AU2013201431B2 - Functional lipid constructs - Google Patents

Abstract

The invention relates to methods for effecting qualitative and quantitative changes in the functional moieties expressed at the surface of cells and multi-cellular structures, and functional lipid constructs for use in such methods. In particular, the invention relates to functional lipid constructs and their use in diagnostic and therapeutic applications, including serodiagnosis, where the functional moiety is a carbohydrate, peptide, chemically reactive group, conjugator or fluorophore.

Description

K01.075AUB FUNCTIONAL LIPID CONSTRUCTS TECHNICAL FIELD 5 The invention relates to methods for effecting qualitative and quantitative changes in the functional moieties expressed at the surface of cells and multi-cellular structures, and functional lipid constructs for use in such methods. 10 In particular, the invention relates to functional lipid constructs and their use in diagnostic and therapeutic applications, including serodiagnosis, where the functional moiety is a carbohydrate, peptide, chemically reactive group, conjugator or fluorophore. 15 BACKGROUND ART The ability to effect qualitative and quantitative changes in the functional moieties expressed at the surface of liposomes, 20 cells and multi-cellular structures provides for a range of diagnostic and therapeutic applications. A functional moiety may be a carbohydrate, peptide, chemically reactive group (e.g. maleimide), conjugator (e.g. biotin) or fluorophore (e.g. fluorescein). 25 The specification accompanying international application number PCT/NZ2005/000052 (publication number WO 2005/090368) describes the preparation of carbohydrate-lipid constructs for use in methods of effecting qualitative and quantitative 30 changes in the level of carbohydrates expressed at the surface of cells and multi-cellular structures. The use of the constructs to prepare quality control cells for use in blood grouping and diagnostics is described. 1 K01.075AUB The specification accompanying international application number PCT/NZ2006/000245 (publication number WO 2007/035116) describes another method for the preparation of carbohydrate 5 lipid constructs where the carbohydrate is the polymer hyaluronic acid. The use of the constructs to modify embryos to promote association with endometrial cells is described. The specification accompanying international application 10 number PCT/NZ2007/000256 (publication number WO 2008/030115) describes the preparation of fluorophore-lipid constructs. The use of the constructs in methods of fluorescently labeling cells is described. 15 Known methods of effecting changes in the peptides expressed at the surface of cells include gene manipulation, chemical modification of membrane peptides, and "cell surface painting" using lipid anchors such as GPI (Legler et al (2004), McHugh et al (1995), Medof et al (1996), Metzner et al (2008), 20 Morandat et al (2002), Premkumar et al (2001), Ronzon et al (2004), Skountzou et al (2007)). In addition to these methods of effecting changes of endogenously expressed peptides, exogenously prepared peptides 25 may be coupled to lipids of the membrane utilising biotin avidin conjugation. Biotin binds to the tetrameric protein avidin with a dissociation constant (KD) of the order 1015 mol/L. This strong binding is exploited in a number of laboratory applications. 30 In these laboratory applications biotin is linked to a molecule such as a carbohydrate or a peptide. The preferential binding of avidins to biotin is exploited in a 2 K01.075AUB number of isolation or separation applications in addition to the coupling of peptides to the lipids of membranes. The specification accompanying international application no. 5 PCT/NZ02/00214 (publication no. WO 03/039074) describes a "two-step method" of localizing an antigen such as a peptide to the surface of cells. In the method the biotinylated glycoside (BioG) is contacted with a suspension of cells for a time and at a temperature sufficient to allow the BioG 10 molecules to incorporate via their diacyl lipid tails into the cell membrane of the cells. An exogenously prepared avidinylated peptide may then be localized to the surface of the BioG modified cells by 15 contacting the peptide with the modified cells. Alternatively, an exogenously prepared biotinylated peptide may be localized to the surface of the modified cells via a biotin-avidin bridge. 20 In either alternative of the "two-step method" the amount of peptide localized to the surface of the cells may be controlled by controlling the concentration, time and temperature at which the BioG molecules are contacted with the suspension of cells to provide the modified cells. However, 25 the utility of the method is limited by the availability and dispersibility of BioG in biocompatible media such a saline. The specification accompanying international application no. PCT/NZ2005/000052 (publication no. WO 2005/090368) describes a 30 "one-step method" of localizing carbohydrate antigen to the surface of cells. The "one-step method" utilizes carbohydrate-lipid constructs that are dispersible in biocompatible media and can therefore be used to prepare 3 KO1.075AUB modified cells without loss of vitality. However, a method of preparing peptide-lipid constructs with comparable dispersibility in biocompatible media and of general applicability to peptides has not been described. 5 Relatively little work has been performed on the coupling of peptides to phospholipids as individual components prior to their incorporation in self-assembling lipid structures, such as liposomes. However, a variety of standard techniques have 10 been described for the covalent coupling of peptides to liposome surfaces. Martin et al (1990) has reviewed methods of attaching moieties including peptides, to the surface of liposomes. 15 Blume et al (1993) describes the coupling of the water soluble Glu-plasminogen to liposomes by the method described by Kung and Redemann (1986). The chemical ECDI (1-ethyl-(3 dimethylaminopropyl) carbodiimide hydrochloride) is used to 20 activate the liposomes prior to incubation of the activated liposome suspension with Glu-plasminogen. Proteo-PEG-coated liposomes with Glu-plasminogen covalently attached to the ends of the distearylyphosphatidylethanolamine (DSPE)-PEG-COOH are provided. 25 Haselgrnbler et al (1995) describes a heterobifunctional crosslinker used to facilitate the preparation of immunoliposomes. The crosslinker is synthesised from a diamine derivative of poly (ethylene glycol) (PEG, average 30 molecular weight 800 dalton (18mer)). The crosslinker has 2 (pyridylthio)propronyl (PDP) and N-hydroxysuccinimide ester (NHS) as functional groups. 4 K01.075AUB Ishida et al (2001) describes the preparation of liposomes bearing polyethylene glycol-coupled trasnferrin. Transferrin was conjugated via the terminal carboxyl residue of DSPE-PEG COOH. The liposomes were proposed as having utility in in 5 vivo cytoplasmic targeting of chemotherapeutic agents or plasmid DNAs to target cells. Massaguer et al (2001) describes the incorporation of a peptide sequence (GGRGRS) and hydrophobic derivatives to the 10 surface of chemically activated liposomes. The incorporation was carried out through the carboxyl group of N-glutaryl dipalmitoyl phosphatidyl choline (NGPE). Massaguer et al (2001) noted that considering potential in 15 vivo applications, where sterility and simplicity would be some of the most important requirements, processes based on chemical reactions on the surface of liposomes involving extra steps would be more difficult to be scaled up at the industrial level. A hydrophobic derivative of the peptide 20 sequence was identified as providing optimal properties for incorporation to the surface of liposomes. Chung et al (2004) describe the antigenic determinant shielding effect of DOPE-PEG incorporated into the membranes 25 of cells and speculated concerning the potential of lipid PEG(n) (s) to regulate biological cell responses and the extension of this concept to the introduction of functional molecules at the end of the PEG chain. 30 Kato et al (2004) describe a method for anchoring of macromolecular proteins into the membranes of living mammalian cells. A dioleylphosphatidylethanolamine (DOPE) derivative coupled with hydrophilic poly (ethylene glycol) (PEG80) was 5 KO1.075AUB used as the synthetic membrane anchor. Peptides were conjugated at the distal terminal of the PEG moiety via an amino-reactive N-hydroxysuccinimide derivative of the synthetic membrane anchor. 5 The PEG80 moiety facilitated solublisation of the synthetic membrane anchor in water. As noted by Kato et al (2004) if the anchor is insoluble in water, undesirable and complicated processes such as liposome preparation and the fusion of 10 liposomes with the cell membrane may be required to anchor the conjugates into the cell membrane. An additional advantage noted by Kato et al (2004) was that synthetic membrane anchors with high hydrophile-lipophile 15 balance values (attributable to PEG spacer with a high number of oxyethylene units) were concluded to have no cytolytic activity. However, difficulties arise in the use of synthetic membrane anchors including a PEG spacer with a high number of oxyethylene units. 20 Firstly, the expression of the conjugative peptide or other endogenous cell surface peptides may be masked by the PEG spacer. Secondly, a PEG spacer with a high number of oxyethylene units may elicit non-specific adherence of protein 25 (including antibodies in certain individuals) and/or the non specific activation of the complement cascade. Winger et al (1996) describes the conjugation of bromoacetylated DSPE with a thiol terminated decapeptide 30 comprising at its C-terminus the minimal human thrombin receptor peptide agonist (HS---SerPheLeuLeuArgAsn). 6 K01.075AUB Hashimoto et al (1986) describes the conjugation of iodoacetylated DSPE with thiolated compounds. A need exists for a general method of preparing peptide-lipid 5 constructs that may be incorporated as individual components in self-assembling lipid structures, such as liposomes, by a "one-step method". The method should desirably provide peptide-lipid constructs that are readily dispersible in biocompatible media and spontaneously incorporate in to the 10 membranes of cells and multi-cellular structures. Peptide-lipid constructs with these characteristics are anticipated to have utility in a range of therapeutic and diagnostic applications, especially serodiagnosis, in addition 15 to the preparation of functionalized liposomes. It is an object of this invention to provide functional-lipid constructs that are dispersible in biocompatible media and spontaneously incorporate into the membranes of cells and 20 multi-cellular structures. It is an object of this invention to provide functional-lipid constructs for use in the preparation of peptide-lipid constructs that are dispersible in biocompatible media and 25 spontaneously incorporate into the membranes of cells and multi-cellular structures. It is an object of this invention to provide peptide-lipid constructs that are dispersible in biocompatible media and 30 spontaneously incorporate into the membranes of cells and multi-cellular structures. These objects are to be read disjunctively with the object to 7 K01.075AUB at least provide the public with a useful choice. DISCLOSURE OF INVENTION 5 In a first aspect the invention provides a functional lipid construct of the structure F-S-L where F is a functional moiety, L is a diacyl or a dialkyl lipid, and S is a spacer covalently linking F to L including the substructure: OM H H H H-(CH2) -N N * 0 1- o 0 10 OM where g is the integer 1, 2 or 3, M is a monovalent cation or substituent, and * is other than H. 15 Preferably, the substructure is: OM H 0 * N N-(CH 2 ) H N I * HiI H g N H~1~ o I o Io OM h h where h is the integer 1, 2, 3 or 4. 20 Preferably, g is the integer 2 and h is the integer 1, 2 or 4. Preferably, M is H or CH 3 . 8 KO1.075AUB Preferably, L is a diacylglycerophospholipid. More preferably, L is phosphatidylethanolamine. Preferably, the structure of the functional lipid construct 5 includes the partial structure: OM OHN O N O NHNN 0 N-O NH h 0 NH 0 OM

>(CH

2 )v NHH 00 \, H O

R

1

R

2 where v is the integer 3, 4 or 5, M' is a monovalent cation, 10 and R 1 and R 2 are independently selected from the group consisting of: alkyl or alkenyl substituents of the fatty acids 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 15 octadecenoic acid, trans-11-octadecenoic acid, cis-11 octadecenoic acid, cis-11-eicosenoic acid or cis-13-docsenoic acid. Preferably, F is a functional moiety selected from the group 20 consisting of: carbohydrate, peptide, chemically reactive group, conjugator or fluorophore. 9 K01.075AUB In a first alternative of the first aspect the invention provides a functional lipid construct of the structure: / R3 O( (C2 2 ) (NNH am C HN to o -NHh O N4 0 NHNH C NH 'O OM N 0 OM' 0 0 R" R 2 5 where F is a carbohydrate, x is the integer 2, 3 or 4, y is the integer 1, 2 or 3, and R 3 is 0 of a substituted hydroxyl of the carbohydrate. 10 [followed by page 111 10 K01.075AUB In a second alternative of the first aspect the invention provides a functional lipid construct of the structure: NZ

(CH

2 ) w OM 0 HN 0h NH h 0 N 0 0 NH N 0 N N O O N -H 2 0N 00O \,O~ O OM

R

1 R 2 5 where F is a peptide, w is the integer 1 or 2, and R 3 is S of a substituted sulfhydryl of a Cys residue of the peptide. [followed by page 12] 10 11 K01.075AUB In a third alternative of the first aspect the invention provides a functional lipid construct of the structure: (C 0 00 -- NI NITNE NH h a0 O O

>-(CH

2 ). '0 O\ O O=< C=< R1 R, 5 where F is the chemically reactive group maleimide and w is the integer 1 or 2. [followed by page 13] 10 12 K01.075AUB In a fourth alternative of the first aspect the invention provides a functional lipid construct of the structure: NH (H) OM O H 00 NH h 0 N H N 0N O aMo (CH ), \,C)

R

1 R2 5 where F is the conjugator biotin and k is the integer 2, 3 or 4. [followed by page 14] 10 13 K01.075AUB In a fifth alternative of the first aspect the invention provides a functional lipid construct of the structure:

NH-R

3 0 / (CH2)z NNH (CH2)x OM 0 HN O N Oh 00 NH h 0 N O NHN 00 N O NH h OMO O NH 'O OM o(CH2) NH

R

1 R 2 5 where F is the fluorophore of fluorescein (or one of its derivatives), x is the integer 2, 3 or 4, z is the integer 3, 4 or 5, and R 3 is C of the thiocyanate substituent of the isothiocyanate derivative of fluorescein (or one of its 10 derivatives). Preferably, the substructure is:

OCH

3 * "N N N ' NL " 0 0 o 0

OCH

3 15 designated MCMG(1); 14 K01.075AUB OCH, OCH, O 0 0 o o o o

OCH

3 OCH 3 designated MCMG(2); OH 0 01 0 *LN N H H H H O 0 0 0 5 OH designated CMG(1); or OH OH H OH OH 10 designated CMG(2). In a second aspect the invention provides a water soluble peptide-lipid construct of the structure F-S-L where S is a 15 spacer linked to F via a sulphide bond and includes the substructure: OM H1 0 * ' N- (C H 2 ) -N OM 20 where g is the integer 1, 2 or 3, M is a monovalent cation or substituent, and * is other than H. 15 KO1.075AUB Preferably, the substructure is: OM 0 01 * . N -(CH 2 ) H N I * HiI H g N H o I o Io 0 K. - 0 OM h h 5 where h is the integer 1, 2, 3 or 4. Preferably, g and h are the integer 2. 10 Preferably, M is H or CH 3 . Preferably, L is a diacylglycerophospholipid. More preferably, L is phosphatidylethanolamine. 15 [followed by page 17] 16 KO1.075AUB Preferably, the structure of the peptide-lipid construct includes the partial structure: OM O HN* NH h 0 N 0 N H _/0-h NHN N O \\ 0 0 _NH) h NHO \>(CH2)v 0 /NH 0 OM' O O O= O= R,

R

2 5 where v is the integer 3, 4 or 5, M' is a monovalent cation, and R 1 and R 2 are independently selected from the group consisting of: alkyl or alkenyl substituents of the fatty acids trans-3-hexadecenoic acid, cis-5-hexadecenoic acid, cis 7-hexadecenoic acid, cis-9-hexadecenoic acid, cis-6 10 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. 15 [followed by page 18] 17 KO1.075AUB Preferably, the structure of the peptide-lipid construct includes the partial structure: N (CH2) O OM 0 HN N H h" O N Nh 00 N O O -(CH2), /NHI \,O O0 o0 O~ O RI R, 5 where w is the integer 1 or 2, and R 3 is S of a substituted sulfhydryl of a Cys residue of the peptide. [followed by page 19] 10 18 K01.075AUB In an embodiment of the second aspect the invention provides a peptide-lipid construct of the structure: (Xaa) xCs (Xaa) y 0 S N

(CH

2 ) w O O HN 0 m N 00 t o O NH h 0 N O 0 NH N N N O0h o /NH O O O NH 0 OM >(CH2)v -NH \, O O o0o o0o R,

R

2 5 where the sum of x and y is greater than 5. Optionally, F is a peptide including a proximal terminal sequence (PTS) selected to promote solubility of the peptide. 10 In a preferment of this option, the PTS of the peptide is selected from the group consisting of: SerLysLysLysLysGly AlaAlaAlaAla GlySerGlySerGly Preferably, the Cys residue is a terminal Cys residue of the 15 peptide (Cys). 19 KO1.075AUB Preferably, the terminal sequence of the peptide is selected from the group consisting of: GlyLysLysLysLysSerCys AlaAlaAlaAlaCys GlySerGlySerGlyCys CysSerLysLysLysLysGly CysAlaAlaAlaAla CysGlySerGlySerGly 5 Preferably, the Cys residue is a terminal Cys residue of the peptide at the carboxy-terminus of the peptide. Preferably, F is a peptide comprising an epitope of antigens selected from the group consisting of: Glycophorin A, 10 Glycophorin B, or mutations thereof (including the MNS blood group system). More preferably, F is a peptide selected from the List of Peptides. Most preferably, F is a peptide selected from the group consisting of: GlnThrAsnAspLysHisLysArgAspThrTyrAlaAlaAlaAlaAlaCys GlnThrAsnAspLysHisLysArgAspThrTyrGlySerGlySerGlyCys GlnThrAsnAspMetHisLysArgAspThrTyrGlySerGlySerGlyCys SerSerGlnThrAsnAspLysHisLysArgAspThrTyrCys ThrTyrProAlaHisThrAlaAsnGluValCys ThrTyrProAlaHisThrAlaAsnGluCys ProAlaHisThrAlaAsnGluValCys SerGlnThrAsnAspLysHisLysArgAspCys CysThrTyrProAlaHisThrAlaAsnGlu 15 20 K01.075AUB Preferably, L is a glycerophospholipid selected from the group consisting of: 1,2-0-dioleoyl-sn-glycero-3 phosphatidylethanolamine (DOPE) and 1,2-0-distearyl-sn glycero-3-phosphatidylethanolamine (DSPE). 5 In an exemplifying first embodiment of the second aspect the invention provides a peptide-lipid construct of the structure: GlnThrAsnAspLysHisLysArgAspThrTyrGlySerGlySerGlyCy (CH2) 0 OH 0 NH NH \~I 0 OP N NH O NH N O NH \ OH O /- o OH 10R~R0 NH O N 0 o-< NH NH 0 0 -NH 0 H F-NH C, R, R2 10 designated DOPE-Ad-CMG (2) -BAla-Mal-PTS-Milt (K) (IX). 21 K01.075AUB In an exemplifying second embodiment of the second aspect the invention provides a peptide-lipid construct of the structure: GlnThrAsnAspLysHisLysArgAspThrTyrSerSerGlnThrAsnAspMetHisLysArgAspThrTyrCys o S (CH2)20 OH 0 NH Q C NH NH 0 NH 0P N 0O OH 0 /----/Ho NH NO 0 N NH 0 F-<\

NH

N NH [folowdb pag 23 O NH 0 OH (CH2J r-NH 101 O O, R, R2 5 designated DOPE-Ad-CMG (2) -BAla-Mal-Milt (K, M) (X) . [followed by page 23] 10 22 K01.075AUB In an exemplifying third embodiment of the second aspect the invention provides a peptide-lipid construct of the structure: AspThrTyrProAlaHisThrAlaAsnGluValSerGluCyI o s O(CH OH O NH 2 C NH NH 0 NH NH OO N O NH O NH NH R 1 R 10 foloweb pag 24 O -NH 0 OH (CH2) 4 0,FNH O OM R1 R2 5 where R1 and R2 are both (CH2)7CHCH(CH2)7 and designated DOPE Ad-CMG (2) -BAla-Mal-Mur (D14C) (XI) . 10 [followed by page 24] 23 K01.075AUB In an exemplifying fourth embodiment of the second aspect the invention provides a peptide-lipid construct of the structure: ValMetTyrAlaSerSerGlyCyI o S (CH2) 2 OH 0 NH 0 PN 0 NH O 0- -/O NH 0 NH OH O /- OH OH NH O O d NH O NH NH o 0 O NH 0 OH O(CH24 -NH O,

R

1 R 2 5 where R 1 and R 2 are both (CH 2

)

7

CHCH(CH

2

)

7 and designated DOPE Ad-CMG(2)-BAla-Mal-Syph(V8C) (XII). 10 In a third aspect the invention provides a method of detecting reactive antibody in the serum of a subject including the steps of: Contacting a sample of the serum with a suspension of 15 cells modified to incorporate a functional lipid construct (F-S-L) of the first or second alternatives of the first aspect of the invention or a peptide-lipid 24 K01.075AUB construct of the second aspect of the invention to provide a mixture; e Incubating the mixture for a time and at a temperature 5 sufficient to allow agglutination; and e Determining the degree of agglutination of the cells in the mixture; 10 where: F is a carbohydrate or peptide comprising an epitope for the reactive antibody. 15 Optionally, the method includes the intermediate step of: e Adding an anti-subject globulin antibody to the mixture prior to determining the degree of agglutination of the cells of the mixture. 20 Preferably, the anti-subject globulin antibody is anti-human globulin (AHG) antibody. Optionally, where F is a peptide, the method includes the 25 preliminary step of: * Adding an amount of the peptide to the sample of the serum; 30 where the amount of the peptide is sufficient to neutralize non-specific agglutination or confirm specificity of the reactive antibody. 25 K01.075AUB Preferably, the reactive antibody is reactive to an antigen selected from the group consisting of: Glycophorin A, Glycophorin B, or mutations thereof (including the MNS blood 5 group system). Preferably, the subject is a human. Preferably, the cells are red blood cells. 10 In a fourth aspect the invention provides a method of preparing a peptide-lipid construct of the second aspect of the invention including the step of: 15 e Reacting a peptide including a Cys residue with a functional lipid construct of the third alternative of the first aspect of the invention. In a claimed fifth aspect the invention provides a method of 20 effecting qualitative and quantitative changes in the functional moieties expressed at the surface of a cell or a multi-cellular structure including the step of: * contacting the cell or multi-cellular structure with a 25 solution of a functional lipid construct of the first aspect of the invention for a time and at a temperature sufficient to allow the construct to incorporate into the cell or multi-cellular structure. 26 K01.075AUB In a sixth aspect the invention provides a method of immobilizing one or more cells or multi-cellular structures including the steps of: 5 Contacting the cells or multi-cellular structures with a solution of constructs of the fourth alternative of the first aspect of the invention for a time and at a temperature sufficient to allow an effective amount of the constructs to incorporate into the cells or multi 10 cellular structures to provide modified cells or multi cellular structures; and Contacting the modified cells or multi-cellular structures with an avidin-coated substrate capable of 15 being reversibly localized to a surface. Preferably, the avidin-coated substrate is selected from the group consisting of: avidin-coated magnetic beads. 20 Preferably, the being reversibly localized to a surface is by application of a magnetic field. [followed by page 28] 27 K01.075AUB In an embodiment of the sixth aspect the invention provides a method of immobilizing one or more cells or multi-cellular structures including the steps of: 5 Contacting the cells or multi-cellular structures with a dispersion of constructs of the structure: 0 N'I NHl HH (C- 1) 3 NH 0 INHC NHN NA 0 NHN #00 NH0 OHd N o NHH

R

1

R

2 10 for a time and at a temperature sufficient to allow an effective amount of the construct to incorporate into the cells or multi-cellular structures to provide modified cells or multi-cellular structures; 15 e Contacting the modified cells or multi-cellular structures with avidin-coated magnetic beads; and e Applying a magnetic field to localize the beads to a surface; 28 K01.075AUB where M' is a monovalent cation and R 1 and R 2 are both

(CH

2 ) 7 CHCH (CH 2 ) 7. 5 In a seventh aspect the invention provides a method of promoting the aggregation of a first and second population of cells including the steps of: * Contacting the first population of cells with a solution 10 of constructs of the fourth alternative of the first aspect of the invention for a time and at a temperature sufficient to allow an effective amount of the constructs to incorporate into the cells to provide modified cells of the first population; 15 * Contacting the second population of cells with a solution of constructs of the fourth alternative of the first aspect of the invention for a time and at a temperature sufficient to allow an effective amount of the constructs 20 to incorporate into the cells to provide modified cells of the first population; e Contacting the modified cells of one of the populations with an excess of avidin; and then 25 e Contacting the modified cells of the first and second populations. 29 K01.075AUB In an embodiment of the seventh aspect the invention provides a method of promoting the aggregation of a first and second population of cells including the steps of: 5 Contacting the first population of cells with a solution of constructs of the structure ~-/ 0 HH (CH-2) 3 O NHH uOH NH 0 1lO N NH) 0 N O OH 0 NHl GM, or~o R- R2 10 for a time and at a temperature sufficient to allow an effective amount of the constructs to incorporate into the cells to provide modified cells of the first population; 15 Contacting the second population of cells with a solution of the constructs for a time and at a temperature sufficient to allow an effective amount of the constructs to incorporate into the cells to provide modified cells of the first population; 20 30 K01.075AUB e Contacting the modified cells of one of the populations with an excess of avidin; and then e Contacting the modified cells of the first and second 5 populations. In all aspects of the invention M is typically H+, but may be replaced by another cation such as Nat, K+ or NH 4 + or monovalent substituent such as CH 3 . The notation M' excludes 10 M being a monovalent substituent such as CH 3 . For the most part amino acid residues of peptides are identified according to Table 3 of Appendix 2 of Annex C of the Administrative Instructions under the Patent Cooperation 15 Treaty dated 7 February 2007 and in accordance with the convention:

H

2 N-XaaXaaXaa......XaaXaaXaa-COOH 20 In Tables the corresponding one-letter codes for amino acid residues may be employed to provide Tables of acceptable dimensions. In the description and claims of the specification the 25 following acronyms, terms and phrases have the meaning provided: "Avidins" means the biotin-binding tetrameric protein produced in the oviducts of birds, reptiles and amphibians and 30 deposited in the whites of their eggs, its biotin-binding homomers and biotin-binding modified forms thereof including EXTRAVIDIN-, NEUTRAVIDIN m and NEUTRALITE m . 31 KO1.075AUB "Biotin-binding" means non-covalent binding to the biotin moiety with a dissociation constant (KD) under biocompatible conditions of the order 10-1- M. 5 "Diagnostic marker" means a molecule, the presence of which in a body fluid of a subject is diagnostic of a phenotype or pathological condition of the subject. "Dispersible in biocompatible media" means capable of forming 10 a stable, single phase system in a medium at a concentration sufficient to effect qualitative and quantitative changes in the functional moieties expressed at the surface of a cell or a multi-cellular structure without loss of vitality. 15 "(or one of its derivatives)" means a chemical modification of the chemical structure to provide a fluorophore with substantially equivalent physico-chemical properties, but modified spectral characteristics. 20 "List of Peptides" means the list of peptides provided at pages 56 to 58 of this specification. "MNS blood group system" means blood group antigens or epitopes of those antigens and mutations which are present on 25 either glycophorin A, glycophorin B or mutations which result in glycophorin A/B hybrids. "pcv" means packed cell volume. 30 "Proximal terminal sequence" means that portion of the peptide sequence proximal to the amino- or carboxy- terminus of the peptide (F). 32 K01.075AUB "Reactive antibody" means an immunoglobulin, the presence of which in a body fluid of a subject is diagnostic of a phenotype or pathological condition of the subject. 5 "RBC" means red blood cells. "Water soluble" means a stable, single phase system is formed when the construct is contacted with water or saline (such as PBS) at a concentration of at least 100 pg/ml and in the 10 absence of organic solvents or detergents. The terms "soluble" and "dispersible" are used synonymously. Exemplifying embodiments of the invention will now be described in detail with reference to the Figures of the 15 accompanying drawings pages. BRIEF DESCRIPTION OF DRAWINGS Figure 1. 'H-NMR spectrum of the construct DOPE-Ad-CMG(I)amine (11) (5 20 mg/ml in D 2 0/CD 3 0D 2:1 5 ppm). Figure 2. MALDI-TOF MS spectrum of DOPE-Ad-CMG(I)- Ala-Mal-Syph(V8C) (XII) (FLEX-PC (Bruker), DHB). 25 Figure 3. 'H-NMR spectrum of the construct DOPE-Ad-CMG(I)-pAla-Mal Syph(V8C) (XII) (7 mg/ml in D 2 0/CD 3 0D 4:1, pH c. 7.5; 600 MHz, 30 'C, 5 ppm). Figure 4. 'H NMR spectrum of Biotin-CMG(2)-Ad-DOPE (I) (2.5 mg/ml in 30 CD 3 0D/D 2 0 1:1, 5 ppm, 600 MHz). Figure 5. ESI-MS spectrum of Biotin-CMG(2)-Ad-DOPE (I) (ThermoFinnigan LCQDecaXP (negative mode, 30% MeOH)). 33 K01.075AUB Figure 6. Fluorescence microscopy of avidin AF labelled red blood cells modified with the construct designated Biotin-CMG(2)-Ad-DOPE (I) (1 mg/mL) and stored for 14 days. 5 Figure 7. Fluorescence microscopy of avidin AF labelled zona-free D3.5pc murine embryos modified with the construct designated Biotin-CMG(2)-Ad DOPE (I) (0.1 mg/mL) (400x magnification). Figure 8. Fluorescence confocal microscopy of an avidin AF labelled 10 Biotin-CMG(2)-Ad-DOPE (I) modified murine embryo. Figure 9. Attachment of streptavidin beads to spermatozoa following modification of the spermatozoa by incubation with the construct designated Biotin-CMG(2)-Ad-DOPE (I). 15 Figure 10. Retention by streptavidin beads of following modification of the RBCs by incubation with the construct designated Biotin-CMG(2)-Ad-DOPE (I). 20 Figure 11. Structure of the construct designated Biotin-CMG(2)-Ad-DOPE. Figure 12. RL95-2 monolayers modified with 20, 100 and 500 pg/mL biotin CMG(2)-Ad-DOPE or media alone in serum-free media (A, C, E and G) and serum-containing media (B, D, F and H). 25 Figure 23. Addition of Avidin Alexa Fluor@ 488 to RL95-2 monolayers modified with biotin-CMG(2)-Ad-DOPE. Fluorescence microscopy images of cells after addition of Avidin Alexa Fluor@ 488 incubated at 37 'C show fluorescence starts to internalize within 4 hr (C) and is present in the 30 cell interior in 24 hr (E) . No internalization was observed when cells were incubated at 4'C (D, F). DETAILED DESCRIPTION 35 The invention resides primarily in conjugating functional moieties to a diacyl or dialkyl lipid (L) via a spacer (S) to provide a construct (F-S-L) that is dispersible in biocompatible media, but will also spontaneously incorporate 34 K01.075AUB into the lipid bilayer of a cell membrane or multi-cellular structure. The invention resides secondarily in the use of the selected 5 structural motif (CMG) in the applications described and the advantages that accrue from using this structural motif and derivatives thereof. Despite the advances in cell surface modification described in 10 the specifications accompanying the international PCT applications referred to under the heading Background Art,the availability of constructs for use in the "one-step method", in particular peptide-lipid constructs, and the availability of BioG for use in the "two-step method", places a limitation 15 on the broad application of these methods. For example, a two-step method of localizing peptide antigen to the surface of cells or multi-cellular structures that avoids the use of BioG, or other conjugates obtained from 20 biological sources, is desirable. Although it was recognized that the biotinylation of the carbohydrate-lipid constructs described in the specification accompanying international application no. PCT/NZ2005/000052 25 provided a substitute for BioG in the "two-step method", it remains desirable to be able to use a biotin-lipid construct that has the favourable properties of these biotinylated carbohydrate-lipid constructs and could be used in the "one step method". 30 In contrast with the preparation of constructs where the function (F) is a carbohydrate, the preparation of constructs where F is a peptide presents additional technical problems. 35 K01.075AUB Firstly, it is desirable for the peptide (F) ligated to the L S or S-L moiety to be dispersible in water such as a buffered solution of solutes, e.g. PBS, or at least a biocompatible 5 solvent. Overcoming this difficulty may require the selection of a proximal terminal sequence (PTS) to promote solubility without modifying the desired biological properties of the construct. 10 Secondly, it is desirable for the peptide-lipid construct to be dispersible in water, or at least a biocompatible buffered solution or serum, according to the requirements of the proposed application (i.e. it is desirable for the construct 15 to be "water soluble" as defined herein). Overcoming this difficulty requires the selection of a spacer (S) to promote solubility of the construct. 20 Thirdly, where the proposed application is the modification of cells such as red blood cells (RBCs) for use in diagnostic applications, or as quality controls in blood group typing, it is required for the construct to be dispersible in a biocompatible buffered solution without participating in 25 antigen-antibody cross reactivity not specific to the diagnostic peptide or blood group type antigen. Satisfying this requirement requires the identification of suitable structural motifs for the spacer (S) and/or proximal 30 terminal sequence (PTS) when the latter is present. Where the application is for use in the modification of the surface of cells or multi-cellular structures (e.g. an embryo) 36 K01.075AUB with a view to promoting the association of the modified cell or modified multi-cellular structure with a target surface (e.g. the endometrium) exposing the cell or multi-cellular structure to solvents or buffered solutions that are not 5 biocompatible must be avoided. Fourthly, the presentation of the peptide of the peptide lipid construct at the surface of the modified cell or multi cellular structure will have an influence on the extent of 10 cross reactivity with diagnostic markers. The ability to localise peptides to the surface of cells or multi-cellular structures via a residue proximal to either the N- or C- terminus of the peptide may allow the naturally 15 occurring configuration of the peptide sequence relative to the cell surface to be approximated. The presentation of the peptide sequence in the tertiary (or quaternary) structure of the parent polypeptide (or protein) 20 may therefore be mimicked. It is contemplated that peptides may be localised to the surface of cells via multiple residues. For example, where both a residue proximal to the amino terminus and a residue proximal to the carboxyl terminus are used to localise the peptide a "looped" configuration of 25 the peptide may be promoted at the surface. The poly-ethylene glycol (PEG) spacer of known peptide-lipid constructs is selected to provide solubility. However, polymers of PEG may interfere with the expression and function 30 of the peptide at the surface. The as yet unpublished specification accompanying international application number PCT/NZ2008/000239 describes 37 K01.075AUB the preparation of peptide-lipid constructs for use in methods of effecting qualitative and quantitative changes in the level of peptides expressed at the surface of cells and multi cellular structures where an oligomer of ethylene glycol is 5 used as a spacer covalently linking lipid of the construct to the peptide moiety. The use of the constructs to prepare cells for use in serodiagnosis is described. In the peptide-lipid constructs of the present invention the 10 structural motif designated CMG is used as a component (S 1 ) of the spacer (S) covalently linking the lipid (L) and peptide (F). Inclusion of this structural motif provides a degree of rigidity to the spacer, distancing the functional moiety (peptide) of the peptide-lipid construct from the surface of 15 the modified cell or multi-cellular structure. It will be recognized that this attribute of the invention may be favourably applied to the development of other functional lipid constructs as demonstrated here with reference to the 20 use of constructs including this structural motif where the functional moiety is a carbohydrate, such as the glycotope of the antigens of the ABO blood grouping, a fluorophore such as fluorescein (or one of its derivatives), or a conjugator, such as biotin. 25 Biotin ([3aS-(3aa, 4@, 6aa)]-hexahydro-2-oxo-1H-thieno[3,4 d]imidazole-4-pentanoic acid) is a water soluble vitamin of the B complex, also referred to as vitamin H. Biotin is a growth factor present in minute amounts in every living cell. 30 The compound plays an indispensible role in numerous naturally occurring carboxylation reactions, including the production of fatty acids. 38 KO1.075AUB Biotin has a solubility (250C) in water of approximately 22 mg/100 mL and approximately 80 mg/100 mL in 95% alcohol. The compound has increased solubility in hot water and in dilute alkali, but is relatively insoluble in other common organic 5 solvents. The ability to localize this functional moiety to the surface of cells and multi-cellular structures provides a number of applications as demonstrated. Whilst not wishing to be bound by theory it is believed that 10 the properties of the functional lipid constructs may be modified and refined to suit particular applications by by selection of the cation (M+) or derivation of the free carboxyl groups of the structural motif to provide modified structural motifs, e.g. by substitution with methyl (CH 3 ; 15 MCMG). The properties of the functional-lipid constructs for use in the claimed methods must be such that they can be readily dispersed in biologically compatible media in the absence of 20 solvents or detergents, but incorporate into the lipid bilayer of a membrane when a solution of the construct is contacted with a suspension of cells or multi-cellular. Peptide-lipid constructs with these potentially conflicting 25 properties are prepared by selection of other components of the spacer (S) in addition to the inclusion of the unmodified (CMG) or modified (e.g. MCMG) structural motif and/or the inclusion of a proximal terminal sequence (PTS) in the peptide (F). 30 The preparation of the peptide-lipid constructs where S is linked to F via a sulphide bond formed with a terminal Cys (Cys) residue of the peptide at the carboxy-terminus of the 39 K01.075AUB peptide is preferred as the peptide is less prone to oxidation. A range of peptides may therefore be prepared as peptide-lipid 5 constructs for use in methods of effecting qualitative and quantitative changes in the levels of peptide expressed at the surface of cells and multi-cellular structures. A particular advantage of the biotin-lipid constructs is that 10 they permit cells or multi-cellular structures to be localized to surfaces with minimal detriment to the biological activity and viability of the cells or multi-cellular structure. Examples of the localization of cells to a surface are 15 provided. It will be noted that where the localization to a surface is achieved by means of avidin-coated magnetic beads the localization is reversible, thereby providing the opportunity to control the selection and positioning of cells on a surface. 20 The utility of the constructs in sub-cellular fractionation and localization of membrane bound organelles to surfaces is contemplated. The utility of the constructs in promoting the aggregation of populations of cells as may be required in the 25 generation of hybridomas is also contemplated. [followed by page 41] 40 K01.075At3B SCHEME I 0 00

H

3 cy N.Y H'Y' + HN 0 CH, 0 0 )- ~ + 0

-CH

3 JDMF

(CH

3 CH,) 3 N 0 HC N~j

CH

3 03 [1CH 3 0H/H,0 NaCH

H

3 C N~9 3CH, 0 H o0 OH JN,N'-Dicyclohexylcarbodiimide 0 0 rK 0 OCH, H H oyNH 3CH 3 0 0 - 0 o 0 41 K01.075AUB It will be understood that for a non-specific interaction, such as the interaction between diacyl- or dialkyl glycerolipids or glycerophospholipids and a membrane, structural and stereo-isomers of naturally occurring lipids 5 can be functionally equivalent. For example, it is contemplated that diacylglycerol 2 phosphate could be substituted for phosphatidate (diacylglycerol 3-phosphate). Furthermore it is contemplated 10 that the absolute configuration of phosphatidate can be either R or S. The structural motif (CMG) may be prepared by the method summarized in Scheme I and Scheme II to provide the 15 substructures designated MCMG(1) and CMG(2). The preparation of the structural motif, the preparation of functional-lipid constructs utilizing this structural motif, and the use of these constructs in chemical and biological 20 applications is described below Preparation of the structural motif designated CMG Materials and Methods 25 Acetone, benzene, chloroform, ethylacetate, methanol, toluene and o-xylene were from Chimmed (Russian Federation). Acetonitrile was from Cryochrom (Russian Federation). DMSO, DMF, CF 3 COOH, Et 3 N, N,N'-dicyclohexylcarbodiimide and N 30 hydroxysuccinimide were from Merck (Germany). Iminodiacetic acid dimethyl ester hydrochloride was from Reakhim (Russian Federation). 42 KO1.075AUB Dowex 50X4-400 and Sephadex LH-20 were from Amersham Biosciences AB (Sweden). Silica gel 60 was from Merck (Germany) . Tetraamine (H 2

N-CH

2

)

4 C x 2H 2

SO

4 was synthesized as described by Litherland et al. (1938). Thin-layer 5 chromatography was performed using silica gel 60 F 2 5 4 aluminium sheets (Merck, 1.05554) with detection by charring after 7%

H

3

PO

4 soaking. Preparation of {[2-(2-tert-butoxycarbonylamino-acetylamino) 10 acetyl]-methoxycarbonylmethyl-amino}-acetic acid methyl ester (SCHEME I) To a stirred solution of (methoxycarbonylmethyl-amino)-acetic acid methyl ester hydrochloride (988 mg, 5 mmol) in DMF (15 15 ml) were added Boc-GlyGlyNos (3293 mg, 10 mmol) and (CH 3

CH

2

)

3 N (3475 pL, 25 mmol) were added. The mixture was stirred overnight at room temperature and then diluted with o-xylene (70 ml) and evaporated. 20 Flash column chromatography on silica gel (packed in toluene, and eluted with ethyl acetate) resulted in a crude product. The crude product was dissolved in chloroform and washed sequentially with water, 0.5 M NaHCO 3 and saturated KCl. 25 The chloroform extract was evaporated and the product purified on a silica gel column (packed in chloroform and eluted with 15:1 (v/v) chloroform/methanol). Evaporation of the fractions and drying under vacuum of the residue provided a colourless thick syrup. Yield 1785 mg, (95%) . TLC: Rf=0.49 (7:1 (v/v) 30 chloroform/methanol). 'H NMR (500 MHz, [D 6 ]DMSO, 30 0C) 6, ppm: 7.826 (t, J=5.1 Hz, 1H; NHCO), 6.979 (t, J=5.9 Hz, 1H; NHCOO), 4.348 and 4.095 (s, 43 KO1.075AUB 2H; NCH 2 COO), 3.969 (d, J=5.1 Hz, 2H; COCH 2 NH), 3.689 and 3.621 (s, 3H; OCH 3 ), 3.559 (d, J=5.9 Hz, 2H; COCH 2 NHCOO), 1.380 (s, 9H; C(CH 3

)

3 ). 5 Preparation of {[2-(2-tert-butoxycarbonylamino-acetylamino) acetyl] -methoxycarbonylmethyl -amino }-acetic acid (SCHEME I) To a stirred solution of {[2-(2-tert-butoxycarbonylamino acetylamino)-acetyl]-methoxycarbonylmethyl-amino}-acetic acid 10 methyl ester (1760 mg, 4.69 mmol) in methanol (25 ml) 0.2 M aqueous NaOH (23.5 ml) was added and the solution kept for 5 min at room temperature. The solution was then acidified with acetic acid (0.6 ml) and evaporated to dryness. 15 Column chromatography of the residue on silica gel (packed in ethyl acetate and eluted with 2:3:1 (v/v/v) i-PrOH/ethyl acetate/water) resulted in a recovered {[2-(2-tert butoxycarbonylamino-acetylamino)-acetyl] methoxycarbonylmethyl-amino}-acetic acid methyl ester (63 mg, 20 3.4%) and target compound (1320 mg). The intermediate product was then dissolved in methanol/water/pyridine mixture (20:10:1, 30 ml) and passed through an ion exchange column (Dowex 50X4-400, pyridine form, 5 ml) to remove residual sodium cations. 25 The column was then washed with the same solvent mixture, the eluant evaporated, the residue dissolved in chloroform/benzene mixture (1:1, 50 ml) and then evaporated and dried under vacuum. Yield of 10 was 1250 mg (74%), white solid. TLC: 30 Rf=0.47 (4:3:1 (v/v/v) i-PrOH/ethyl acetate/water). 'H NMR (500 MHz, [D 6 ]DMSO, 30 0C), mixture of cis- and trans conformers of N-carboxymethylglycine unit c.3:1. Major 44 KO1.075AUB conformer; 6, ppm: 7.717 (t, J=5 Hz, 1H; NHCO), 7.024 (t, J=5.9 Hz, 1H; NHCOO), 4.051 (s, 2H; NCH 2

COOCH

3 ), 3.928 (d, J=5 Hz, 2H; COCH 2 NH), 3.786 (s, 2H; NCH 2 COOH), 3.616 (s, 3H; OCH 3 ), 3.563 (d, J=5.9 Hz, 2H; COCH 2 NHCOO), 1.381 (s, 9H; C(CH 3

)

3 ) 5 ppm; minor conformer, 6 = 7.766 (t, J=5 Hz, 1H; NHCO), 7.015 (t, J=5.9 Hz, 1H; NHCOO), 4.288 (s, 2H; NCH 2

COOCH

3 ), 3.928 (d, J=5 Hz, 2H; COCH 2 NH), 3.858 (s, 2H; NCH 2 COOH), 3.676 (s, 3H;

OCH

3 ), 3.563 (d, J=5.9 Hz, 2H; COCH 2 NHCOO), 1.381 (s, 9H;

C(CH

3

)

3 ) 10 Preparation of {[2-(2-tert-Butoxycarbonylamino-acetylamino) acetyl]-methoxycarbonylmethyl-amino}-acetic acid N oxysuccinimide ester (Boc-Gly 2 (MCMG1y)Nos) (SCHEME I) 15 To an ice-cooled stirred solution of {[2-(2-tert butoxycarbonylamino-acetylamino)-acetyl] methoxycarbonylmethyl-amino}-acetic acid (1200 mg, 3.32 mmol) and N-hydroxysuccinimide (420 mg, 3.65 mmol) in DMF (10 ml) was added N,N'-dicyclohexylcarbodiimide (754 mg, 3.65 mmol). 20 The mixture was stirred at 00C for 30 min, then for 2 hours at room temperature. The precipitate of N,N'-dicyclohexylurea was filtered off, washed with DMF (5 ml), and filtrates evaporated to a minimal 25 volume. The residue was then agitated with (CH 3

CH

2

)

2 0 (50 ml) for 1 hour and an ether extract removed by decantation. The residue was dried under vacuum providing the active ester (1400 mg, 92%) as a white foam. TLC: Rf=0.71 (40:1 (v/v) acetone/acetic acid). 30 'H NMR (500 MHz, [D 6 ]DMSO, 30 0C), mixture of cis- and trans conformers of N-carboxymethylglycine unit c. 3:2. 45 KO1.075AUB Major conformer; 6, ppm: 7.896 (t, J=5.1 Hz, 1H; NHCO), 6.972 (t, J=5.9 Hz, 1H; NHCOO), 4.533 (s, 2H; NCH2COON), 4.399 (s, 2H; NCH2COOCH 3 ), 3.997 (d, J=5.1 Hz, 2H; COCH 2 NH), 3.695 (s, 5 3H; OCH 3 ), 3.566 (d, J=5.9 Hz, 2H; COCH 2 NHCOO), 1.380 (s, 9H; C (CH 3 ) 3 ) Minor conformer; 6, ppm: 7.882 (t, J=5.1 Hz, 1H; NHCO), 6.963 (t, J=5.9 Hz, 1H; NHCOO), 4.924 (s, 2H; NCH2COON), 4.133 (s, 10 2H; NCH 2

COOCH

3 ), 4.034 (d, J=5.1 Hz, 2H; COCH 2 NH), 3.632 (s, 3H; OCH 3 ), 3.572 (d, J=5.9 Hz, 2H; COCH 2 NHCOO), 1.380 (s, 9H; C (CH 3 ) 3 ) The active ester (1380 mg) was dissolved in DMSO to provide a 15 volume of 6 ml and used as a 0.5 M solution (stored at -18 C). [followed by page 47] 20 46 KO1.075AUB SCHEME II HC 0 H HN N + 0 N CH 0 0r N NJ CHC3 H3C _0C

OH

3 0 0 0 NN NH 0 H

H

3 C CF3COOH 0CH 3 0 H H H

H

2 N ~ N N N NH 2 + 0~ 0

CH

3

H

3 C 0 O 3 C N 0K N N^N O-H 3 0 OCCH H3C CH3 H3,0CF3COOH 0 H 0H 0 0 HsC%) _)k H H2NIA N AN NIr N N NH2 000 0 Kto- 00 + l CH 3 + H3 1 0 0 0 0 0 C

H

3 C CH 3

CH

2

N/H

2 0 IICH 3 OH OH

H

2 N N NN N 0H 0 0 0 0 OHO 0 OH OH 47 KO1.075AUB Preparation of {[2-(2-tert-Butoxycarbonylamino-acetylamino) acetyl]-methoxycarbonylmethyl-amino}-acetic acid methyl ester To the stirred solution of (methoxycarbonylmethyl-amino) 5 acetic acid methyl ester hydrochloride (988 mg, 5 mmol) in DMF (15 ml) Boc-GlyGlyNos (3293 mg, 10 mmol) and Et 3 N (3475 pl, 25 mmol) were added. The mixture was stirred overnight at room temperature (r.t.), 10 then diluted with o-xylene (70 ml) and evaporated. Flash column chromatography on silica gel (packed in toluene and eluted with ethyl acetate) resulted in crude product. The crude product was dissolved in chloroform and washed 15 sequentially with water, 0.5 M NaHCO 3 and saturated KCl. The chloroform extract was evaporated, and the product was purified on a silica gel column (packed in chloroform and eluted with chloroform/methanol 15:1). 20 Evaporation of fractions and vacuum drying of residue resulted in a colorless thick syrup of (3) (1785 mg, 95%). TLC: Rf = 0.49 (chloroform/methanol 7:1). 25 'H NMR (500 MHz, [D 6 ]DMSO, 30 0C) 5 = 7.826 (t, J = 5.1 Hz, 1H; NHCO), 6.979 (t, J = 5.9 Hz, 1H; NHCOO), 4.348 and 4.095 (s, 2H; NCH 2 COO), 3.969 (d, J = 5.1 Hz, 2H; COCH 2 NH), 3.689 and 3.621 (s, 3H; OCH 3 ), 3.559 (d, J = 5.9 Hz, 2H;

COCH

2 NHCOO), 1.380 (s, 9H; CMe 3 ) ppm. 30 48 KO1.075AUB Preparation of {[2-(2-tert-Butoxycarbonylamino-acetylamino) acetyl]-methoxycarbonylmethyl-amino}-acetic acid To the stirred solution of {[2-(2-tert-Butoxycarbonylamino 5 acetylamino)-acetyl]-methoxycarbonylmethyl-amino}-acetic acid methyl ester (1760 mg, 4.69 mmol) in methanol (25 ml) 0.2 M aqueous NaOH (23.5 ml) was added. The solution was kept for 5 min at r.t., then acidified with acetic acid (0.6 ml) and evaporated to dryness. 10 Column chromatography of the residue on silica gel (packed in ethyl acetate and eluted with iPrOH/ethyl acetate/water (2:3:1)) resulted in recovered (3) (63 mg, 3.4%) and crude target compound (1320 mg). 15 The crude target compound was dissolved in methanol/water/pyridine mixture (20:10:1, 30 ml) and passed through an ion-exchange column (Dowex 50X4-400, pyridine form, 5 ml) to remove residual Na cations. 20 The column was washed with the same mixture, eluant evaporated, dissolved in chloroform/benzene mixture (1:1, 50 ml) then evaporated and dried in vacuum to provide a yield of pure (10) was 1250 mg (74%), white solid. 25 TLC: Rf = 0.47 (iPrOH/ethyl acetate/water (4:3:1)). 'H NMR (500 MHz, [D 6 ]DMSO, 30 0C) of mixture of cis- and trans- conformers of N-carboxymethyl-glycine unit c.3:1. 30 Major conformer: 5 = 7.717 (t, J = 5 Hz, 1H; NHCO), 7.024 (t, J = 5.9 Hz, 1H; NHCOO), 4.051 (s, 2H; NCH 2 COOMe), 3.928 (d, J = 5 Hz, 2H; COCH 2 NH), 3.786 (s, 2H; NCH 2 COOH), 3.616 (s, 3H; 49 KO1.075AUB OCH), 3.563 (d, J = 5.9 Hz, 2H; COCH 2 NHCOO), 1.381 (s, 9H; CMe 3 ) ppm. Minor conformer: 5 = 7.766 (t, J = 5 Hz, 1H; NHCO), 7.015 (t, 5 J = 5.9 Hz, 1H; NHCOO), 4.288 (s, 2H; NCH 2 COOMe), 3.928 (d, J = 5 Hz, 2H; COCH 2 NH), 3.858 (s, 2H; NCH 2 COOH), 3.676 (s, 3H;

OCH

3 ), 3.563 (d, J = 5.9 Hz, 2H; COCH 2 NHCOO), 1.381 (s, 9H; CMe 3 ) ppm. 10 Preparation of {[2-(2-tert-Butoxycarbonylamino-acetylamino) acetyl]-methoxycarbonylmethyl-amino}-acetic acid N oxysuccinimide ester Boc-Gly2(MCMG1y)Nos To an ice-cooled stirred solution of {[2-(2-tert 15 Butoxycarbonylamino-acetylamino)-acetyl] methoxycarbonylmethyl-amino}-acetic acid (1200 mg, 3.32 mmol) and N-hydroxysuccinimide (420 mg, 3.65 mmol) in DMF (10 ml) N,N'-dicyclohexylcarbodiimide (754 mg, 3.65 mmol) was added. The mixture was stirred at 0 0C for 30 min, then for 2 h at 20 r.t.. The precipitate of N,N'-dicyclohexylurea was filtered off, washed with DMF (5 ml) and the filtrates evaporated to a minimal volume. 25 The residue was agitated with Et 2 0 (50 ml) for 1 h. An ether extract was removed by decantation, and the residue dried in vacuum to yield the target compound (1400 mg, 92%) as a white foam. 30 TLC: Rf = 0.71 (acetone/acetic acid 40:1). 50 K01.075AUB 'H NMR (500 MHz, [D 6 ]DMSO, 30 0C), mixture of cis- and trans conformers of N-carboxymethyl-glycine unit c. 3:2. Major conformer: 5 = 7.896 (t, J = 5.1 Hz, 1H; NHCO), 6.972 5 (t, J = 5.9 Hz, 1H; NHCOO), 4.533 (s, 2H; NCH 2 COON), 4.399 (s, 2H; NCH 2 COOMe), 3.997 (d, J = 5.1 Hz, 2H; COCH 2 NH), 3.695 (s, 3H; OCH 3 ), 3.566 (d, J = 5.9 Hz, 2H; COCH 2 NHCOO), 1.380 (s, 9H; CMe 3 ) ppm. 10 Minor conformer: 5 = 7.882 (t, J = 5.1 Hz, 1H; NHCO), 6.963 (t, J = 5.9 Hz, 1H; NHCOO), 4.924 (s, 2H; NCH 2 COON), 4.133 (s, 2H; NCH 2 COOMe), 4.034 (d, J = 5.1 Hz, 2H; COCH 2 NH), 3.632 (s, 3H; OCH 3 ), 3.572 (d, J = 5.9 Hz, 2H; COCH 2 NHCOO), 1.380 (s, 9H; CMe 3 ) ppm. 15 Preparation of DOPE-Ad-CMG(I)amine (SCHEME III) DOPE-Ad-CMG(2)amine was prepared from {[2-(2-tert butoxycarbonylamino-acetylamino)-acetyl] 20 methoxycarbonylmethyl-amino}-acetic acid N-oxysuccinimide ester Boc-Gly 2 (MCMGly)Nos according to Scheme III. [followed by page 52] 51 0 I I I 0 0 0 0 0 0 0 0 II I 0O O 0 + z 0 C 0~0

H+

-ol U0 0~ f< f< o= o= y0 17 o >o o o o -3 0% 0 0> a- C) C-, C C- C) O 0 0o 0 0 0 0 0~ C)I C) -'o o 0 0 z)C J-z C)C U) 0 0~ 0~ O 0 f -o C{0C 0{C 0 o o 0 X + 00 + Y, 0 0 o~2 o.Q ok~syC 0 5 0 0) K01.075AUB Preparation of functional lipid constructs (F-S-L)(SCHEME IV) Preparation of DOPE-Ad-CMG (2) -OAla-Mal-Milt (K,M) (X) 5 The construct DOPE-Ad-CMG(2)-BAla-Mal-Milt(K,M) (X) was prepared according to Scheme IV. DOPE-Ad-CMG(2)amine was treated with 5-fold excess of 3 maleimidopropionic acid oxybenztriazol ester (12) in i-PrOH 10 water. Conversion of DOPE-Ad-CMG(2) into was somewhat low maleimido derivative (about 70%), presumably due to fast hydrolysis of the intermediate promoted by the amount of organic base, 15 diisopropylethylamine, required to be added to keep DOPE-Ad CMG(2) in solution. The maleimido-derivative was isolated in 40% yield after gel permeation chromatography on Sephadex LH-20 (i-PrOH-water, 20 1:2). Initially, the conjugation of the maleimido-derivative with peptide was attempted using i-PrOH-TRIS buffer, pH 8 (1:2), but the intermediate appeared to be almost insoluble in this 25 medium. However, addition of pyridin (1 pl/mg of intermediate) resulted in immediate dissolution of reactants and a surprisingly clean and substantially complete conversion. Notably, although no reducing agent was used to prevent 30 oxidative deactivation of the peptide, MS analysis of the whole reaction mixture revealed no traces of S-S dimer. 54 K01.075AUB The desired construct (X) was purified on a Sephadex LH-20 column. A solubility problem was again encountered as fractions containing (X) were slightly opaque. 5 This would appear to indicate that the amount of base added to the eluent was insufficient to keep compounds properly charged and soluble in the concentration range of 1-5 mg/ml. The structure of purified construct (X) was unambiguously 10 established by NMR and MS spectra. NMR spectrum revealed the expected peptide: DOPE ratio as deduced from the signal ratio for the most characteristic aromatic and olefin protons. 15 According to MS data, almost half of the final product (X) spontaneously formed pyroglutamyl derivative ([M-17]+ ion). In MALDI MS spectra of (X) peaks corresponding to unmodified 20 peptide are present while the related peaks are absent in ESI MS spectrum of the same substance. This is ascribed to facile fragmentation at the thiosuccinimide bond (retro-Michael reaction) under MALDI ionization conditions (destructive technique). 25 The general method of preparing peptide-lipid constructs was applied with minor modification to the preparation of constructs including peptides (F) selected from the following List of Peptides: 30 55 U Ul) Ul) U)0 U U) U U u) u U N N N N U ~D Q4'C F-L U CD >i U : DU (1 ) H H 0() c-H COU -H U U) U) FZ U) CD CD FZ Ui i1 H , U) co H- d ~ C 0) ) H -H Cd co c)-Ho-H U Cl) (a) c Z H 'c -H4 -H Cd Cdd -H -H - H 4-) 4-) r).0 (1) co co -H 0- -H ( 0)~~~~- I) -- H - H - H ~ -H rH >1 >1 (0C C 0 H 0 C - >1 ' 0 H4 4) I CO H 02 Z- ',4H H u (D CD4 ',4 >C (H >1~ H H H >1 i > -- Cd E- CD CD-H ~> H a 41.) ~ ) U) c) >i 4J H H : 0 >1 >1 H H, S >1 rH rH rH c rH rH4 0) r-H FI1 F:I1 U) Hd o o ro cd H >, -1 iH 0 0 ) ', H H, 1 N- 'J 4J N >1 >1 >1 >1 Cl) CU) C') Cl) CO U) ) !) U) U) c) U) I ) Uo I) Uo Uo c) I co co C) ) C) C) C C) C C) C C) C C) C C) ) C) Co N N N N N N N - l - 04 -H4 - - -4 414 (D (d 4 D U) > >1 >, - >1 >1 >, L, L, Cd CO -H - -sH O's ) Q-Q U)) U) 04 04 04 04 0- ) U) >i- d >1 - -H - -H >1i U) U) U) -H U) U) U) - - 9 H -4 - U) 0) O3) O3) U) 0) 0) CT U) U) 04 >i~ S U) 'L Uf) -H - - - i 4 - - >1 >1 U) r-i U) Cd ) Cd ) Uf) U) U) 0, U) U) U) U) U) 0) -- H H 2 >i > i i U >1 >1 >1 -H -H - 4 C~ Cd4 (0 0, U) U)oU c U) C)OU U) U) U) ) > 42- '91 -H ) -H -H -H UO -H -H -H > 1 > H o ) 0 f) r. U) 4J~ 4J~ - U) 4J 4J 04 4 U) >i 4 - H 4 -H Uf) >i (D (D 9, >i C) CD U ) - - Cd 4 - Cd 04 04Q Q Q4 Q4 04 co U) > - >i 4 U) )o U) U) U) U) U) U) >1 '9 42 0 4 0 r. r. 4 r . r - - 04 ) 5 - 4 2-- U) U) U) D U) U) U) m ) -H 4 04 H 4 0 C gI g CO) g' 9 H H 0I D C U) >U d)~Cd ~ ~ 4 2 )rH 4 41-) (D D CD (D CD CD ) H 0I~ a) (D) (D) Cl Cl) Cl) Cl) Cl) H H1 H- H c) U) >1 -H U) c) CD CD Cl) Cl) CO U ) ) U >1 >1 > 1 > 6C 1 C) , C) 0 C) 0C -H - -H C) ) I-H 0) 4-) Cd > C) 0I 0) C) ~ C) 9 C)4 0) ', 0 o1 U) ~ > '-H >1 'H >1 0 04 ', (H- ' '-H C) ' C H 'g- () >1 p) H ) CF 4I 00 r'-4 4 C) H .1-Id .1-)4 >1 0 tH 0H 0 , 0 >1 F4 a) FCCd >11 a) 0 C) '--I (2) 0 K01.075AUB The use of the peptide-lipid constructs in methods for effecting qualitative and quantitative changes in the levels of peptide expressed at the surface of cells and multi cellular structures was illustrated with reference to 5 serodiagnosis. In the following table cross-reactivity of polyclonal sera and monoclonal antibodies of known specificities and red blood cells (RBCs) modified with the construct DOPE-Ad-CMG(I)-BAla 10 Mal-Mur(D14C) (XI) (2 hours, 37 0C) is summarized. [followed by page 601 59 K01.075AUB Reagent ID Type EIA/Miltenberger Specificity Reactive with MUT-T peptides by 2 T217 Human group AB serum EIA 3 T165 Human group 0 serum Reactive with MUR peptides by EIA 4 T702 Hmangrou B srum Reactive with MUT-M peptides by 4 T7202 Human group B serum 6 Reactive with MUT-T peptides by 6 T6025 Human group A serum EIA 7 T8445 Human group 0 serum Uncertain 8 T5896 Human group 0 serum Uncertain 9 MIII Monoclonal antibody Reactive with Mi III red cells 10 Mia Monoclonal antibody Reactive with Mi III red cells Reactive with Mur positive red 11 Mur Monoclonal antibody cells IgG monoclonal 12 Gam Reactive with Mi III red cells antibody 13 BoxH Human serum Uncertain 14 TAP1 Human group 0 serum Presumed MUT-K specificity 15 TAP2 Human serum Presumed MUR specificity Transformed Untransformed Antibody ID Specificitiescells T165 (serum Mur 10 0 T6025 serum K+Mur 5 0 4) lu T8445 serum Mur+Hil+Tsen 5 0 T5896 serum M+K+(Mur) 0 0 Japan MoAb Mur 0 0 ) I T4130 serum Hil+Tsen 0 0 T217 serum T 0 0 T7202 serum M 0 0 0 T8012 serum M+K+T 0 0 Japan MoAb Mi III (1:10) 0 0 t Box Hill serum? 0 0 Japan MoAb Mia (1:50) 0 0 E119 KBL 7201 MoAb Mia GAMMA(1:100) 0 0 60 KO1.075AUB Modification of red blood cells with peptide-lipid constructs Red blood cells are modified by mixing 1 part by volume of 5 washed packed red blood cells with 1 part by volume of peptide-lipid construct dispersed at a concentration of 10 to 1000 pg/ml in cell media (Celpresolm). The suspensions are either: 10 1. incubated for 2 hours at 37 0C before being washed and suspended in a cell medium for serological analysis at a concentration of 0.8 to 3% (Method 1); or 15 2. incubated for 3 to 4 hours at room temperature (circa 25 0C) followed by 18 hours at 4 0C before being washed and suspended in a cell medium for serological analysis at a concentration of 0.8 to 3% (Method 2). 20 Tube serology testing of modified red blood cells Serological reactions are graded or scored by either of two established systems (0 or '-' = no agglutination, 1+ or 3 = very weak agglutination, 2+ or 5 = weak agglutination, 3+ or 8 25 = moderate strong agglutination, 4+ or 10/12 = strong agglutination) Serological platforms used are Tube (addition of reagents and reactants into plastic or glass serology tubes and after 30 appropriate incubations, washing and centrifugation observing reactions macroscopically by eye and a 1oX magnification eyepiece and scoring) and BioVueM (addition of reactants into cassettes containing beads (including some reactants) and 61 K01.075AUB after appropriate incubations and centrifugation observing the reaction patterns trapped within the Gel matrix). BioVue is the serological column agglutination platform of Ortho Clinical Diagnostics. Diamed is the serological column 5 agglutination platform of Diamed AG. Serum samples were available from 47 blood donors of negative antibody screen status. These samples were designated "negative samples", but not determined not to have anti 10 Miltenberger antibodies). Three serum samples known to have Miltenberger related antibodies T217, T6025, T5896. These samples were designated "positive samples", but not determined to have anti 15 antibodies against the peptide of the peptide of the construct designated DOPE-PEG 6 -BAla-Mal-Milt (K) (MOO). A suspension of 3 % modified RBCs was prepared in PBS and 30 pl of the suspension mixed with 30 pl serum sample. The 20 mixtures were then incubated for 45 min at 37 0C. Following incubation the RBCs were centrifuged for 10 s in an Immufuge (setting: "high") and observed for agglutination before being washed 3 times with PBS. 25 After washing one drop of Epiclone- anti-human globulin (AHG) was added and the tubes then centrifuged for 10 s in an ImmufugeM (setting: "high"). Tubes were then read and serology scores recorded. 30 Comments on the observed serology scores are provided in the legends to the following tables. 62 K01.075AUB Age of modified Concentration of RBCs DOPE-PEG 6 -PAla-Mal-Milt (K) (MOO) (mg/ml) (days) 1.0 0.5 0.25 Serum (n = 47) (n = 21) (n = 21) Negative AHG+ AHG- AHG+ AHG- AHG+ AHG samples 1 46 0 21 0 21 Table 1. Summary of reactivity of samples of serum from 47 blood donors 5 not expected to have anti-Miltenberger activity ("negative samples") . AHG+ means sample reacted by the anti-human globulin test. AHG- means sample is unreactive. RBCs were modified with the peptide-lipid construct designated

DOPE-PEG

6 -@Ala-Mal-Milt(K) at the concentrations indicated. Sera were tested against modified RBCs following 3 days storage. 10 Age of modified Concentration of RBCs DOPE-PEG 6 -PAla-Mal-Milt (K) (MOO) (mg/ml) (days) Serum 1.0 0.5 0.25 3 T217 2+ 1+ 3 T6025 4+ 4+ 4+ 3 T5896 24 T217 n.t. 24 T6025 2+ 2+ n.t. 24 T5896 n.t. Table 2. Results by tube serology of 3 serums known to contain antibodies against antigens of the Miltenberger complex. Score results show sample reactivity by the anti-human globulin test, 1+ = weak, 2+ = medium, 3+ = 15 medium/strong, 4+ = strong, - means sample is unreactive. RBCs were modified with the peptide-lipid construct at the concentrations indicated. Sera were tested against modified RBCs following 3 days and 24 days storage. (n.t. - not tested). 63 K01.075AUB Age of modified Concentration of RBCs DOPE-PEG 6 -BAla-Mal-Milt (K) (MOO) (mg/ml) (days) Serum 1.0 0.5 0.25 3 T217 1+ 3 T6025 1+ 2+ 1+ 3 T5896 24 T217 24 T6025 2+ 2+ 1+ 24 T5896 Table 3. Results by Diamed column serology of 3 serums known to contain antibodies against the Miltenberger complex. Score results show sample 5 reactivity by the anti-human globulin test, 1+ = weak, 2+ = medium, 3+ = medium/strong, 4+ = strong, - means sample is unreactive. RBCs were modified with the peptide-lipid construct at the concentrations indicated. Sera were tested against modified RBCs following 3 days and 24 days storage. 10 64 CC) o .

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0 U-) 000 4-) 0 -I 4- H 4 H' -1 00 0 )HO ( O4- -O 0,() o r 0 01 r OC ) 0-H1 rO> tOCj -H) ) H 4 () 0 0- 4- 0 -1 0Q 01 4-)0 00O )- o Fi) k- -H 4-H .04 04-H 04 i 0 ~ 4-) U 4-) U ) ) 4-) 0 4- 0 (1) 1-D -H 0 - H (1 0 ro -H!(- ) -1 -)H -H-H) ( ......... -H 440HqH 0 - 4 ) 0O 4)~ t3) 4-)H M H o\003 C)Q C) H 04-) 1 1 4-) (D 4Q -H 0- H- ) ril 0n r, 4)-H k 0>1 0 4 4)U $4 0) CO- 4-00 44 4)0 (4) 0 0 0-H ) 4- > r)0 1 4-) - 0 4- 4- -0 r,-) 4-) 0 -4) 0 - 0 0 z U - 0 H -0 Q) 4- ) -1 C) Ln C) c > 0 0 (1) ZH rd r - 4 Co C) 0 - U 0 0 -H 1 ri4) C > (,~ rl4-0 OH44 Cl) 0000-HC)0O 4-0- )0r- 1 ~ -H0C U. 0 - H O 0H 4-) ) -0- 1 1 C4)r 4-) r, 4 -)H4) >1 (1) r > 0 -H 0- C1 ) 0 r 1 -4H H~ OC) 0 :M H r 0 4-))0-0 -H-H3 04 -)50 4C) 0C-) 0 rlH4U) H- 1 (1) 0) 04 4-) , r- r, >a ro ) H 0 u r 0 4-) Ii * -44-) - H SO - C 0T Ca-4 (I4r- 0 (1 N a - - 4 U CN 4) -0 () 4- - K01.075AUB 22 17 24 Sample No. (50 (200 (200 Interpretation pg/ml) pg/ml) pg/ml) 488-6 10 K 9327986660 8 3 0 K 9325490091 5 5 0 K 9328791834 5 3 0 K 621-3 5 K 922390844-5 0 12 0 Mur 9322338631 0 10 0 Mur 914146821-8 0 10 0 Mur 932809044-1 0 8 0 Mur 942433813-3 0 8 0 Mur 942404708-4 0 5 0 Mur 942421413-0 0 5 0 Mur 942223755-1 0 5 0 Mur 942442720-2 0 5 0 Mur 927619701-8 0 3 0 Mur 912485657-9 0 3 0 Mur 926190919-0 0 3 0 Mur 9328154853 0 10 3 Mur + Hil 9328118428 0 10 5 Mur + Hil 9425256505 0 8 8 Mur + Hil 942433855-3 0 8 8 Mur + Hil 942753165-4 0 8 8 Mur + Hil 9424292604 0 8 5 Mur + Hil 9427455417 0 5 5 Mur + Hil S-3 0 3 5 Mur + Hil 942448627-8 0 0 5 Hil 942423002-4 0 0 3 Hil 942762589-1 0 0 3 Hil 71 K01.075AUB 22 17 24 Sample No. (50 (200 (200 Interpretation pg/ml) pg/ml) pg/ml) 9424248012 0 0 0 other 942761516 0 0 0 other 942761397 0 0 0 other 9427613497 0 0 0 other 927175131-4 0 0 0 other 932467774-5 0 0 0 other 927299700-1 0 0 0 other 926555294-1 0 0 0 other 932360876-4 0 0 0 other 927516053-2 0 0 0 other 942404708-4 0 0 0 other 589-6 0 other Table 11. Positive serum reactivity. Miltenberger negative red cells were modified with the peptide-lipid construct M22 at a transformation concentration of 50 pg/ml, M17 at a transformation concentration of 200 pg/ml, M24 at a transformation concentration of 200 pg/ml, and tested against natural Mi III antibody reactive human serums in the field to determine rates of reactivity. Equal volumes of packed RBCs and a solution containing 50 pg/ml of the construct were contacted for 3 hours at room temperature and then 18 hours at 4 0 C. The three different constructs of MUT, MUR and HIL were able to discriminate most natural Mi III reactive polyclonal antibodies into specific reactivity profiles. Twelve serums were unreactive with the modified cells, suggesting they may have specificity against another Mi III antigen. [followed by page 73] 72 K01.075AUB TAP 1 Serum RBCs Saline AHG Natural Mi III positive cells R2R2, 11297161 8 10 RlRz 11291347 8 10 Abtectcell III 8245009 10 Peptide-lipid Construct (pg/ml) Modified cells M22 (MUT) 200 0 10 M22 (MUT) 100 0 8 M22 (MUT) 50 0 8 M22 (MUT) 20 0 8 M22 (MUT) 10 0 5 M22 (MUT) 0 0 0 M17 (Mur) 0 0 M24 (Hil) 0 0 Antibody Screen/ID panel Abtectcell III 0 Batch 2223005 Cells I - III Phenocell B Batch 2653046 Cells 1-11 0 Table 12. Anti-MUT serum reactivity. Miltenberger negative RBCs were modified with the peptide-lipid constructs M22 at concentrations ranging from 10 to 200 pg/ml, M17 at a concentration of 50 pg/ml and M24 at a concentration of 50 pg/ml. The modified cells were tested against a natural Mi III antibody reactive Taiwan human serum (TAP1) detected in the field to determine reactivity profile. Reactivity was compared against natural Mi III antigen positive cells. TAP1 (Taiwan Miltenberger antibody 73 KO1.075AUB positive sample). TAP1 serum was shown to contain both IgG and IgM antibodies (the latter being saline reactive) directed against natural MiIII positive cells. The lack of reactivity against Abtectcell m and Phenocellm antibody screening and identification panels concludes no other antibodies against red cells are present. Reactivity with M22 modiifed cells over transformation concentrations of 10 to 200 pg/ml (and not with untransformed cells - 0 pg/ml) concludes the presence of an antibody directed against MUT. The failure of the M22 modified cells to detect a saline reaction, suggests the assay is sensitive to the IgG class of antibody and not IgM - a clinically favourable result. The lack of reactivity with the M17 and M24 transformed cells concludes and absence of antibodies to the MUR and HIL mutations. [followed by page 75] 74 K01.075AUB TAP 2 Serum RED CELLS BioVue AHG Natural Mi III positive cells Mi III PDN No. 54 5 Mi IV No;4131850 10 Mi VI 1523A 8 Mi ?x ID: CBR 8 Vw No: 9422184 0 Peptide-lipid construct (pg/ml) M28 (MUR) 100 10 M22 (MUT) 100 0 Antibody Screen panel Abtectcell III Batch 2223009 Cells I - III 0 Table 13. Anti-MUT serum reactivity. Miltenberger negative red cells were transformed with the peptide-lipid constructs M28 at a concentration of 100 pg/ml and M22 at a concentration of 100 pg/ml and tested against a natural Mi III antibody reactive Taiwan human serum (TAP2) detected in the field to determine reactivity profile. Reactivity was compared against natural Mi III antigen positive cells. TAP 2 (Taiwan Miltenberger antibody positive sample). TAP2 serum was shown to contain IgG antibodies antibodies directed against natural Mi III positive cells. The lack of reactivity against Abtectcell T M antibody screening and identification panels concludes no other antibodies against red cells are present. Reactivity with M28 modified cells and not with M22 modiifed cells concludes the presence of an antibody directed against the MUR peptide. 75 44 1 1 1 1 1 1 D I O 0 rD + + + + + 004 0 -1 E-1 E-1 LnL . + 3+ Vm I I I I i F ' Fl Fl cnc CN cn CZ CZ CZ CZ Z o $4 4 .4 + a 4 U) N N r'2-i o Ma 0 0 H E-i E-i E-i cn Cl LUD m t-0 _n_1 4 I-i S 0 t-0( K LC) cm >C0 4-1 -C w 0 -1 r rd M - f1.4 4-) 0 -- S-] 0) r C -- 0-) 0 0 4 F F] M 4-) r, I ( 9 0) 0) > 0 ( C D- ( 0 -l 4 0 -H r S-] f4) 4- ) r > 4 0-) H , r 4-1 o) r 0 ) - - 1 -H H > 1 -) > 0) A r, 4- C 4 -) - (9- 0 l ) . ' -H 0 r u a ) E- 4-4-14-) r, D r CO f1 r, U 4 o) a -a W - ) 4- > -- ,I >1 4- > ,) r V . ) -1 r) 0) CO - 4) U 0 0 C E 0) o co -- -aU d o4- 1 4) ) 0 O C O 0 >14 -- X >(0 -- V oA r - -a1 > 4 - 0) 0) > 0 M r a0 4 41 C) 0 A 0 o , o U rd 4) o 0) O4 -1 4-) 4- 4) r or- D U) o W 'A S- C) 0 4-) Z ) o >1 4-) 0 0) - 0 4-) U l U) 4 N) LO -H l 0 4-) 0) CY) 4-) :> S A -- 1 'A- T z -A 4-) ) M ( (4-] 4-) U) 0) -HN - r 0 m 4-) CY) 00 0) U9 >1 (9 0 ,- A 0) 0 U Y) -- l I r d - 4-) 0) C\] > 0 rd C,4 m -- l 'd 0) U) A- (%J-- U) rl S -0)4-) 0) -H ro -- 0) "O U) o 4-) U) o r CO 4r) (9 c S- - - Q S - 0) 0 U) (9 U) 4-) 0) 0) 4-) ' ) ' 0 U) r d 0 ) o -a rd E-1- 0) 0 4-) 0 4) - 0 - H U) 4I) - I > A-l U) 0) d ~A 0) 4-) r, a 7A > , 0 M - 0) -H 0 0) -- (0 F]A -- 4-) 4-) U) 'A l A-l 0) (0 I '-l U) 'Al A 00 0 C rd CY) 7d 4-) S-] 0 f1 -- (0 -- l U) 0)- ) > 4-) 0) U) 1 l' O C 4-) U) r F] - 0) 0) ' 0 4 - H 0 0 >i (4-1 0) 0 4-) 0 4-) r, ' O o e o A 'Ao 4-) U) * a - c U) 0 Al o 4- 'A1 0) -a0 4-) 0 -A 4-) -A 'A 0 4-) o 4-) -- 0) >1 a M r 0) Z - - rd - a 0 4-) S o o r - 0) 9 0) e (1 H d 'd H ) rd ,Q r (4-1 0) 0) r 0cC 0 A- 4-l -- 4-) 0) 0) 0 4-) -- 4-l r, > 4-) 0) 4-) 4-) -- ro O- 0) 7d ' 4 -) l A 0) > 'd 0 0 - A A o A F] 0 4l-1 M) (0 * S- A 0 4-)(1 * * U) V. W) 'A- >0)l >1 - 3 4-) > 4-) 0 fn r -a - - A -1 z U) >- r > - > H 0 -a -a ro -- -a .Q H 4-) 4-) M - 4-) E-1 U Lc) E- ro ro r U) r '- K01.075AUB MUT Peptides Reactivity 34 MUTK4 S S Q T N D K H K R 5 22 MUTK3 S Q T N D K H K R D 8 36 MUTK6 S Q T N D K H K R D T 8 35 MUTK5 Q T N D K H K R D T 5 33MUTK1 Q TN D K H K RD T 3 37 MUTK7 T N D K H K R D T 8 HIL peptides 40 Hil 2 E E E T G E T G Q L 41 Hil 3 E T G E T G Q L vIH Table 15. Anti-MUT serum (TAP1) reactivity. Miltenberger negative cells were modified with the peptide-lipid constructs M22, M33, M34, M35, M36, M37, M40 and M41 at a concentration of 50 pg/ml (2 hours, 37 'C). 5 Modified cells were tested against Taiwan Mi III antibody positive serum TAP1 to determine its MUT reactivity profile. TAP 1 (Taiwan Miltenberger antibody positive sample). The TAP1 serum is able to recognize some, but not all, peptide variations of MUT. The lack of reactivity with M40 and M41 (HIL peptide) modified cells and the untransformed cells is expected. 10 Panel Serum #7 (T8445) 30 Mur 9 D T Y P A H T A N E 8 17 Mur 2 T Y P A H T A N E V 10 28 Mur 6 T Y P A H T A N E 8 27 Mur 5 T Y PA H T A N 25 Mur 4 Y P A H T A N E 3 26 Mur 7 Y P A H T A N E V 10 31 Mur 10 Y P A H T A N E V S 8 19 Mur 3 P A H T A N E V 8 29 Mur 8 P A H T A N E V S 8 Table 16. Anti-MUR serum (TAP1) reactivity. Miltenberger negative cells were modified with the peptide-lipid constructs M17, M19, M25, M26, M27, M28, M29, M30 and M31 at a concentration of 50 pg/ml (2 hours, 37 'C). 15 Modified cells were tested against antibody positive serum #7 from the Miltenberger Antibody Positive Panel to determine their MUR reactivity profile. 78 - 4 4 C4- U) C) C)LC) 0 > o\O o\O - 1 0) 0) .- - -- -- -- 4 0 -U U 4- ) C CD C) ~ -- l -- l 0 0 0 4-H -- l r---l 0 4-) 4-) 4 du U C C) C) C) C Cr 4 0 0) (0 0) S0) 0 S U) S4- -H r0 -- U) 0 0) 0) 0 4-) r, U~ (' .4 F F ---- l 4-) U ro o -H Cfl o\o 0) T 0) (0 a 00 U) 0)CU) l 0' (0 U)0U(4-1 r, o 4-] 0) l r (4- 1 4-) ( 0 w m rl 0 4I-) 4-) 0 'd T . 0) - 0 1 -H 1 o\OLO , O C)) C) 0' f U) I d O SLC) ) Cfl 1) 0 0) (0 4 U) 1 T 0 0] 4-1 (%J C) C) C) C) C) .l -,-l W) '1 4-1 r, 0 -) -0) - > 0- 0 O.4 1 -ad - r- T a) 0)) 40) E-1 0,H A C) F) T P 0) (0 rr .4-) -Z - - - - 4 ) U ) U ) - -] C)o OCY S ccU) U) c -H C')

----

l ---- ---- --- -- - ro ol 'Ao0C D 0d C )4-) Fq! u 'A 0 A CO CO 0 0) S-] 0 CY) > r S L C O) o w 4-) -,-l U) OLC) > 0 C H IC A 0- 0C r, U) 4-) -1 C- U) 4-) -- U) - 'HLC) 0) U) ) 0 z I > U) C l z --- ---- - -A- ) O -4-) C ~ 4 -)S Ar Cfl C') U) 0 -A 'A U) F]( 4- S- 0) S-] rd- 'A 00 _ H 0 -H U O oQo -H . 04 U) S-I l 'ALfl' ---- d 0 0 r00 - u 0 0) 0) 0 rl 0 D~ 'AM 4- U) d T_ 3 rI U Ic -- 0)r ) OO ) -H0 o o0 - -1 0)l L 4 - - - - - - - - - ) - ) > (0 M a- U 0) 0 0) - l S0) N 0)0u > 0) I- -- 1 ' 4-) U) CD ril r--) -,-l] . )Q - U) r V o w r- 0 d 0) -- ] O o0 CY NN) Y - - 4 r KO.075AUB False positive reactions with M17 Score 12-10 8-5 3 0 (n=102) 17 18 3 65 17% 18% 3% 64% Table 18. False positive MUR M17 construct reactions with 102 negative serums. Cells modified with the peptide-lipid construct M17 were tested 5 against 102 negative serum samples. Cells modified with the peptide-lipid construct M17 give the most "false positive" reactive construct showing up to 36% false positive rate with negative serums. 10 T series reactives (n=58) 2 4 31 44 61 18 21 28 55 42 63 62 7 20 48 22 39 23 30 positive M17 10 8 8 10 10 12 12 12 12 10 10 8 8 8 8 8 5 3 3 33% M28 10 8 8 10 10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 9% M30 10 8 8 10 10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 9% 39 T negative samples were negative with all 3 constructs. Larger series = 102 15 T18 T21 T28 T55 T78 T92 30 Mur 9 D T Y P A H T A N E - - - - - 17 Mur 2 T Y P A H T A N E V 12 12 12 12 12 12 28 Mur 6 T Y P A H T A N E - - - - - 27 Mur 5 T Y A H TAN - - - - - 25 Mur 4 Y P A H T A N E - - - - 5 26 Mur 7 Y P A H T A N E V 5 10 12 10 10 12 31 Mur 10 Y P A H T A N E V S - 10 12 - 8 12 19 Mur 3 P A H T A N E V - - - 12 - 29 Mur 8 P A H T A N E V S - - 5 - - 8 5 samples reacted with all 3 constructs 80 K01.075AUB Panel Serum #7 False (T8445) Positivity 30 Mur 9 D T Y P A H T A N E 8 17 Mur 2 T Y P A H T A N E V 10 +++ 28 Mur 6 T Y P A H T A N E 8 27 Mur 5 T Y PA H T A N 25 Mur 4 Y P A H T A N E 3 26 Mur 7 Y P A H T A N E V 10 +++ 31 Mur 10 Y P A H T A N E V S 8 ++ 19 Mur 3 P A H T A N E V 8 + 29 Mur 8 P A H T A N E V S 8 + Table 19. "M17-false-positive" negative serum reactivity against other Mur constructs. The 6 most false positive negative serums reactive against cells modified with the peptide-lipid construct M17 were tested against 5 cells modified by contacting with the peptide-lipid constructs M19, M25, M26, M27, M28, M29, M30 and M31 at a concentration of 50 pg/ml (2 hour 37 0 C). The modified cells were tested in BioVue"m AHG cards. Cell modified with the peptide-lipid construct M17 provided the most "false positive" reactions with negative serums. The reactivity of the 6 most false 10 positive samples when tested against other modified cells shows that some are unreactive (M28, M27), some are poorly reactive or show a single discrete reactivity (M5, M29, M19) while others are more reactive (M31, M26). Minor changes in amino acid sequence can influence the rate of false positive reactivity. Cells modified with the constructs M30 and M28 show 15 both specificity and low non-specificity. Ml modified cells M2 cells vs serum Identity of sera #4 #5 #6 #2 #6 #8 Serum alone 5 5 10 8 8 8 Serum + peptide 0 0 0 0 2 j 0 Table 20. Sera reactive with RBCs modified to incorporate the M1 peptide 20 lipid construct or M2 peptide-lipid construct constructs by contacting the cells with a 500 pg/ml dispersion of the construct (Method 1) were "neutralised" with the peptide QTNDKHKRDTY and retested against the modified cells. Sera were neutralized by adding 10 pL of 1 mg/ml solution of peptide to a 50 pL volume of sera and incubating for 30 minutes at 37 25 0 C. Testing was performed using BioVuem cards. 81 K01.075AUB M13 modified cells Identity of sera #3 #42 #37 #34 Serum alone 8 8 8 8 Serum + peptide 0 0 0 0 Table 21. Sera reactive with RBCs modified to incorporate the M13 peptide-lipid construct by contacting the cells with a 500 pg/ml 5 dispersion of the construct (Method 1) were "neutralised" with the peptide SSQTNDKHKRDTY and retested against the modified cells. Sera were neutralized by adding 10 pL of 1 mg/ml solution of peptide to a 50 pL volume of sera and incubating for 30 minutes at 37 'C. Testing was performed using BioVue cards. 10 PAC74 TAP1 Pre-neutralisation serology (cells modified 8 8 with the peptide-lipid construct M22) Post-neutralisation serology (cells modified Neutraliser F Linker with the peptide-lipid construct M22) nil nil nil 8 8 M22 peptide SQTNDKHKRDC nil 8 M28 peptide TYPAHTANEC nil 8 8 M22 molecule SQTNDKHKRDC CMG(2) - Cys-CMG-DE Ac-C CMG(2) - 8 VMYASSG? 8 8 Table 22. Cells were modified by the peptide-lipid construct M22 (2 hours, 37'C) and two positive reactions were identified. Neutralisation 15 experiments were then performed. A volume of 40 pl of plasma was incubated with 10 pl of peptide or Ac-C at a concentration of 1.0 mg/ml for 30 minutes at 37 'C. The standard AHG test in BioVuem was then performed. The false positive reaction for PAC74 was confirmed as a reaction not neutralised by addition of peptide. The true positive reaction for TAP1 20 confirmed as reaction neutralised by peptide and the whole construct, but not the construct bearing only acetylated cysteine. 82 K01.075AUB PAC70 PAC71 PAC72 TAP2 Pre-neutralisation serology (cells modified 8 5 5 8 with the peptide-lipid construct M28) Post-neutralisation serology (cells modified Neutraliser F group Linker with the peptide-lipid construct M22) saline nil nil 8 5 5 8 M22 peptide SQTNDKHKRDC nil 8 - - 3 M28 peptide TYPAHTANEC nil - - - 8 Cys-CMG-DE Ac-C CMG(2) 8 3 3 5 Atri-CMG-DE GalNAca3[Fuca2]Gal3 CMG(2) 8 3 5 8 Atri adipate-DE GalNAca3[Fuca2]Gal3 adipate 8 3 5 8 VMYASSG? 8 5 5 8 Table 23. Cells were modified by the peptide-lipid construct M28 (2 5 hours, 37'C) and four positive reactions were identified. Neutralisation experiments were then performed. A volume of 40 pl of plasma was incubated with 10 pl of peptide or Ac-C at a concentration of 1.0 mg/ml for 30 minutes at 37 'C. The standard AHG test in BioVue T m was then performed. The neutralisations of PAC70, 71 and 72 with the M28 peptide suggests 10 specificity. The fact that the unrelated peptide M22 was also able to cause neutralisation of serums PAC71 and PAC72, together with reductions in score with other unrelated structures, revises the results for these two sera as being false positive reactivity. The fact that PAC70 does not react with Miltenberger positive cells suggests that although an antibody 15 appears to be present to the peptide sequence it is not blood group specific. In contrast although TAP2 was not fully inhibited by peptide, the substantial reduction in score suggests specificity, although it is possible that specificity may be present with a low level of non specificity as suggested by the reaction score reduction against Cys-CMG 20 DE. 83 KO1.075AUB Consideration of the MUT peptide reactivities presented in the foregoing in Tables shows that peptides M22, M36 and M37 all showed superior sensitivity and specificity towards a human polyclonal antibody panel when compared with sequence 1, the 5 sequence identified in the prior art (Reid and Lomas-Francis (2004)). Modification of red blood cells with peptide-lipid constructs with peptide in alternative configurations 10 Peptide-lipid constructs comprising CMG(2) and the following peptides were prepared: ThrTyrProAlaHisThrAlaAsnGluCys (M44) and 15 CysThrTyrProAlaHisThrAlaAsnGlu (M45) The termini of the peptides were formylated and amidated to provide "capped" peptides. The reactivity of random Miltenberger antibody positive samples and three false 20 positive antibody negative samples were tested against RBCs modified to incorporate these "capped" peptide-lipid constructs (50 pg/mL, 2 hours at 37 C). The reactivity was assessed and recorded in Table 24. The 25 capping of the peptide did not affect reactivity with positive samples, nor did it appear to affect reactivity with false positive antibody-negative samples. However, linkage of the peptide via a Cys residue located at the amino terminus (as opposed to the carboxy terminus) appeared to reduce the 30 likelihood of reactivity with the false-positive antibody negative samples and improve reactivity with the known antibody positive samples. 84 K01.075AUB Whilst not wishing to be bound by theory it is speculated that the presentation of the peptide by cells modified to incorporate M45 may be more analogous to the presentation of the corresponding peptide sequence expressed by the naturally 5 occurring antigen. Not Natural Sample M44 M45 modified MiIII cells 71 10 10 0 8 67 8 10 0 5 65 10 8 0 12 Antibody positive 55 8 8 0 10 serums 61 8 5 0 8 942-433813-3 0 5 0 12 68 0 0 0 10 False- PAC302 5 0 0 0 positive antibody PAC332 12 0 0 0 negative PAC340 serums 5 0 0 0 Table 24. 10 Modification of cells and multi-cellular structures with biotin-lipid constructs The modification of red blood cells (RBCs) and murine embryos 15 by the construct designated Biotin-CMG(2)-Ad-DOPE was demonstrated using Avidin-Alexafluor (Avidin AF). Materials 20 A stock solution of the construct designated Biotin-CMG(2)-Ad DOPE (100 pL) was prepared in water at a concentration of 10 mg/mL. A stock solution of Avidin-Alexafluor (Avidin-AF) was prepared in sterile phosphate buffered saline (PBS) at a concentration of 2 mg/mL. A stock solution of biotinylated 85 K01.075AUB gangliocide (BioG) was prepared in sterile PBS at a concentration of 5 mg/mL. Red Blood Cells 5 Dilution series of the construct designated Biotin-CMG(2)-Ad DOPE and BioG (positive control) were prepared at concentrations of 0.001, 0.1, 0.1 and 1 mg/mL with Celpresol

T

. O group red blood cells (RBCs) were modified by incubation of 10 15 pL of packed RBCs and 5 pL of a dilution of the construct designated biotin-CMG(2)-Ad-DOPE or BioG. Incubations were performed in a plastic ependorf tube of nominal volume 1.5 mL for 2 hours at 370C in a water bath. 0 group RBCs were incubated with a solution of BioG at a concentration of 0.33 15 mg/mL as a positive control. Incubated RBCs were washed 3 times with PBS in a mini centrifuge. Fluorescent labeling of the washed, modified RBCs was performed by adding 10 pL of a solution of Avidin-AF at a 20 concentration of 0.1 mg/mL. The RBCs were then incubated in the dark for 1 hour at 37 C in a water bath and washed 3 times with PBS. 85% solution of the washed, fluorescent labeled RBCs were 25 viewed on a slide with cover slip under a fluorescent microscope at 488 nm and photographic exposure of 1.903 (Figure 6). The intensity of the fluorescent signal was recorded using a scoring system of 0 (no fluorescence observed) to 4 (maximum fluorescence observed). 30 An aliquot of the washed, modified RBCs obtained from incubation with the construct designated biotin-CMG(2)-Ad-DOPE at a concentration of 1 mg/mL was retained and stored at 14 C 86 KO1.075AUB for 14 days. Retention of the construct by the RBCs was assessed by incubation with Avidin-AF as before. The assessment of the fluorescence is recorded in Table 25. 5 Dilution 1 0.1 0.01 0.001 0 (mg/mi) Biotin-CMG(2)-Ad-DOPE 4 2 1 0 0 Day 0 Biotin-CMG(2)-Ad-DOPE 4 Day 14 BioG 3 2 0 0 0 Day 0 Table 25. Murine Embryos 10 Murine embryos (morula/early blastocyst stage, 3.5 day) were incubated in 50 microlitre microdrops in blastassist" culture media. Embryos were incubated with construct designated biotin-CMG(2)-Ad-DOPE at a concentration of 0.1, 1 or 2 mg/mL 15 or BioG at a concentration of 0.5 mg/mL (positive control). The zona pellucida of the embryos was removed by treatment with 0.5% pronase (4 minute incubation) and washing 3 times in embryo handling media prior to introduction into the 20 microdrops. Each microdrop was equilibrated 5% C02 at 37 C overnight prior to introduction of the embryos. Microdrops containing embryos and the construct designated biotin-CMG(2)-Ad-DOPE were incubated for 2 hours in 5% C02 at 25 37 C. Microdrops containing embryos and BioG were incubated for 40 minutes in 5% C02 at 37 C. 87 K01.075AUB Following incubation each group of embryos was washed 3 times in handling media and transferred to 50 microlitre microdrops containing 2 mg/mL Avidin-AF microdrops for fluorescent labelling. The microdrops containing transferred embryos were 5 incubated at 37 C for 30 minutes in the dark. Each group of embryos was then washed 3 times in handling media and mounted on a glass microscope slide for viewing. Embryos were viewed under a fluorescent microscope at 488 nm. 10 The intensity of the fluorescence was recorded using a scoring system of 0 (no fluorescence) to 4 (maximum fluorescence). Biotin- Biotin- Biotin CMG(2) -Ad- CMG(2) -Ad- CMG(2) -Ad- BioG Media alone DOPE DOPE DOPE 0.5 mg/mL 0.1 mg/mL 1 mg/mL 2 mg/mL 2 1 1 3 1 n=21 n=19 n=19 n=19 n=19 Table 26. 15 Immobilization of spermatozoa and cells The immobilization of spermatozoa and red blood cells (RBCs) was demonstrated by use of the construct designated Biotin 20 CMG(2)-Ad-DOPE and streptavidin beads (Dynabeads® M-280). 88 K01.075AUB Materials A stock solution of the construct designated Biotin-CMG(2)-Ad DOPE (100 pL) was prepared in water at a concentration of 10 5 mg/mL and diluted in culture media (Medicult 10310060A) to provide a test dilution at 0.1 mg/mL. The spermatozoa in fresh semen (less than one day old) were assessed for motility (80%, grade 3 (fast, forward 10 progression) by 10-fold dilution in culture medium (Medicult 10310060A; pre-incubated for a minimum of 2 hours at 37 0C in a 5% C02 atmosphere). Spermatozoa counts (91.5 x 10 6 /mL) were performed by 10-fold dilution in deionised water. 15 Spermatozoa were washed and isolated by layering 1.1 mL of fresh semen over a gradient of SpermGrad 125 (Vitrolife 10099; 2 mL of 40% solution over 2 mL of 80% solution in a 15 mL round bottom tube) and centrifuging at 500 x g for 20 min. 20 The bottom layer of the gradient (c. 0.7 mL was transferred to 4 mL round bottom tubes and c. 2 mL flushing (handling) media (Medicult 10840125A) added. The tube was centrifuged at 300 x g for 10 min and the spermatozoa washed two more times (mixing by tube inversion). 25 Samples of washed spermatozoa were incubated overnight at 37 0C in a 5% C02 atmosphere. Spermatozoa counts (c. 25 x 10 6 /mL) were performed post overnight incubation by 10-fold dilution in deionised water. 30 89 K01.075AUB Spermatozoa A volume of 100 pl of the test dilution of the construct designated Biotin-CMG(2)-Ad-DOPE (I) was added to each of four 5 0.6 mL ependorf tubes (A-D) and 100 pL of culture media added to one 0.6 mL ependorf tube (E). Open tubes were incubated at 37 OC in a 5% C02 atmosphere prior to addition of c. 70 pL spermatozoa (c. 25 x 10 6 /mL) to 10 each tube and incubation for 120 min (A), 60 min (B), 30 min (C), 10 min (B) and 120 min (E). Following incubation a couple of drops of flushing media were added and the tubes centrifuged at 300 x g for 5 min. The 15 spermatozoa were washed two more times with flushing media and before being re-suspended in culture media to a final volume of 100 pL. Streptavidin beads at a concentration of c. 6.25 x 10 6 /100pL 20 were diluted 35 times in BSA plus flushing media to provide a ratio of 0.1 beads/spermatozoa when mixed in equal volume with a diluted suspension of the modified spermatozoa. A volume of 5 pL of a diluted suspension of the modified 25 spermatozoa was mixed on a slide with 5 pL of a diluted suspension of streptavidin beads and covered with a coverslip. The mixture was observed under a microscope at 400x magnification. Figure 9 provide a photomicrograph of the 30 mixture provided following incubation with 0.1 mg/mL of the construct designated Biotin-CMG(2)-Ad-DOPE for 60 min (B). 90 K01.075AUB The assessment of the attachment of streptavidin beads to modified spermatozoa is recorded in Table 27. Number of beads attached to Biotin-CMG(2) -Ad-DOPE Incubation spermatozoa treatment time (mi) Immediate 30 min A 120 10 -12 10-15 (with cross-linking) B 460 2, 4, 8 8 C 30 1-3 3 D 10 1 1 E (Control) 120 0 0 5 Table 27. Spermatozoa were observed to retain motile capacity despite attachment of beads (no acrosome reaction was evident) with a preference of attachment to motile spermatozoa. 10 Red Blood Cells A dilution of the construct designated Biotin-CMG(2)-Ad-DOPE was prepared at a concentration of 1 mg/mL with Celpresol. 15 A volume of 60 pL washed A group red blood cells (RBCs) was modified by incubation with 20 pL of the dilution of the construct at 37 0C for 2 hours. The modified RBCs were washed twice in PBS and once in 20 Celpresolm as described above. A 2% cell suspension of washed cells (modified or control) was prepared in CelpresolM and cell concentration (150 x 10 6 /mL) determined using a haemocytometer. Similarly the concentration of a suspension streptavidin beads (134 x 10 6 /mL) was determined. 25 91 KO1.075AUB A volume of 50 pL of the suspension of streptavidin beads was added to the wells of a 96-well plat with a Neodymium (Rare Earth) Super magnet (Magnets NZ Limited) affixed to the base. A volume of 50 pL of a suspension of RBCs was added to provide 5 a bead to RBC ratio of c. 1:1 and incubated at room temperature for 1 hour to allow RBCs to settle. The wells were washed 3 x with PBS, aspirating the washing solution with a pipette. The washed wells were observed under 10 a microscope and the RBCs determined to be retained (Figure 10). Separation of populations of cells 15 A 0.5 mg/ml solution of the construct designated Biotin CMG(2)-Ad-DOPE was prepared in Celpresol T m and a volume of 10 pl used to modify 30 pl packed cell volume of group 0 RBCs in a 1 ml eppendorf tube to provide a first population of cells. Unmodified group A RBCs were used as a second population of 20 cells. Both populations of cells were incubated for 37 0C for 2 hrs in a water bath and then washed 2x in PBS and lx in Celpresol using an Immufuge II (low, 1 min). The concentration of cells in each suspension was made up to 2% by adding 1.5 mL. 25 The suspensions of RBCs were mixed with avidinylated magnetic Dynabeads at an approximate ratio of RBC:bead of 1 and incubated for 10 min at room temperature on a gyrator. Samples of the first and second populations of RBCs were then 30 mixed in equal volumes (35 pl each) in an ependorf tube for two minutes. 92 KO1.075AUB The contents of the ependorf were transferred to the well of a 96-well plate and a magnet was applied to the underside of the well for 1 minute. The supernatant was carefully removed with the magnet applied and without disruption of the beads. The 5 blood grouping of the cells of the supernatants were then assessed by applying 30 pl of supernatant and 30 pl anti-A antibody to a DynamedTM gel card. Cards were spun for 10 min in a centrifuge. Retention of the 0 group RBCs by the magnet was demonstrated by the absence of a pellet of group 0 cells. 10 Modification of cell layers with biotin-lipid constructs The modification of monolayers of the cell line RL95-2 (established from a human endometrial adenocarcinoma (ATCC HTB 15 CRL 1671)) in serum-free and serum-containing media was evaluated. D-MEM/F12 (Gibco 11320-033, Invitrogen NZ) containing 1% penicillin/streptomycin (Gibco 15140-122, Invitrogen NZ) was 20 used as a serum-free medium. D-MEM/F12 10% FBS (Gibco 10091 130, Invitrogen NZ) containing 1% penicillin/streptomycin and 5 pg/mL insulin (Gibco 12585-014, Invitrogen NZ) was used as a serum-containing medium. 25 A suspension of the cell line RL95-2 was diluted in pre-warmed serum-containing media to the required concentration e.g. 4 x 105 cells/mL. A 25 pL volume of the suspension was used to seed the required wells in a Terasaki tray so that each treatment was performed in duplicate. The plates were 30 incubated overnight in a 5% C02, 37 0C incubator until the monolayer was approximately 60% confluent. 93 K01.075AUB Dilutions of Biotin-CMG(2)-Ad-DOPE were prepared and 12 pl volumes added to wells containing washed cell layers to provide final concentrations of 20, 100 or 500 pg/mL. Trays were incubated at 37 0C, 5% C02 for 120 min. 5 The cells were then washed and a 12 pl volume of a 0.1 mg/mL solution of Avidin Alexa Fluor@ 488 added. The cells were then incubated at room temperature for a further 30 minutes in the dark. 10 The monolayers were finally washed 3 times with PBS, the trays inverted and photographed using an Olympus BX51 fluorescent microscope at 200x magnification, exposure time 475 ms (Figure 12). 15 When the construct is inserted in serum-free media at 20, 100 and 500 pg/mL, a homogenous intense fluorescent signal is observed in the cell membrane that intensifies with increasing concentration of the construct (Figure 12A, C and E). When 20 the construct is inserted in serum-containing media, weakened fluorescence is observed at the same concentrations (Figure 12B, D and F). These results imply that optimal insertion of construct into the cell membranes requires serum-free media. 25 When the construct is inserted in serum-free media at 20, 100 and 500 pg/mL, an homogenous intense fluorescent signal is observed in the cell membrane that intensifies with increasing concentration of construct. The intensity of the fluorescence also increased with increasing insertion time (Table 28). 30 These results imply that optimal insertion of construct into cell membranes occurs with increased concentration of construct and/or increased insertion time. 94 K01.075AUB Mean Fluorescence* IInsertion time Concentration of biotin-CMG(2)-Ad-DOPE 10 30 60 120 (pg/mL) 20 1+ 1 -2+ 2-3+ 3-4+ 100 2+ 3 3+ 4+ 500 3+ 3-4+ na na Media alone 0 0 0 0 Table. 28. Optimal insertion of construct into cell membranes occurs with increased concentration of construct and/or increased insertion time. 5 'na' denotes "not assessed". When avidin Alexa Fluor@ 488 was added to construct modified RL95-2 cells and incubated at 37 0C for 4 and 24 hr the fluorescence gradually shifted from the cell surface to the 10 cell interior (Figure 13). No internalization was observed when cells were incubated at 40C. Fluorescence was detected in construct modified RL95-2 cells 15 24 hr post-insertion when cultured in serum-free media, albeit with reduced fluorescence from T=O (Table 29). However, when cells were cultured in serum-containing media a fluorescence score of 1+ was detected at the highest concentration of construct (500 mg/mL), but not at lower. These results imply 20 that the construct is optimally retained in cell membranes 24 hr post-insertion when cultured in serum-free media, but not in serum-containing media. 95 K01.075AUB Mean Fluorescence* Concentration of biotin-CMG(2)-Ad-DOPE T=0 T=24 hr (pg/mL) - Serum-free Serum-containing 20 3-4+ 1-2+ 0 100 4+ 2-3+ 0 500 4+ 1+ Media alone 0 0 10 Table 29. Retention of construct 24 hours post-insertion. The construct was detected in cells cultured for 24 hr post-insertion in serum-free 5 media, but was only detected at the highest concentration in serum containing media. 'na' denotes "not assessed". Modification of antigen presentation 10 The amount of construct used in the manufacture of quality controls cells as described in the specification accompanying international application no. PCT/NZ2005/000052 (publication no. WO 2005/090368) is a determinant of the cost of 15 manufacture. It was anticipated that presentation of antigen at a distance from the immediate milieu of the cell surface may promote recognition by cross-reactive antibody and subsequent 20 agglutination. The estimated distances from the cell surface for an antigen (F) of a functional lipid construct (F-S-L) where the spacer (S) includes the structural motif of the present invention are 7.2 nm (CMG(2)) and 11.5 nm (CMG(4)). These distances compare with 1.9 nm for the antigen of a 25 construct (F-S-L) where the spacer is one described in the specification accompanying international application no. 96 KO1.075AUB PCT/NZ2005/000052 (publication no. WO 2005/090368), i.e. Atr sp-Ad-DOPE (I). To test the hypothesis solutions of four different 5 trisaccharide (At)-lipid constructs were prepared as 50 pM, 10 pM and 5 pM solutions in Celpresol. Modified red blood cells were then prepared by contacting 0.6 mls (pcv) of washed RBCs with 0.6 mls of the relevant solution. The mixtures were incubated at 37 0C for 2 hours and then washed 3 times to 10 provide a modified cell suspension. The modified cell suspensions were prepared in 0.8% in Celpresol LISSTM suitable for BioVue serology cards. A volume of 0.05 mls of CSL monoclonal anti-A (026129801) followed by 15 0.05 mls of modified cell suspension was applied to each card. Reactions were recorded following centrifugation in a BioVue cassette centrifuge (Table 29). A comparison of the recorded reaction for molar equivalents of 20 the Atr-lipid constructs demonstrates that less CMG(2) or CMG(4) construct is required to provide an equivalent serological result. There was no significant gain with CMG(2) compared with CMG(4). There was a minor, but not significant change for MCMG (2). 25 Although the invention has been described by way of examples it should be appreciated that variations and modifications may be made to the claimed methods without departing from the scope of the invention. As noted it will be understood that 30 for a non-specific interaction, such as the interaction between the diacyl- or dialkyl- glycerolipid portion of the functional-lipid constructs and a membrane, structural and 97 KO1.075AUB stereo-isomers of naturally occurring lipids can be functionally equivalent. Where known equivalents exist to specific features, such 5 equivalents are incorporated as if specifically referred to in this specification. For example, it is contemplated that diacylglycerol 2-phosphate could be substituted for phosphatidate (diacylglycerol 3-phosphate) and that the absolute configuration of phosphatidate could be either R or 10 S. [followed by page 991 98 C14 0 1 1 1 C') I I Ln i I m' I I CN r-I C') I I Lncy) I Ic Ln ')m Irn 0 OD CD C' ) C ) CC a C o ~ co m Io'-4- o n c) L V D 0 C D c (N CD c D C 0 0 0 CN mN 00 DI0 C ) C) (N C w 4 m -i- i - iH 00 4 (N Q (N ( Q ( w C 14~ co cC) N CD CD N D N D C 0 N. (D N (N Q ( (N C) (N (~ D CjN (D 14 04 N (Dco N (N CD (N (N C) (N (N C) ( (N CD (N) (\ N CD (N (N C) (N (N CD r, E 0 0 00 00 00 It 0 .'4 r4 qA0 -i- -H (N U). C.H C.! 4-)N d4 JQj V4 0 U) I ) K01.075AUB REFERENCES Blume et al (1993) Specific targeting with poly(thylene glycol)-modified liposomes coupling of homing devices to the ends of the polymeric chains 5 combines effective target binding with long circulation times. Biochimica et Biophysica Acto, 1149: 180-184 Chung et al (2004) Casual Cell Surface Remodelling Using Biocompatible Lipid-poly(ethylene glycol) (n): Development of Stealth Cells and 10 Monitoring of Cell Membrane Behaviour in Serum-supplemented Conditions. J Biomed. Mater. Res, Part A, 70A/2:179-185 Haselgrnbler et al (1995) Sythesis and Applications of a New Poly(ethylene glycol) Derivative for the Crosslinking of Amines with Thiols. 15 Bioconjugate Chem, 6: 242-248 Hashimoto et al (1986) Iodacetylated and biotinylated liposomes: Effect of spacer length on sulfhydryl ligand binding and avidin precipitability. Biochim Biophys Acta, 856: 556-565. 20 Holmberg et al (2005) The Biotin-streptavidin interaction can be reversibly broken using water at elevated temperatures, Electrophoresis, 26 (3), 501 to 510. 25 Ishida et al (2001) Liposomes Bearing Polytheneglycol-Coupled Transferrin with Intracellular Targeting Property to the Solid Tumors In Vivo. Pharmaceutical Research, 18 (7): 1042-1048 Kato et al (2004) Rapid Proprotein anchoring into the membranes of 30 mammalian cells using olial chain and polyethylene glycol derivatives. Kinsky et al (1983) An alternative procedure for the preparation of immunogenic liposomal model membranes. J Immunol Method, 65: 295-306 35 Kung and Redemann (1986) Synthesis of carboxyacyl derivatives of phosphatidylethanolamine and use as an efficient method for conjugation of protein to liposomes. Biochim Biophys Acta, 862: 435-439 100 K01.075AUB Legler et al (2004) Differential insertion of GPI-anchored GFPs into lipid rafts of live cells The FASEB Journal, Online article 10.1096/fj.03-1338fje 5 Mannino et al (1993) Liposomes as adjuvants for peptides: Preparation and use of immunogenic peptide-phospholipid complexes. Liposome Technology: 167-184 Martin et al (1990) Liposomes a Practical Approach, 163-182 10 Martin and Papahadjopoulos (1982) Irreversible coupling of immunoglobulin fragments to preformed vesicles. An improved method for liposome targeting. J Biol Chem, 257: 286-288 15 Massaguer et al (2001) Synthesis of RGD Containing Peptides. Comparative Study of their Incorporation to the Surface of 5-Fluoruridine Loaded Liposomes. Journal of Liposome Research, 11(I):103-113 McHugh et al (1995) Construction, purification, and functional 20 incorporation on tumor cells of glycoplipid-anchored human B7-1 (CD80) Proc. Natl. Acad. Sci. USA, 92: 8059-8063 Medof et al (1996) Cell-surface engineering with GPI-anchored proteins The FASEB Journal, 10: 574-586 25 Metzner et al (2008) Association of glycosylphosphatidylinositol-anchored protein with retroviral particles The FASEB Journal, Online article fj.08-108217 30 Morandat et al (2002) Cholesterol-dependent insertion of glycosylphosphatidylinositol-anchored enzyme Biochimica et Biophysica Acta, 1564: 473-478 New (1992) Liposomes: A Practical Approach 35 Premkumar et al (2001) Properties of Exogenously Added GPI-Anchored Proteins Following Their Incorporation Into Cells Journal of Cellular Biochemistry, 82: 234-245 101 K01.075AUB Reid and Lomas-Francis (2004) The Blood Group Antigen facts book. Elsevier Academic Press, Amsterdam, 2nd ed. Ronzon et al (2004) Insertion of Glycosylphosphatidylinositol-Anchored 5 Enzyme into Liposomes The Journal of Membrane Biology, 197: 169-177 Shek and Heath (1983) Immune response mediated by liposome-associated protein antigens III Immunogenicity of bovine serum albumin covelantly coupled to vesicle surface. Immunology, 50: 101-106 10 Skountzou et al (2007) Incorporation of Glycosylphosphatidylinositol Anchored Granulocyte-Macrophage Colony-Stimulating Factor or CD40 Ligand Enhances Immunogenecity of Chimeric Simian Immunodeficiency Virus-Like Particles Journal of Virology, 81, 3: 1083-1094 15 Winger et al (1996) Lipopeptide conjugates: biomolecular building blocks for receptor activating membrane-mimetic structures. Biomaterials, 17: 437-441 20 [followed by page 103 - Claims] 102

Claims (1)

11-octadecenoic acid, cis-11-octadecenoic acid, cis 11-eicosenoic acid or cis-13-docsenoic acid. 20 8) The method of claim 7 where F is a functional moiety selected from the group consisting of: carbohydrate, peptide, conjugator or fluorophore. 105 KO1.075AUB 9) The method of claim 8 where the structure of the functional lipid construct is: (CH1 2 ) OM OH 0 0 N h NN 0C \\N O ~NH OHN o O of of 5 R- R 2 where F is a carbohydrate, x is the integer 2, 3 or 4, y is the integer 1, 2 or 3, and R 3 is 0 of a substituted hydroxyl of the carbohydrate. 10 106 KO1.075AUB 10) The method of claim 8 where the structure of the functional lipid construct is: O R3 N (CH 2 ) w OM 0 HN O O NH h ON 0 NHN NHNH \\ 0 OM O NH O OM (CH2)v NH O OM R, R 2 5 where F is a peptide, w is the integer 1 or 2, and R 3 is S of a substituted sulfhydryl of a Cys residue of the peptide. 10 107 K01.075AUB 11) The method of claim 8 where the structure of the functional lipid construct is: NH H N OM O H 00 NH h 0 N H N 0N O (CH ), aM P, R , 5 where F is the conjugator biotin and k is the integer 2, 3 or 4. 108 KO1.075AUB 12) The method of claim 8 where the structure of the functional lipid construct is: ,NH-R 3 0 /-(CH2)z j-NH (CH 2 )x o >jN OM 0 HN O O 0 00 NH NH h O-N Op-O NH N-0-N NHNH 00 0 NH 'O OM \(CH2) NH P S O OM O=< O=< R, R 2 5 where F is the fluorophore of fluorescein (or one of its derivatives), x is the integer 2, 3 or 4, z is the integer 3, 4 or 5, and R 3 is C of the thiocyanate substituent of the isothiocyanate derivative of fluorescein (or one of its derivatives). 10 13) The method of claim 10 where the terminal sequence of the peptide is selected from the group consisting of: GlyLysLysLysLysSerCys; AlaAlaAlaAlaCys; GlySerGlySerGlyCys; CysSerLysLysLysLysGly; CysAlaAlaAlaAla; and CysGlySerGlySerGly, 109 KO1.075AUB and the Cys residue is a terminal Cys residue of the peptide at the carboxy-terminus of the peptide. 14) The method of claim 13 where F is a peptide comprising 5 an epitope of antigens selected from the group consisting of: Glycophorin A, Glycophorin B, or mutations thereof (including the MNS blood group system). 10 15) The method of claim 14 where F is a peptide selected from the List of Peptides where (Xaa)z is selected from the group consisting of: GlyLysLysLysLysSerCys; AlaAlaAlaAlaCys; GlySerGlySerGlyCys; CysSerLysLysLysLysGly; CysAlaAlaAlaAla; and CysGlySerGlySerGly. 15 16) The method of claim 15 where F is a peptide selected from the group consisting of: CysThrTyrProAlaHisThrAlaAsnGlu; GlnThrAsnAspLysHisLysArgAspThrTyrAlaAlaAlaAlaAlaCys; GlnThrAsnAspLysHisLysArgAspThrTyrGlySerGlySerGlyCys; GlnThrAsnAspMetHisLysArgAspThrTyrGlySerGlySerGlyCys; SerSerGlnThrAsnAspLysHisLysArgAspThrTyrCys; ThrTyrProAlaHisThrAlaAsnGluValCys; ThrTyrProAlaHisThrAlaAsnGluCys; 110 K01.075AUB ProAlaHisThrAlaAsnGluValCys; and SerGlnThrAsnAspLysHisLysArgAspCys. 17) The method of any one of claims 1 to 16 where L is 1,2-0-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) or 1,2-0-distearyl-sn-glycero-3 5 phosphatidylethanolamine (DSPE). 18) The method of any one of claims 1 to 17 where the cell is a human cell. 10 19) The method of claim 18 where the cell is a human red blood cell. 20) A cell produced by the method of any one of claims 1 to 19. 15 111