis or thrombosis and occlusion of blood flow leading to the possible death of the recipient. 
         [0004]    A need clearly exists for a method of neutralizing circulating antibody, at least transiently, to assist in the treatment or prevention of hyperacute reactions and delayed vascular rejections arising from ABO-incompatible transfusions and transplantations. It is to be anticipated that if such a need can be met, that similar methods may be employed to treat or mitigate hyperacute reactions and delayed vascular rejections arising from other glycotope-incompatible transfusions and transplantations. 
         [0005]    It is an object of the invention to provide a method of neutralizing circulating antibody. It is an object of the invention to provide a method to be used in mitigating the risk of a clinically significant reaction to an incompatible blood transfusion. It is an object of the invention to provide a method to be used in mitigating the risk of rejection by a subject of an incompatible allograft or xenograft. It is an object of the invention to provide a method to be used in treating a subject with a clinically significant reaction to an incompatible blood transfusion. These objects are to be read disjunctively with the object of to at least provide a useful choice. 
       DISCLOSURE OF INVENTION 
       [0006]    In a first aspect the invention provides a method to be used in neutralizing one or more populations of circulating antibody in a subject mammal including the step of:
       Administering to the subject mammal an amount of synthetic antigen-lipid construct (F—S-L);
 
where the antigen (F) is reactive with the one or more populations of circulating antibody.
       
 
         [0008]    Preferably, the administering to the subject mammal is by intravascular injection of a dispersion of the amount of synthetic antigen-lipid construct (F—S-L). More preferably, the administering to the subject mammal is by intravenous injection of a dispersion of the amount of synthetic antigen-lipid construct (F—S-L). 
         [0009]    Preferably, the administering to the subject mammal is prior to transfusion or transplantation. More preferably, the administering to the subject mammal is prior to transfusion or transplantation of donor cells, tissues or organs expressing the antigen (F). Most preferably, the administering to the subject mammal is prior to transfusion or transplantation of donor cells, tissues or organs predetermined to express the antigen (F). 
         [0010]    Preferably, the amount of synthetic antigen-lipid construct (F—S-L) is effective to neutralize the one or more populations of circulating antibody. 
         [0011]    Preferably, the amount of synthetic antigen-lipid construct (F—S-L) is in the form of a formulation that excludes plasma. 
         [0012]    Preferably, the method is to provide at least a transient tolerance of the transfusion or graft expressing the antigen in the subject mammal. 
         [0013]    Preferably, the antigen-lipid construct (F—S-L) is of the structure: 
         [0000]      F—S 1 —S 2 -L
 
         [0000]    where
       F is the epitope of a carbohydrate antigen (glycotope);   S 1 —S 2  is a spacer (S) linking F to L; and   L is a lipid selected from the group consisting of diacyl- and dialkyl-glycerolipids, including glycerophospholipids.       
 
         [0017]    Preferably, F, S 1 , S 2  and L are covalently linked. 
         [0018]    Preferably, F is selected from the group consisting of: glycotopes of blood group antigens. More preferably, F is selected from the group consisting of: GalNAcα3(Fucα2)Galβ-; Galα3(Fucα2)Galβ-; GalNα3(Fucα2)Galβ-; Fucα2Galβ-; Galβ4GlcNAcβ3(Galβ4GlcNAcβ6)Galβ-; Galβ4GlcNAcβ3-; Galβ4Glcβ-; Galβ3GlcNAcβ-; Galβ3(Fucα4)GlcNAcβ-; Fucα2Galβ3(Fucα4)GlcNAcβ-; GalNAcα3(Fucα2)Galβ3(Fucα4)GlcNAcβ-; Galα3(Fucα2)Galβ3(Fucα4)GlcNAcβ-; Galβ4(Fucα3)GlcNAcβ-; Fucα2Galβ4(Fucα3)GlcNAcβ-; NeuAcα2-3Galβ3(Fucα4)GlcNAcβ-; NeuAcα2-3Galβ4(Fucα3)GlcNAcβ-; GalNAcβ4(NeuAcα2-3)Galβ4-; Galβ3GalNAcα-; NeuAcα2-3Galβ4-; NeuAcα2-6Galβ4-; Galα4Galβ4-; GalNAcβ3Galα4Galβ4-; Galα4Galβ4GlcNAcβ3-; Galβ3GalNAcβ3Galα4-; NeuAcα2-3Galβ3GalNAcβ3Galα4-; Galα3Galβ-; GalNAcα3GalNAcβ3Galα4-; and GalNAcβ3GalNAcβ3Galα4-. Most preferably, F is selected from the group consisting of: glycotopes of the ABO blood group antigens. 
         [0019]    Preferably, S 1 —S 2  is selected to provide an antigen-lipid construct that is dispersible in a biocompatible medium. More preferably, S 1 —S 2  is selected to provide an antigen-lipid construct that is dispersible in water and preferentially partitions into the plasma relative to naturally occurring glycolipids. 
         [0020]    Preferably, the antigen-lipid construct (F—S-L) is of the structure: 
         [0000]      F—S 1 —S 2 -L
 
         [0000]    where
       F is a mono-, di-, tri- or oligo-saccharide;   S 1  is 2-aminoethyl, 3-aminopropyl, 4-aminobutyl, or 5-aminopentyl;   S 2  is —CO(CH 2 ) 2 CO—, —CO(CH 2 ) 3 CO—, —CO(CH 2 ) 4 CO— or —CO(CH 2 ) 5 CO—; and   L is a diacyl- or dialkyl-glycerophospholipid.       
 
         [0025]    Preferably, L is selected from the group consisting of: diacylglycerolipids, phosphatidate, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl inositol, phosphatidyl glycerol, and diphosphatidyl glycerol derived from one or more of trans-3-hexadecenoic acid, cis-5-hexadecenoic acid, cis-7-hexadecenoic acid, cis-9-hexadecenoic acid, cis-6-octadecenoic acid, cis-9-octadecenoic acid, trans-9-octadecenoic acid, trans-11-octadecenoic acid, cis-11-octadecenoic acid, cis-11-eicosenoic acid or cis-13-docsenoic acid. More preferably, the lipid is derived from one or more cis-desaturated fatty acids. Most preferably, L is selected from the group consisting of: 1,2-O-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), 1,2-O-distearyl-sn-glycero-3-phosphatidylethanolamine (DSPE) and rac-1,2-dioleoylglycerol (DOG). 
         [0026]    In preferred embodiments of the first aspect of the invention the antigen-lipid construct includes the substructure: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    where n is the integer 2, 3, 4 or 5 and * is other than H. More preferably, n is the integer 4. 
         [0027]    In a preferred embodiment of the first aspect of the invention the antigen-lipid construct (F—S-L) is the structure: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    designated A tri -sp 1 -sp 2 -DOPE (I). 
         [0028]    Preferably, the subject mammal is a human. 
         [0029]    In a second aspect the invention provides a pharmaceutical preparation comprising a dispersion of synthetic antigen-lipid construct (F—S-L) for use in the method of the first aspect of the invention where the antigen-lipid construct is of the structure: 
         [0000]      F—S 1 —S 2 -L
 
         [0000]    and
       F is the epitope of a carbohydrate antigen (glycotope);   S 1 —S 2  is a spacer linking F to L; and   L is a lipid selected from the group consisting of diacyl- and dialkyl-glycerolipids, including glycerophospholipids.       
 
         [0033]    Preferably, F, S 1 , S 2  and L are covalently linked. 
         [0034]    Preferably, the dispersion of synthetic antigen-lipid construct (F—S-L) is at a concentration of at least 10 μmol. More preferably, the dispersion of synthetic antigen-lipid construct (F—S-L) is at a concentration of at least 15 μmol. Most preferably, the dispersion of synthetic antigen-lipid construct (F—S-L) is at a concentration of at least 17.5 μmol. 
         [0035]    Preferably, the dispersion of synthetic antigen-lipid construct (F—S-L) is a dispersion in human red cell transfusion medium. 
         [0036]    In a third aspect the invention provides the use of an amount of synthetic antigen-lipid construct (F—S-L) in the manufacture of a preparation for neutralizing one or more populations of circulating antibody in a subject mammal. 
         [0037]    In a first preferred embodiment the preparation is a medicament for administration to a human prior to transfusion to mitigate the risk of a clinically significant reaction to a donor antigen. 
         [0038]    In a second preferred embodiment the preparation is a medicament for administration to a human prior to transplantation to mitigate the risk of a clinically significant reaction to a donor antigen. 
         [0039]    Preferably, the antigen-lipid construct (F—S-L) is of the structure: 
         [0000]      F—S 1 —S 2 -L
 
         [0000]    where
       F is the epitope of a carbohydrate antigen (glycotope);   S 1 —S 2  is a spacer linking F to L; and   L is a lipid selected from the group consisting of diacyl- and dialkyl-glycerolipids, including glycerophospholipids.       
 
         [0043]    Preferably, F is selected from the group consisting of: glycotopes of blood group antigens. More preferably, F is selected from the group consisting of: GalNAcα3(Fucα2)Galβ-; Galα3(Fucα2)Galβ-; GalNα3(Fucα2)Galβ-; Fucα2Galβ-; Galβ4GlcNAcβ3(Galβ4GlcNAcβ6)Galβ-; Galβ4GlcNAcβ3-; Galβ4Glcβ-; Galβ3GlcNAcβ-; Galβ3(Fucα4)GlcNAcβ-; Fucα2Galβ3 (Fucα4)GlcNAcβ-; GalNAcα3(Fucα2)Galβ3(Fucα4)GlcNAcβ-; Galα3(Fucα2)Galβ3(Fucα4)GlcNAcβ-; Galβ4(Fucα3)GlcNAcβ-; Fucα2Galβ4(Fucα3)GlcNAcβ-; NeuAcα2-3Galβ3(Fucα4)GlcNAcβ-; NeuAcα2-3Galβ4(Fucα3)GlcNAcβ-; GalNAcβ4(NeuAcα2-3)Galβ4-; Galβ3GalNAcα-; NeuAcα2-3Galβ4-; NeuAcα2-6Galβ4-; Galα4Galβ4-; GalNAcβ3Galα4Galβ4-; Galα4Galβ4GlcNAcβ3-; Galβ3GalNAcβ3Galα4-; NeuAcα2-3Galβ3GalNAcβ3Galα4-; Galα3Galβ-; GalNAcα3GalNAcβ3Galα4-; and GalNAcβ3GalNAcβ3Galα4-. Most preferably, F is selected from the group consisting of: glycotopes of the ABO blood group antigens. 
         [0044]    Preferably, S 1 —S 2  is selected to provide an antigen-lipid construct that is dispersible in a biocompatible medium. More preferably, S 1 —S 2  is selected to provide an antigen-lipid construct that is dispersible in water and preferentially partitions into the plasma relative to naturally occurring glycolipids. 
         [0045]    Preferably, L is selected from the group consisting of: diacylglycerolipids, phosphatidate, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl inositol, phosphatidyl glycerol, and diphosphatidyl glycerol derived from one or more of trans-3-hexadecenoic acid, cis-5-hexadecenoic acid, cis-7-hexadecenoic acid, cis-9-hexadecenoic acid, cis-6-octadecenoic acid, cis-9-octadecenoic acid, trans-9-octadecenoic acid, trans-11-octadecenoic acid, cis-11-octadecenoic acid, cis-11-eicosenoic acid or cis-13-docsenoic acid. More preferably, the lipid is derived from one or more cis-desaturated fatty acids. Most preferably, L is selected from the group consisting of: 1,2-O-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), 1,2-O-distearyl-sn-glycero-3-phosphatidylethanolamine (DSPE) and rac-1,2-dioleoylglycerol (DOG). 
         [0046]    In the preferred embodiments of the third aspect of the invention the antigen-lipid construct (F—S-L) preferably includes the substructure: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    where n is the integer 2, 3, 4 or 5 and * is other than H. More preferably, n is the integer 4. 
         [0047]    In the preferred embodiments of the third aspect of the invention it is most preferred that the antigen-lipid construct (F—S-L) is the structure: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    designated A tri -sp 1 -sp 2 -DOPE (I). 
         [0048]    In a fourth aspect the invention provides a method of modifying the profile of circulating antibody in the plasma of a subject mammal comprising the step of:
       Administering to the subject mammal an amount of synthetic antigen-lipid construct of the structure F—S 1 —S 2 -L;
 
where
   F is the epitope of a carbohydrate antigen (glycotope) reactive with one or more populations of the circulating antibody;   S 1 —S 2  is a spacer linking F to L; and   L is a lipid selected from the group consisting of diacyl- and dialkyl-glycerolipids, including glycerophospholipids.       
 
         [0053]    Preferably, the administering to the subject mammal is by intravascular injection of a dispersion of the amount of synthetic antigen-lipid construct. More preferably, the administering to the subject mammal is by intravenous injection of a dispersion of the amount of synthetic antigen-lipid construct. 
         [0054]    Preferably, F is selected from the group consisting of: glycotopes of blood group antigens. More preferably, F is selected from the group consisting of: GalNAcα3(Fucα2)Galβ-; Galα3(Fucα2)Galβ-; GalNα3(Fucα2)Galβ-; Fucα2Galβ-; Galβ4GlcNAcβ3(Galβ4GlcNAcβ6)Galβ-; Galβ4GlcNAcβ3-; Galβ4Glcβ-; Galβ3GlcNAcβ-; Galβ3(Fucα4)GlcNAcβ-; Fucα2Galβ3(Fucα4)GlcNAcβ-; GalNAcα3(Fucα2)Galβ3(Fucα4)GlcNAcβ-; Galα3(Fucα2)Galβ3(Fucα4)GlcNAcβ-; Galβ4(Fucα3)GlcNAcβ-; Fucα2Galβ4(Fucα3)GlcNAcβ-; NeuAcα2-3Galβ3(Fucα4)GlcNAcβ-; NeuAcα2-3Galβ4(Fucα3)GlcNAcβ-; GalNAcβ4(NeuAcα2-3)Galβ4-; Galβ3GalNAcα-; NeuAcα2-3Galβ4-; NeuAcα2-6Galβ4-; Galα4Galβ4-; GalNAcβ3Galα4Galβ4-; Galα4Galβ4GlcNAcβ3-; Galβ3GalNAcβ3Galα4-; NeuAcα2-3Galβ3GalNAcβ3Galα4-; Galα3Galβ-; GalNAcα3GalNAcβ3Galα4-; and GalNAcβ3GalNAcβ3Galα4-. Most preferably, F is selected from the group consisting of: glycotopes of the ABO blood group antigens. 
         [0055]    Preferably, S 1 —S 2  is selected to provide an antigen-lipid construct that is dispersible in a biocompatible medium. More preferably, S 1 —S 2  is selected to provide an antigen-lipid construct that is dispersible in water and preferentially partitions into the plasma relative to naturally occurring glycolipids. 
         [0056]    Preferably, L is selected from the group consisting of: diacylglycerolipids, phosphatidate, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl inositol, phosphatidyl glycerol, and diphosphatidyl glycerol derived from one or more of trans-3-hexadecenoic acid, cis-5-hexadecenoic acid, cis-7-hexadecenoic acid, cis-9-hexadecenoic acid, cis-6-octadecenoic acid, cis-9-octadecenoic acid, trans-9-octadecenoic acid, trans-11-octadecenoic acid, cis-11-octadecenoic acid, cis-11-eicosenoic acid or cis-13-docsenoic acid. More preferably, the lipid is derived from one or more cis-desaturated fatty acids. Most preferably, L is selected from the group consisting of: 1,2-O-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), 1,2-O-distearyl-sn-glycero-3-phosphatidylethanolamine (DSPE) and rac-1,2-dioleoylglycerol (DOG). 
         [0057]    In preferred embodiments of the fourth aspect of the invention the antigen-lipid construct (F—S-L) includes the substructure: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    where n is the integer 2, 3, 4 or 5 and * is other than H. More preferably, n is the integer 4. 
         [0058]    In a preferred embodiment of the fourth aspect of the invention the antigen-lipid construct (F—S-L) is the structure: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    designated A tri -sp 1 -sp 2 -DOPE (I). 
         [0059]    In the description and claims of this specification the following acronyms, terms and phrases are intended to have the meaning indicated: 
         [0060]    “Clinically significant” means an adverse event that requires intervention by a clinician or hospitalization. 
         [0061]    “Dispersible” means a stable, single phase system is formed when the antigen-lipid construct is contacted with an aqueous vehicle such as serum or plasma in the absence of organic solvents or detergents, and the term “dispersion” has a corresponding meaning. 
         [0062]    “Donor” means the subject from which the cells, tissues or organs are obtained. 
         [0063]    “Glycotope” means the epitope of a carbohydrate antigen. 
         [0064]    “Incompatible” means:
       in reference to an antigen, an antigen for which a clinically significant humoral and/or cell mediated specific immune response occurs or is occurring; and   in reference to a blood transfusion, a blood transfusion where the plasma of the recipient contains antibodies to an antigen of the donor at a sufficiently high titre to cause a clinically significant humoral and/or cell mediated specific immune response.       
 
         [0067]    “Intravascular” means delivery into the vascular fluids (blood, lymph) of the body, and includes intravenous delivery. 
         [0068]    “Neutralise” means that the recipient does not experience a clinically significant response following the transfusion of blood or transplantation of cells expressing antigen for which the recipient expresses antibody, and the term “neutralising” has a corresponding meaning. 
         [0069]    “pcv” denotes packed cell volume. 
         [0070]    “Plasma” means the colourless fluid part of blood or lymph, in which corpuscles or fat globules are suspended. 
         [0071]    “hRBC” denotes human red blood cells. 
         [0072]    “mRBC” denotes mouse red blood cells. 
         [0073]    “rRBC” denotes rabbit red blood cells. 
         [0074]    “Saline” means a solution of one or more salts. 
         [0075]    “Serum” means the amber-coloured, protein-rich liquid which separates out when blood coagulates. 
         [0076]    “Synthetic” means made by chemical synthesis. 
         [0077]    The term “antigen” is to be understood in a functional sense, i.e. structures capable of eliciting an antibody mediated immune response. 
         [0078]    Exemplary embodiments of the invention will now be described in detail with reference to the Figures of the accompanying drawings pages. 
     
    
     
       BRIEF DESCRIPTION OF FIGURES 
         [0079]      FIG. 1 . Urine samples (30 minutes post infusion with murine A kodecytes): L to R—ID#4605, ID#8553, ID#5234, ID#7999 (control). 
           [0080]      FIG. 2 . Schematic representation of control and trial series described under the heading Administration of compatible and incompatible modified red blood cells to mice. 
       
    
    
     DETAILED DESCRIPTION 
       [0081]    The antigen-lipid constructs of the invention are administered by intravascular injection. It is a requirement of this mode of administration that the antigen-lipid constructs are delivered in a biocompatible medium. Antigen-lipid constructs that are dispersible in biocompatible media at a concentration sufficient to allow administration at a rate sufficient to neutralise one or more populations of circulating antibody or induce at least a transient tolerance of the antigen are required. It is a particular advantage of the synthetic antigen-lipid constructs of the invention that there is no significant activation of the complement cascade. 
         [0082]    Antigen-lipid constructs with these required properties are prepared by covalently joining the epitope of the carbohydrate antigen (glycotope) to a diacyl or dialkyl lipid via a spacer. The spacer is selected to impart the required dispersibility on the antigen-lipid construct. When administered into the vascular system the antigen-lipid construct is anticipated to partition between the plasma and membranes of the cells of the circulatory and lymphatic systems (red blood cells (RBCs), lymphocytes), but with a greater propensity to partition into the plasma than a naturally occurring glycolipid (glycosphingolipid) counterpart. Whilst not wishing to be bound by theory it is anticipated that it is this ability of the antigen-lipid constructs to partition between both the plasma and membranes of cells of the vascular system that provides for the observed neutralization of circulating antibody. 
         [0083]    In one application it is proposed that the antigen-lipid constructs are administered by intravenous injection or infusion to mitigate the risk of a hemolytic response arising from the presence of reactive circulating antibody in an incompatible recipient of transfused blood. Reactive circulating antibody to one or more of the following antigens may be present in a recipient: 
         [0000]    
       
         
               
               
               
             
           
               
                   
               
               
                   
                 Antigen 
                 Glycotope 
               
               
                   
               
             
             
               
                   
                 A 
                 GalNAcα3(Fucα2)Galβ-R 
               
               
                   
                 B 
                 Galα3(Fucα2)Galβ-R 
               
               
                   
                 Acquired B 
                 GalNα3(Fucα2)Galβ-R 
               
               
                   
                 H 
                 Fucα2Galβ-R 
               
               
                   
                 I 
                 Galβ4GlcNAcβ3(Galβ4GlcNAcβ6)Galβ-R 
               
               
                   
                 i 
                 Galβ4GlcNAcβ3-R 
               
               
                   
                 Lactosyl 
                 Galβ4Glcβ-R 
               
               
                   
                 Lec 
                 Galβ3GlcNAcβ-R 
               
               
                   
                 Lea 
                 Galβ3(Fucα4)GlcNAcβ-R 
               
               
                   
                 Leb 
                 Fucα2Galβ3(Fucα4)GlcNAcβ-R 
               
               
                   
                 ALeb 
                 GalNAcα3(Fucα2)Galβ3(Fucα4)GlcNAcβ-R 
               
               
                   
                 BLeb 
                 Galα3(Fucα2)Galβ3(Fucα4)GlcNAcβ-R 
               
               
                   
                 X 
                 Galβ4(Fucα3)GlcNAcβ-R 
               
               
                   
                 Y 
                 Fucα2Galβ4(Fucα3)GlcNAcβ-R 
               
               
                   
                 Sialyl Lea 
                 NeuAcα2-3Galβ3(Fucα4)GlcNAcβ-R 
               
               
                   
                 Sialyl Lex 
                 NeuAcα2-3Galβ4(Fucα3)GlcNAcβ-R 
               
               
                   
                 Cad 
                 GalNAcβ4(NeuAcα2-3)Galβ4-R 
               
               
                   
                 T 
                 Galβ3GalNAcα-R 
               
               
                   
                 Sialyl 
                 NeuAcα2-3Galβ4-R 
               
               
                   
                 Sialyl 
                 NeuAcα2-6Galβ4-R 
               
               
                   
                 Pk 
                 Galα4Galβ4-R 
               
               
                   
                 P 
                 GalNAcβ3Galα4Galβ4-R 
               
               
                   
                 P1 
                 Galα4Galβ4GlcNAcβ3-R 
               
               
                   
                 SSEA-3 
                 Galβ3GalNAcβ3Galα4-R 
               
               
                   
                 LKE 
                 NeuAcα2-3Galβ3GalNAcβ3Galα4-R 
               
               
                   
                 Galili 
                 Galα3Galβ-R 
               
               
                   
                 Forssman 
                 GalNAcα3GalNAcβ3Galα4-R 
               
               
                   
                 paraForssman 
                 GalNAcβ3GalNAcβ3Galα4-R 
               
               
                   
               
             
          
         
       
     
         [0084]    It is contemplated that antigen-lipid constructs (F—S-L) where F is one of the foregoing glycotopes may be prepared and used in the methods of the invention. The antigen-lipid constructs (F—S-L) would be of the following generalized structures:
       GalNAcα3(Fucα2)Galβ-S 1 —S 2 -L (e.g. A tri -sp 1 -sp 2 -DOPE (I))   Galα3(Fucα2)Galβ-S 1 —S 2 -L   GalNα3(Fucα2)Galβ-S 1 —S 2 -L   Fucα2Galβ-S 1 —S 2 -L   Galβ4GlcNAcβ3(Galβ4GlcNAcβ6)Galβ-S 1 —S 2 -L   Galβ4GlcNAcβ3-S 1 —S 2 -L   Galβ4Glcβ-S 1 —S 2 -L   Galβ3GlcNAcβ-S 1 —S 2 -L   Galβ3(Fucα4)GlcNAcβ-S 1 —S 2 -L   Fucα2Galβ3(Fucα4)GlcNAcβ-S 1 —S 2 -L   GalNAcα3(Fucα2)Galβ3(Fucα4)GlcNAcβ-S 1 —S 2 -L   Galα3(Fucα2)Galβ3(Fucα4)GlcNAcβ-S 1 —S 2 -L   Galβ4(Fucα3)GlcNAcβ-S 1 —S 2 -L   Fucα2Galβ4(Fucα3)GlcNAcβ—S 1 —S 2 -L   NeuAcα2-3Galβ3(Fucα4)GlcNAcβ-S 1 —S 2 -L   NeuAcα2-3Galβ4(Fucα3)GlcNAcβ-S 1 —S 2 -L   GalNAcβ4(NeuAcα2-3)Galβ4-S 1 —S 2 -L   Galβ3GalNAcα-S 1 —S 2 -L   NeuAcα2-3Galβ4-S 1 —S 2 -L   NeuAcα2-6Galβ4-S 1 —S 2 -L   Galα4Galβ4-S 1 —S 2 -L   GalNAcβ3Galα4Galβ4-S 1 —S 2 -L   Galα4Galβ4GlcNAcβ3-S 1 —S 2 -L   Galβ3GalNAcβ3Galα4-S 1 —S 2 -L   NeuAcα2-3Galβ3GalNAcβ3Galα4-S 1 —S 2 -L   Galα3Galβ3-S 1 —S 2 -L   GalNAcα3GalNAcβ3Galα4-S 1 —S 2 -L   GalNAcβ3GalNAcβ3Galα-4-S 1 —S 2 -L
 
where S 1 —S 2 -L are as defined under the heading Disclosure of Invention.
       
 
         [0113]    A phenomenon documented in transfusion medicine is the ability of Lewis glycolipids present in plasma to temporarily neutralise circulating antibodies (Mollison et al (1972)). Lewis antibodies are potent haemolytic antibodies which can result in severe haemolytic transfusion reactions. Individuals who have no option other than to undergo a transfusion with Lewis (blood group antigen) incompatible blood are firstly infused with 1 unit of Lewis positive plasma. This neutralises the circulating antibody and is immediately followed by the transfusion of incompatible red blood cells. The extent to which the administered antigen-lipid construct incorporates non-specifically in to the outer membranes of cells will depend in part on the amount administered and the dosage form, e.g. a pharmaceutically acceptable preparation that is a dispersion, or a pharmaceutically acceptable preparation that is a suspension of liposomes. 
         [0114]    A pilot study in rabbits demonstrated it was possible to drastically reduce the antibody titre of one animal from 1:512 to 1:8 after one infusion of 20 mg of antigen-lipid construct 4 hours post infusion. In another animal, the antibody titre dropped from 1:256 to 1:2 after one infusion of 36 mg of antigen-lipid construct 24 hours post infusion. Furthermore, in a third animal, the subject mammal&#39;s antibody titre was still significantly reduced from 1:32 to 1:8, 5 days after one infusion of 30 mg of antigen-lipid construct. Further studies were subsequently performed to confirm circulating antibody in subject mammals can be neutralised by intravenous injection of synthetic antigen-lipid constructs. The sustainability of neutralization over a short period of time was also assessed. 
         [0115]    Without inferring any limitation on the general applicability of the proposed method of neutralizing one or more populations of circulating antibody, a synthetic antigen-lipid construct of the following structure: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    and designated A tri -sp 1 -sp 2 -DOPE (I) was used. It will be recognized that synthetic antigen-lipid constructs including different epitopes may be used alone or in combination in the proposed method to neutralize one or more populations of reactive circulating antibody. 
       Experimental Procedures 
     Preparation of Constructs 
       [0116]    The preparation of synthetic antigen-lipid constructs where the epitope of the antigen-lipid construct is a carbohydrate (glycotope) is described in the specification accompanying international application no. PCT/NZ2005/000052 (publication no. WO 2005/090368). 
       Rabbit Studies 
     Administration of the Antigen-Lipid Construct 
       [0117]    Blood was collected from the saphenous vein of two rabbits (rabbit #1; rabbit #2) prior to administration of the antigen-lipid construct. The antibody titre of the blood collected from each rabbit was measured using hRBCs. Prior to the infusion a volume of blood equal to the volume of dispersion to be infused was withdrawn from the subclavian vein. The antigen-lipid construct was administered to each rabbit as a 20 mg/ml dispersion in saline by intravenous infusion into the subclavian vein. The antigen-lipid construct was administered at a rate of 10 mg per 100 g bodyweight of the rabbit. The rabbits were monitored post-infusion and observed for adverse reaction to the administration of the antigen-lipid construct. No adverse reactions were observed. 
         [0000]    Determination of In Vivo Modification of rRBCs 
         [0118]    Blood was collected from the saphenous vein of each rabbit one day following the administration of the antigen-lipid construct and the rRBCs separated from the serum. The separated rRBCs were washed three times and assessed for antigen expression using the DIAMED™ card platform. 30 μl of a 1.6% (pcv) rRBC suspension and 30 μl of anti-A monoclonal antibody were mixed and the cards spun and read. Rabbit #1 had a nil anti-A titre and rabbit #2 had a titre of 1:16 prior to infusion as measured against type A hRBCs. The rRBC agglutination scores as measured in the DIAMED™ card platform are presented in Table 1. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Comparison of agglutination scores for rRBC pre-infusion, 
               
               
                 rRBC from rabbit infused with antigen-lipid construct 
               
               
                 (post infusion), rRBC modified in vitro by contacting 
               
               
                 with antigen-lipid construct and A hRBC. 
               
             
          
           
               
                   
                 rRBC 
                 rRBC 
                   
                   
               
               
                   
                 pre- 
                 post- 
                 modified 
                 A 
               
               
                   
                 infusion 
                 infusion 
                 rRBC* 
                 hRBC 
               
               
                   
               
             
          
           
               
                 Pre-immunisation serum 
                 IgM 
                 0 
                 0  
                 0  
                 0  
               
               
                   
                 IgG 
                 0 
                 0  
                 0  
                 0  
               
               
                 Post-immunization/pre- 
                 IgM 
                 0 
                 4+ 
                 4+ 
                 4+ 
               
               
                 infusion serum 
                 IgG 
                 0 
                 4+ 
                 4+ 
                 4+ 
               
               
                 Monoclonal 
                 IgM 
                 0 
                 4+ 
                 4+ 
                 4+ 
               
               
                 anti-A 
               
               
                   
               
             
          
         
       
     
         [0119]    Intravenous administration of the antigen-lipid construct at a rate of 10 mg per 100 g bodyweight is effective to change the level of antigen expressed at the surface of circulating rRBCs with no observable consequences on the health of the subject rabbit. 
       Modification of the Titre of Reactive IgM Antibody in Plasma 
       [0120]    Prior to immunisation a sample of blood was taken from the marginal ear vein of a rabbit. Indicator cells were prepared by contacting the rRBCs with a dispersion of the antigen-lipid construct at a concentration of 2 mg/ml. A rabbit (id#250) was immunized by subcutaneous injection of blood group A saliva mixed with Titremax™ adjuvant. Two weeks after immunization a sample of blood was taken from the marginal ear vein of the rabbit. The antibody titre of the serum of the blood obtained from the rabbit pre- and post-immunization was determined. Three weeks after immunization 2 ml of a dispersion of the antigen-lipid construct at a concentration of 25 mg/ml was infused into the marginal ear vein of the rabbit and the subject monitored for any signs of an adverse reaction for 4 hours. At 2 hours and 24 hours post-infusion a sample of blood was collected from the marginal ear vein of the rabbit and the serum separated. Serial dilutions of the serum collected prior to infusion with the antigen-lipid construct 2 hours post-infusion and 24 hours post-infusion were prepared. The antibody titre was determined by mixing 30 μl of each dilution and 30 μl of a 5% (pcv) suspension of rRBC modified with the antigen-lipid construct. The agglutination score for each mixture was read by tube serology. The titre of antibodies determined for the dilutions of sera are presented in Table 2. 
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Titres of anti-A IgM in rabbit serum pre- and post-infusion 
               
               
                 measured by agglutination with rRBC cells modified in vitro 
               
               
                 by contacting with the antigen-lipid construct. 
               
             
          
           
               
                   
                 Serum dilution 
               
             
          
           
               
                   
                 1:2 
                 1:4 
                 1:8 
                 1:16 
                 1:32 
               
               
                   
               
             
          
           
               
                   
                 Pre-immunisation 
                 0  
                 0  
                 0 
                 0 
                 0 
               
               
                   
                 Post-immunisation &amp; 
                 4+ 
                 3+ 
                  2+ 
                 w 
                 0 
               
               
                   
                 pre infusion 
                   
                   
                   
                   
                   
               
               
                   
                 2 hours post infusion 
                 0  
                 0  
                 0 
                 0 
                 0 
               
               
                   
                 24 hours post 
                 3+ 
                 w 
                 0 
                 0 
                 0 
               
               
                   
                 infusion 
                   
                   
                   
                   
                   
               
               
                   
                 Anti-A control 
                 4+ 
                 4+ 
                  3+ 
                  2+ 
                 w 
               
               
                   
               
             
          
         
       
     
         [0121]    Normal behaviour of the subject rabbit was observed within 10 minutes of the infusion of the antigen-lipid construct and no adverse reactions were observed. The antigen-lipid construct was able to be infused into the subject rabbit post-immunization with no observable adverse reaction. A reduction in the measurable titre of circulating antibody is observed at 2 hours following intravenous infusion with the antigen-lipid construct and a partial reduction in the titre was observed 24 hours following intravenous indicating a transient neutralisation of circulating antibody (IgM). 
       Modification of the Titre of Reactive IgG and IgM Antibody in Plasma 
       [0122]    Modified rRBC (indicator cells) and an immunized rabbit were prepared as described above. Four weeks following immunization a sample of blood was taken from the saphenous vein of the rabbit and the rRBC and serum separated. The antibody titre of the serum was determined. Five weeks following immunization a volume of blood equal to the volume of the dispersion of the antigen-lipid construct to be infused was withdrawn from the subclavian vein of the subject rabbit. A dispersion of the antigen-lipid construct at a concentration of 20 mg/ml in saline was administered by intravenous infusion into the subclavian vein within circa 5 minutes. The antigen-lipid construct was administered at a rate of 10 mg per 100 g bodyweight. The subject rabbit was monitored for any adverse reaction to the infusion for 4 hours. Samples of blood were taken from the saphenous vein of the subject rabbit 5 minutes, 20 hours, and 48 hours following the infusion and the rRBC separated from the serum. The rRBCs were washed 3 times. Serial dilutions of the serum collected 5 minutes, 20 hours and 48 hours following infusion were prepared and the titre of antibody determined. For each dilution 30 μl was incubated with 30 μl of a 5% (pcv) of indicator cells and incubated as a suspension for 15 minutes as room temperature (RT). Each mixture was then centrifuged and the degree of agglutination determined. The samples were then re-suspended and incubated for a further 60 minutes at 37° C. and examined for agglutination and haemolysis. The samples were then washed 3 times in PBS and incubated with 30 μl anti-rabbit IgG antibody, centrifuged and examined for the presence of anti-A IgG. The lowest serum dilution providing a serology reading with the indicator cells of greater than 1+ are presented in Table 3. The agglutination scores of rRBC of the samples of blood obtained from the rabbit prior to and following infusion with the antigen-lipid construct are provided in Table 4. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 In vivo neutralization of circulating 
               
               
                 anti-A IgM and anti-A IgG antibodies 
               
             
          
           
               
                   
                 5 minutes 
                 5 minutes 
                 20 hours 
                 48 hours 
               
               
                   
                 pre-infusion 
                 post-infusion 
                 post-infusion 
                 post-infusion 
               
               
                   
               
               
                 IgM anti-A 
                 1:8 
                 0 
                 0 
                 0 
               
               
                 RT 
                   
                   
                   
                   
               
               
                 IgM anti-A 
                 1:4 
                 0 
                 1:2 
                 1:2 
               
               
                 37° C. 
                   
                   
                   
                   
               
               
                 IgG anti-A 
                  1:16 
                 1:4 
                 1:4 
                 1:8 
               
               
                   
               
             
          
         
       
     
         [0123]    Referring to Table 3 an infusion of the antigen-lipid construct appeared to neutralize the circulating anti-A antibody and the serum remained at least partially neutralized for 48 hours after infusion. The initial room temperature (RT) reactive anti-A IgM titre of 1:8 was completely neutralised and remained at this level 48 hours post infusion. The initial reactive anti-A IgG titre of 1:16 was neutralised to 1:4 for 20 hours, then increased to 1:8 after 48 hours indicating a transient neutralization of the circulating antibody. Referring to Table 4 rRBC&#39;s collected 5 minutes and 20 hours post an intravenous infusion of FSL A (10 mg/100 g body weight) were successfully transformed to express the A antigen. At 48 hours post infusion the synthetic molecules were not detected on the RBC membrane. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 In vivo modification of rRBC by infusion of antigen-lipid construct. 
               
               
                 Agglutination scores of rRBC using monoclonal anti-A antibody 
               
             
          
           
               
                   
                 5 minutes 
                 5 minutes 
                 20 hours 
                 48 hours 
               
               
                   
                 pre-infusion 
                 post-infusion 
                 post-infusion 
                 post-infusion 
               
               
                   
               
               
                   
                 0 
                 4+ 
                 3+ 
                 0 
               
               
                   
               
             
          
         
       
     
         [0124]    In vivo modification of rRBC to express a serologically detectable level of A antigen is observed following intravenous infusion of the antigen-lipid construct. The modification is concomitant with a reduction in the titre of circulating anti-A antibody of both the IgG and IgM classes. No adverse effects on the health of the subject rabbit are observed. 
       Rat Studies 
     Standard Immunisation Method 
       [0125]    Rats were immunised in a one-step procedure according to protocols published for TiterMax® adjuvant. The rat was placed on its feet and was injected intramuscularly behind each hind quadricep with a 25 gauge needle. The following preparations were injected:
       1. A 100 μl mixture of TiterMax® and natural glycolipid or synthetic antigen-lipid construct; or   2. Cells incorporating synthetic antigen-lipid constructs.       
 
         [0128]    The rat was restrained on a towel on top of a table in a 25° C. room. It is well established that laboratory rats tolerate TiterMax® adjuvant well without the common injection site reactions that result from the Fruends adjuvant (Bennet 1992). 
       Blood Sampling to Assess Antibody Titres 
       [0129]    The rat was anesthetized using the standard mixture of [8.75 mg Ketamine: 1.25 mg xylazine] per 100 g of rat body weight administered via the intraperitoneal (IP) route on a table in a 25° C. room. The rat was placed in a dorsal recumbency on a clean towel above the heating pad and a heating lamp positioned above the rat to prevent hypothermia during anesthesia. Pedal reflex test was then administered to test for complete state of anesthesia. The rat tail was immersed in warm water for 5-10 seconds to dilate the lateral tail vein. The site of needle insertion is prepared with a swab of 70% alcohol. Less than 0.5 ml of blood was drawn per sampling, which was less than 10% of total circulating blood volume recommended. A 25 gauge needle and 1 ml syringe was used for blood sampling. 
       Blood Sampling for RBC Serology 
       [0130]    The rat was restrained by placing it in a restraint tube with its tail exposed. A small nick or needle prick was made on the distal end of the rat&#39;s tail and one drop of blood was collected using a heparinised capillary tube. Pressure was applied to the insertion site to stop bleeding. This blood was then washed and stored at 4° C. for up to 1 month for serology testing. 
       Infusions of Synthetic Antigen-Lipid Construct Solution 
       [0131]    Two standard methods were used. The rat was anesthetized using the method described for blood sampling. 0.5 ml of synthetic antigen-lipid construct per 100 g of rat body weight was slowly infused over a period of 2-5 minutes into either the:
       The lateral tail vein using the same technique as described in the blood sampling procedure; or   The submandibular vein.       
 
         [0134]    The submandibular vein method was used if blood was unable to be obtained from the lateral tail vein. A small 1 to 1.5 cm skin incision was made over the ventral neck area, slightly right of the center. The right submandibular vein was clearly visualised as it passed under the jaw bone. A small portion of the vein, approximately 5 mm section was carefully freed from all underlying tissue by using forceps. A small 25 gauge needle was carefully inserted into the isolated portion of the vein and 0.5 ml of synthetic antigen-lipid construct solution was infused slowly over 3-5 minutes. After the infusion, the incision site was closed with surgical suture or wound clips. The animals were closely monitored post-infusion for the first hour, then hourly afterwards. 
       Euthanasia 
       [0135]    The animal was placed in a carbon dioxide partially filled chamber and sealed in for 10 minutes to ensure asphyxiation. Neck dislocation was then performed to complete the euthanasia process. All general animal manipulation and housing were within purpose built animal facilities providing a controlled environment for ventilation, temperature and light. The temperature is maintained by a thermostatically controlled air-conditioning and heating unit to obtain a desired ambient temperature of 21 to 23° C. A time-controlled light provides 12 hour light/dark cycles. 
       Mice Studies 
       [0136]    A murine model of intravascular hemolytic transfusion reaction has been developed and used to demonstrate the principle of neutralization of circulating antibody by infusion of synthetic antigen-lipid constructs. 
       Preparation of Modified Murine Red Blood Cells (Murine A Kodecytes) 
       [0137]    An amount of A tri -sp 1 -sp 2 -DOPE (I) was diluted in sterile saline to provide a solution of A tri -sp 1 -sp 2 -DOPE (I) at a concentration of 1 mg/mL. Mice red blood cells (RBCs) were washed 3 times in phosphate buffered saline (PBS) by centrifugation at 2700 rpm for 3 minutes. Equal volumes of the solution of A tri -sp 1 -sp 2 -DOPE (I) and washed, packed mRBCs were mixed and incubated in glass tubes at 37° C. for 120 minutes with gentle agitation. Control cells were prepared by mixing equal volumes of packed human A RBCs and PBS, or packed naïve mice RBCs and PBS. Following incubation cells for infusion were washed three times in PBS and resuspended in SAG-M at a concentration (v/v) of 40:60 or 60:40. The transformation of mRBCs by A tri -sp 1 -sp 2 -DOPE (I) (murine A kodecytes) was confirmed by agglutination using monoclonal anti-A reagent. Briefly, 30 μL of a 5% suspension of RBCs in PBS was incubated with 30 μL of monoclonal anti-A reagent at room temperature for 5 minutes. The incubated cells were then centrifuged and read for agglutination. The same procedure was repeated for the control cells (human A RBCs and naïve mice RBCs). 
         [0000]    Infusion with Murine A Kodecytes 
         [0138]    Three control mice (ID#001, ID#002, ID#003) were infused with 40 μL of murine kodecytes with an agglutination score of 4+. The serum reactivity against murine A kodecytes was assessed post infusion. No serum reactivity was detected. The reactivity of serum with A glycolipids of RBCs was also negative (Table 5). 
       Elicitation of A Antibody Expression 
       [0139]    C57/B6 black mice were administered concentrated saliva comprising A antigen (human A Le a-b+  saliva) in combination with TITREMAX™ gold adjuvant to elicit production of anti-A antibodies. A volume of 100 μl of an emulsion of 1 part saliva to 1 part TITREMAX™ was injected subcutaneously behind the head or in the flank of an anesthetized mouse. An electronic identification tag was used to identify each mouse. The administration was repeated three times at three week intervals. The sera of mice (ID#4117, ID#4977) were assessed for reactivity against murine A kodecytes. The elicitation of reactivity was demonstrated by agglutination of the murine A kodecytes. The reactivity of the sera with A glycolipids of RBCs was also confirmed (Table 5). Three mice (ID#7698, ID#1144, ID#9499) were transfused with a 40 μl volume of murine A kodecytes in sterile saline via the subclavian vein. The sera of the mice were assessed for reactivity against murine A kodecytes and with A glycolipids of RBCs as above. Reactivity for each serum sample was confirmed in at least one assay. Levels of free hemoglobin in the serum or plasma for each mouse were assessed at 30 minutes following infusion with the murine A kodecytes. Free hemoglobin was observed (Table 5). Three mice (ID#4605, ID#5234, ID#8553) were transfused with a larger 60 μl volume of murine A kodecytes in sterile saline via the subclavian vein. Increased levels of free hemoglobin in the serum or plasma of each mouse were observed at 30 minutes following infusion. In addition hemoglobin was observed in the urine of each mouse (Table 5). Three mice (ID#4422, ID#3032, ID#6440) were infused with a 200 μl volume of a 20 mg/mL dispersion of A tri -sp 1 -sp 2 -DOPE (I) in sterile saline via the subclavian vein (4 mg A tri -sp 1 -sp 2 -DOPE (I) per mouse). No free hemoglobin was detectable in the serum or plasma of each mouse 30 minutes following infusion. No free hemoglobin was detectable in the urine. No reactivity of the sera of each mouse with A glycolipids of RBCs was observed. Notwithstanding these observations the in vivo transformation of murine RBCs (A antigen positive as determined by monoclonal anti-A antibody) was demonstrated 1 to 3 hours following infusion. The presence of in vivo transformed A kodecytes in the absence of serum reactivity appears to demonstrate the ability of infused A tri -sp 1 -sp 2 -DOPE (I) to neutralize circulating anti-A antibody for at least a transient period. It is suggested that the administration of synthetic antigen-lipid constructs may therefore be employed to mitigate the risk of clinically significant transfusion reactions attributable to circulating antibody. At about 3 days post infusion the red blood cells had become A antigen negative and the mice survived the procedure with no observations of adverse effects. 
       Preparation of Modified Murine Red Blood Cells 
       [0140]    Blood was collected from mice, centrifuged and the serum removed. The red blood cells (RBCs) were washed three times in PBS and pooled. Equal volumes of washed, packed RBCs and dispersions of F—S-L construct(s) in phosphate buffered saline (PBS) were mixed and incubated for two hours at 37° C. with gentle, intermittent mixing. 
         [0141]    The modified RBCs were then washed three times in PBS and resuspended in a human red cell transfusion medium (sucrose, adenine, glucose, mannitol solution). Modification of the murine RBCs was confirmed by agglutination using:
       1. monoclonal anti-A reagent for red blood cells modified with the F—S-L construct where F was the blood group A trisaccharide; and   2. monoclonal anti-B reagent for RBCs modified with the F—S-L construct where F was the blood group B trisaccharide.       
 
         [0144]    Where the RBCs had been modified with F—S-L construct where F was biotin, modification was confirmed by fluorescence microscopy using Avidin-Alexafluor™ 488. Briefly, a volume of 2 μL Avidin-Alexafluor™ 488 that had a concentration of 0.1 mg/mL in PBS was added to a 2 μL volume of packed modified RBCs. The suspension was incubated for 20 minutes and then washed three times in the human red cell transfusion medium. In order to prevent clumping the final pellet of packed modified RBCs was incubated for 2 minutes at 37° C. in the presence of a 2 μL volume of biotin at a concentration of 1 mg/mL in PBS. The mixture was then centrifuged to remove excess biotin and the pellet of modified RBCs resuspended in the human red cell transfusion medium at a ratio of about 1:10 (v/v). The fluorescent signal (1.9 ms) was observed using a 40× objective (Olympus BX51 Fluorescence microscope). 
       Administration of Compatible and Incompatible Modified Red Blood Cells to Mice 
       [0145]    Mice immunized with blood group A substance were weighed and electronically tag checked and anaesthetized. A series of animals were immunized in parallel with the test and control series animals and their antibody status confirmed by thin layer chromatography. All immunized animals (n=4) were confirmed as anti-A positive against human blood group A RBCs and against F—S-L construct where F was the blood group A trisaccharide and blood group A type 2 glycolipid by solid phase analysis. The modified RBCs (F—S-L constructs where F was either the blood group A or B trisaccharide) administered to test animals provided a maximum 4+ reaction score with the corresponding anti-A or anti-B reagent. Either four or five immunized animals were used in each of the test and control series. A description of the test and control series is provided in Table 6 and illustrated schematically in  FIG. 2 . 
         [0146]    A volume of 200 μL of a suspension of modified RBCs in the human red cell transfusion medium (20:180(v/v)) was injected into the subclavian vein of the test animal. The time of administration was noted and after three minutes a haematocrit tube blood collection was taken by a needle prick in the subclavian vein. A volume of about 50 μL of blood was collected from the test animal into anticoagulant in an eppendorf tube. For subsequent sampling of blood at time intervals of 2, 8, 24, 48, 72, 96 and 120 hours after administration the tails of the test animals where anaesthetised with EMLA cream for about 5 minutes. The tails were then warmed under a heat lamp for up to two minutes and blood obtained by tail vein incision. Collection of blood from each test animal was ceased when no fluorescence attributable to conjugation of Avidin-Alexofluor™ 488 to modified RBCs was detectable in 10 high power fields (circa 10 4  RBCs). The post administration blood collection time (hours) at which fluorescence attributable to modified RBCs was no longer detectable is provided in Table 7. 
       Interpretation 
       [0147]    The time of collection at which fluorescence attributable to modified RBCs was no longer detectable is taken to correspond to the survival time of the modified RBCs in the circulatory system of the test animal. Incompatible modified RBCs when administered to test animals without a prior infusion of the modifying F—S-L construct were indicated to have a survival time in the circulatory system of the test animal of no longer than 48 hours. Where an infusion of the modifying F—S-L construct was administered to the same test animals prior to administration of the corresponding modified RBCs the survival time of the administered RBCs was comparable with the survival time of the compatible modified RBCs. Compatible RBCs modified with F—S-L construct(s) where F was either the blood group B trisaccharide or biotin had a survival time in the circulatory system of the test animals of greater than 120 hours. The survival of these compatible modified RBCs in the circulatory system beyond 120 hours was not determined. 
         [0148]    Although the invention has been described by way of exemplary embodiments it should be appreciated that variations and modifications may be made without departing from the scope of the invention. Furthermore where known equivalents exist to specific features, such equivalents are incorporated as if specifically referred to in this specification. 
         [0000]    
       
         
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                   
                 TABLE 5 
               
               
                   
                   
               
               
                   
                 001 
                 002 
                 003 
                 4117 
                 4977 
                 7698 
                 1144 
                 9499 
                 4605 
                 5234 
                 8553 
                 4422 
                 3032 
                 6440 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                 Elicitation of A 
                 x 
                 x 
                 x 
                 ✓ 
                 ✓ 
                 ✓ 
                 ✓ 
                 ✓ 
                 ✓ 
                 ✓ 
                 ✓ 
                 ✓ 
                 ✓ 
                 ✓ 
               
               
                 antibody 
               
               
                 expression 
               
               
                 Volume of A tri - 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 200  
                 200  
                 200  
               
               
                 sp 1 -sp 2 -DOPE (I) 
               
               
                 infused (μL) 
               
               
                 Volume of murine 
                 40  
                 40  
                 40  
                 0 
                 0 
                 40  
                 40  
                 40  
                 60  
                 60  
                 60  
                 0 
                 0 
                 0 
               
               
                 A kodecytes 
               
               
                 infused (μL) 
               
               
                 Reactivity of in 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 ++++ 
                 ++++ 
                 ++++ 
               
               
                 vivo transformed 
               
               
                 A kodecytes 
               
               
                 In vitro serum 
                 0 
                 0 
                 0 
                 +++ 
                 ++ 
                 + 
                 0 
                 ++ 
               
               
                 reactivity 
               
               
                 against murine A 
               
               
                 kodecytes 
               
               
                 Free hemoglobin 
                   
                   
                   
                   
                   
                 ++ 
                 ++ 
                 + 
                 +++ 
                 +++ 
                 +++ 
                 0 
                 0 
                 0 
               
               
                 (plasma/sera) 
               
               
                 Free hemoglobin 
                   
                   
                   
                   
                   
                   
                   
                   
                 ++++ 
                 ++++ 
                 Death 
                 0 
                 0 
                 0 
               
               
                 (urine) 
               
               
                 Reactivity of 
                 −ve 
                 −ve 
                 −ve 
                 +ve 
                 +ve 
                 +ve 
                 +ve 
                 +ve 
                   
                   
                   
                   
                 −ve 
                 −ve 
               
               
                 sera with RBC A 
               
               
                 glycolipids 
               
               
                 Direct 
                   
                   
                   
                   
                   
                   
                   
                   
                 0 
                 0 
                 0 
                 0 
               
               
                 antiglobulin 
               
               
                 test (DAT) with 
               
               
                 anti-murine IgG 
               
               
                   
               
               
                 The absence of an entry means not determined. 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
             
           
               
                 TABLE 6 
               
               
                   
               
               
                 Control and trial series. 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Series 1a 
                 Test series. 20 uL of murine kodecytes (modified with FSL-A and FSL-biotin) were infused into 4 
               
               
                   
                 mice each with anti-A. Blood was sampled at 2, 8, 24 and 48 hours and following staining with 
               
               
                   
                 avidin-Alexafluor were observed for the presence of kodecytes in 10 high power fields 
               
               
                   
                 (magnification 400×) under fluorescent microscopy 
               
               
                 Series 1b 
                 Test series. Two weeks later Series 1a mice were reused. 200 μL of FSL-A (20 mg/mL) was first 
               
               
                   
                 infused as a solution followed by 20 μL of murine kodecytes (modified with FSL-A and FSL- 
               
               
                   
                 biotin). Blood was sampled at 2, 8, 24, 48, 72, 96, and 120 hours and following staining with 
               
               
                   
                 avidin-Alexafluor 488 were observed for the presence of kodecytes in 10 high power fields 
               
               
                   
                 (magnification 400×) under fluorescent microscopy 
               
               
                 Series 2 
                 Control series. 20 uL of murine kodecytes (modified with FSL-B and FSL-biotin) were infused into 
               
               
                   
                 5 mice, each with anti-A. Blood was sampled at 2, 8, 24, 48, 72, 96, and 120 hours and following 
               
               
                   
                 staining with avidin-Alexafluor 488 were observed for the presence of kodecytes in 10 high power 
               
               
                   
                 fields (magnification 400×) under fluorescent microscopy 
               
               
                 Series 3 
                 Control series. 20 uL of murine kodecytes (modified with FSL-GB3 and FSL-biotin) were infused 
               
               
                   
                 into 5 mice each with anti-A. Blood was sampled at 2, 8, 24, 48, 72, 96, and 120 hours and 
               
               
                   
                 following staining with avidin-Alexafluor 488 were observed for the presence of kodecytes in 10 
               
               
                   
                 high power fields (magnification 400×) under fluorescent microscopy 
               
               
                 Series 4 
                 Control series. 20 uL of murine kodecytes (modified only with FSL-biotin) were infused into 5 
               
               
                   
                 mice each with anti-A. Blood was sampled at 2, 8, 24, 48, 72, 96, and 120 hours and following 
               
               
                   
                 staining with avidin-Alexafluor 488 were observed for the presence of kodecytes in 10 high power 
               
               
                   
                 fields (magnification 400×) under fluorescent microscopy 
               
               
                 Series 5 
                 Control series. 20 uL of murine kodecytes (modified only with FSL-biotin) were infused into 1 
               
               
                   
                 mouse without anti-A. Blood was sampled at 2, 8, 24, 48, 72, 96, and 120 hours and following 
               
               
                   
                 staining with avidin-Alexafluor 488 were observed for the presence of kodecytes in 10 high power 
               
               
                   
                 fields (magnification 400×) under fluorescent microscopy 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 7 
               
               
                   
               
               
                   
                   
                 Immunised 
                 20 μL A + 
                 20 μL B + 
                 20 μL Gb3 + 
                 20 μL 
                 Infusion with F-S-L 
                   
               
               
                   
                   
                 with blood 
                 biotin 
                 biotin 
                 biotin 
                 Biotin (only) 
                 (where F is blood 
               
               
                   
                 Number of 
                 group A 
                 modified 
                 modified 
                 modified 
                 modified 
                 group A trisaccharide 
                 Modified RBCs 
               
               
                 SERIES 
                 animals 
                 substance 
                 RBCs 
                 RBCs 
                 RBCs 
                 RBCs 
                 (glycotope)) 
                 survival (hours) 
               
               
                   
               
             
             
               
                  1a 
                 4 
                 + 
                 + 
                 − 
                 − 
                 − 
                 − 
                 8, 24, 48, 48 
               
               
                  1b 
                 4 
                 + 
                 + 
                 − 
                 − 
                 − 
                 + 
                 &gt;120, &gt;120, &gt;96, &gt;120 
               
               
                 2 
                 5 
                 + 
                 − 
                 + 
                 − 
                 − 
                 − 
                 &gt;120, &gt;120, &gt;120, &gt;120, &gt;120 
               
               
                 3 
                 5 
                 + 
                 − 
                 − 
                 + 
                 − 
                 − 
                 &gt;120, &gt;120, &gt;120, &gt;120, &gt;120 
               
               
                 4 
                 5 
                 + 
                 − 
                 − 
                   
                 − 
                   
                 &gt;120, &gt;120, &gt;120, &gt;120, &gt;120 
               
               
                 5 
                 1 
                 − 
                 − 
                 − 
                 − 
                 + 
                 − 
                 &gt;120 
               
               
                   
               
             
          
         
       
     
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       [0000]    
       
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