AU4093400A - Model membrane systems - Google Patents

Description

WO 00/64471 PCT/AUOO/00397 MODEL MEMBRANE SYSTEMS FIELD OF THE INVENTION 5 The present invention relates generally to anchoring molecules to model and biological membrane systems, and to the use of anchored molecules in assays of inter molecular interactions and to modify biological responses. In one form, the present invention provides the basis of a novel method for the screening of drugs and other agents which affect intermolecular interactions. In another form, the invention provides a means of modifying 10 the properties of biological and/or synthetic membranes and liposomes for the purpose of altering immunity when used as vaccines, or for the targeting of drugs and other agents to specific cells or tissues when administered in vivo for either therapeutic purposes or for modifying physiological responses or function. 15 BACKGROUND OF THE INVENTION Bibliographic details of the publications numerically referred to in this specification are collected at the end of the description. 20 In biological systems such as cells, bacteria or viruses, surface biomolecules or receptors often exist as molecular structures consisting of two or more molecular components called subunits; these subunits may be identical, or molecularly distinct. The binding of natural ligand molecule(s) to receptor subunits may induce non-covalent aggregation or oligomerization of these receptor components. The oligomerization event is often an 25 essential part of the mechanism by which the receptor can transduce transmembrane signals for triggering the induction of biological responses by the ligand molecule(s). In addition, the ability of certain receptors, or components thereof, to aggregate spontaneously may affect their ability to interact with ligands. Ligand molecules may be growth factors, cytokines, hormones, proteins, glycoproteins, polysaccharides, or any surface exposed or sub-cellular 30 component of a cell, viral or subviral particle, or other infectious agent, which can bind to the receptor.

WO 00/64471 PCT/AUOO/00397 -2 By virtue of their ability to undergo multimeric interactions, oligomerized receptors often have the potential to interact stably with ligands of low binding affinity. However, for many receptors the affinity of self-association or interaction with ligands is not high enough to allow detection using conventional binding techniques, which often require covalent 5 labelling, solubilization with detergents, or immobilization of the receptor or ligand onto the solid sensing surface of an optical biosensor. These methods are suited to the study of relatively high affinity interactions and they generally rely on the ability of the molecules to interact in solution or to maintain stable interaction after cell disruption. Since the effective receptor/ligand concentration in solution is reduced compared to that on the two-dimensional 10 surface of a cell (where molecules can oligomerize or cluster and interact stably with other molecules through multimeric interactions) these methods are limited in their ability to detect interactions of low affinity. Atomic force microscopy (also known as scanning probe microscopy) allows 15 three-dimensional imaging and measurement of structures ranging in size from atomic dimensions to microns, and is revolutionary in its ability to resolve structures never seen before (1). The development of optical biosensors has permitted the monitoring of the interaction between macromolecules in real time (2). To date, both of these techniques generally have been used with the receptor or the ligand molecule covalently attached to or 20 immobilized onto a solid surface (1-4). Recently a technique has been described in which the linkage of a recombinant hexa-histidine-tagged protein with nitrilotriacetic acid (NTA) is used to reversibly immobilize a hexa-histidine-tagged protein onto the solid sensing surface of a BlAcore 25 surface plasmon resonance biosensor (5). The formation of a hybrid octadecanethiol/phospholipid membrane on the BIAcore sensing surface also has been described (6), enabling analysis of the binding of streptavidin to biotinylated phosphatidylethanolamine in the bilayer. In addition, the immobilization of histidine-tagged biomolecules to bilayer membranes via chelator lipids like NTA-dioctadecylamine has been 30 demonstrated by epifluorescence microscopy and film balance techniques (7-8). These prior art techniques do not permit an analysis of an interaction of receptor domains under WO 00/64471 PCT/AUOO/00397 -3 conditions that mimic a cellular or sub-cellular membrane that allows lateral mobility of the receptors, and that allows for the possibility of studying receptor oligomerization and multimeric interactions with ligands. Furthermore, these prior art techniques do not permit screening for drugs or other agents that may influence such molecular interactions. 5 There is a need, therefore, to develop new techniques to anchor molecules to membranous materials for use, for example, in devices for drug screening, in vaccine preparations, as therapeutic agents, as agents for modifying immunological responses and for targeting drug delivery systems.

WO 00/64471 PCT/AUOO/00397 -4 SUMMARY OF THE INVENTION Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of 5 a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers. One aspect, the present invention provides a method of anchoring receptor domains onto a planar supported membrane comprising amphiphilic molecules arranged in a layer, wherein 10 a proportion of the amphiphilic molecules have been modified by a covalent attachment of a metal chelating group such that some of the metal chelating groups are oriented toward the outside surface of said membrane; which method comprises the step of interacting a receptor domain which is covalently attached to a polypeptide tag with said membrane for a time and under conditions sufficient for said polypeptide tag to attach to said membrane via the 15 outwardly facing metal chelating residues of said membrane, such that the receptor domains are capable of lateral movement, dimerization or oligomerization, and interaction with a ligand molecule. Another aspect, the present invention provides a method of anchoring receptor domains onto 20 a lipid membrane such that the receptor domains are capable of lateral movement, said method comprising of: (i) forming a membrane on an appropriate planar support by either: 25 (a) contacting and incubating the support for a period of time with a solution in an organic solvent of amphiphilic molecules wherein a proportion of the molecules have been modified by covalent attachment of a metal chelating group, then forming a membrane by the addition of water or an aqueous buffer; or 30 (b) forming a suspension of micelles (e.g. liposomes) from amphiphilic WO 00/64471 PCT/AUOO/00397 -5 molecules wherein a proportion of the amphiphilic molecules have been modified by a covalent attachment of a metal chelating group, then interacting said micelle (e.g. liposome) suspension with an appropriate planar support for a time and under conditions sufficient to allow a membrane layer 5 to form in which some metal chelating residues attached to the amphiphilic molecules of said micelle (e.g. liposome) suspension are oriented toward the outside surface of said membrane; and (ii) interacting the receptor domain(s) to be anchored which is covalently attached to a 10 polypeptide tag with said membrane for a time and under conditions sufficient for said polypeptide tag to attach via the metal chelating linkage to the outwardly-facing metal chelating residues of said membrane. Another aspect the present invention contemplates a method of anchoring receptor domains 15 onto a lipid membrane such that said receptor domains are capable of lateral movement to facilitate interactions with biomolecules in the membrane, said method comprising of: (i) forming a membrane on an appropriate planar support by either: 20 (a) contacting and incubating the support for a period of time with a solution in an organic solvent of amphiphilic molecules wherein a proportion of the molecules have been modified by covalent attachment of a metal chelating group, then forming a membrane by the addition of water or an aqueous buffer; or 25 (b) forming a suspension of micelles (e.g. liposomes) from a first phospholipid and a second phospholipid wherein the second phospholipid has been modified by a covalent attachment of a nitrilotriacetic acid (NTA), and then interacting said micelle (e.g. liposome) suspension with an appropriate planar 30 support for a time and under conditions sufficient to allow a phospholipid membrane layer to form in which some NTA residues attached to the second WO 00/64471 PCT/AUOO/00397 -6 phospholipid of said micelle (e.g. liposome) suspension are oriented toward the outside surface of said membrane; and (ii) interacting the receptor domain(s) to be anchored which is covalently attached to a 5 polypeptide tag with said membrane for a time and under conditions sufficient for said polypeptide tag to attach via the NTA-metal chelating linkage to the outwardly-facing NTA residues of said membrane. Yet another aspect of the present invention provides a method of assaying interactions 10 between a receptor and a ligand, comprising the steps of: (A) anchoring a receptor to a lipid membrane by: (i) forming a membrane on an appropriate planar support by either: 15 (a) contacting and incubating the support for a period of time with a solution in an organic solvent of amphiphilic molecules wherein a proportion of the molecules have been modified by covalent attachment of a metal chelating group, then forming a membrane by 20 the addition of water or an aqueous buffer; or (b) forming a suspension of micelles (e.g. liposomes) from amphiphilic molecules wherein a proportion of the amphiphilic molecules have been modified by a covalent attachment of a metal chelating group, 25 and then interacting said micelle (e.g. liposome) suspension with an appropriate planar support for a time and under conditions sufficient to allow a membrane layer to form in which some metal chelating residues attached to the amphiphilic molecules of said micelle (e.g. liposome) suspension are oriented toward the outside surface of said 30 membrane; and WO 00/64471 PCT/AUOO/00397 -7 (ii) interacting the receptor domain(s) to be anchored which is covalently attached to a polypeptide tag with said membrane for a time and under conditions sufficient for said polypeptide tag to attach via the metal chelating linkage to the outwardly-facing metal chelating residues of said membrane; 5 (B) allowing said receptor molecules to interact and/or oligomerize on the membrane; and (C) contacting said anchored receptor with an effective concentration of ligand for a time 10 and under conditions sufficient for binding to occur and detecting said binding. Still yet another aspect of the present invention contemplates a method of assaying interactions between a receptor and a ligand comprising the steps of: 15 (A) anchoring a receptor to a lipid layer by: (i) forming a membrane on an appropriate planar support by either: (a) contacting and incubating the support for a period of time with a 20 solution in an organic solvent of amphiphilic molecules wherein a proportion of the molecules have been modified by covalent attachment of a metal chelating group, then forming a membrane by the addition of water or an aqueous buffer; or 25 (b) forming a suspension of vesicles from a first phospholipid and a second phospholipid wherein said second phospholipid has been modified by covalent attachment of a metal chelating group such as nitrilotriacetic acid (NTA), and then interacting said micelle (e.g. liposome) suspension with an appropriate planar support such as glass 30 or mica for a time and under conditions sufficient to allow a hydrocarbon phospholipid membrane layer to form on the support in WO 00/64471 PCT/AUOO/00397 which some of the NTA residues attached to the second phospholipid of said micelle (e.g. liposome) suspension are oriented toward the outside surface of said membrane; and 5 (ii) interacting the receptor domain to be anchored which is covalently attached to a polypeptide tag with said membrane for a time and under conditions sufficient for said polypeptide tag to attach via the NTA-chelating linkage to the outwardly-facing NTA residues of said membrane; 10 (B) allowing said receptor molecules to interact and/or oligomerize on the membrane; and (C) contacting said anchored receptor with an effective concentration of ligand for a time and under conditions sufficient for binding to occur and detecting said binding. 15 Even yet another aspect of the present invention there is provided a method of engrafting molecules onto liposomes said method comprising: (i) preparing a suspension of liposomes with chelator lipid incorporated, and with or 20 without an encapsulated drug or agent; (ii) incubating the liposomes with a recombinant protein or target molecule bearing an appropriate metal affinity tag; and 25 (iii) if necessary, removing excess protein by washing, filtering or other washing means and suspending them in an appropriate solution. Another aspect of the present invention, there is provided a method of "pasting" recombinant molecules directly onto biological membranes said method comprising: 30 (i) preparing a suspension of chelator lipid or liposomes containing the chelator lipid; WO 00/64471 PCT/AUOO/00397 -9 (ii) incubating a suspension of cells or biological membranous structures with a suspension of the chelator lipid; (iii) washing away excess or unincorporated lipid; 5 (iv) incubating the membranous structures with a solution of recombinant protein or target molecule possessing an appropriate metal affinity tag; and (v) washing away excess or unbound soluble protein, and suspending the structures in a 10 solution suitable for administration in vivo. Still yet another aspect of the present invention contemplates a method for altering the immunogenicity of a target cell or membranous component thereof, said method comprising anchoring a molecule to the membrane of said target cell by: 15 (i) preparing a suspension of chelator lipid or liposomes containing the chelator lipid; (ii) incubating a suspension of cells or membranous structures with a 20 suspension of the chelator lipid; (iii) washing away excess or unincorporated lipid; (iv) incubating the membranous structures with a solution of said 25 molecule to be anchored; and (v) washing away excess or unbound soluble molecule, and suspending the structures in a solution suitable for administration in vivo. 30 Even yet another aspect of the present invention contemplates a method of targeting a liposome or a membranous structure to a particular cell or tissue, said method comprising WO 00/64471 PCT/AUOO/00397 - 10 anchoring or engrafting a molecule having a binding partner on the particular cell or tissue to be targeted by: (i) preparing a suspension of chelator lipid or liposomes containing the 5 chelator lipid; or (ii) incubating a suspension of cells or membranous structures with a suspension of the chelator lipid to incorporate the lipid; 10 (iii) washing away excess or unincorporated lipid; (iv) incubating the liposomes or membranous structures with a solution of molecules to be anchored; and 15 (v) washing away excess or unbound soluble molecule, and suspending the liposomes or structures in a solution suitable for administration in vivo. Another aspect of the present invention contemplates a method of treatment, said method 20 comprising administering to a subject an effective amount of a liposome preparation or membranous material comprising an active material and optionally an anchored or engrafted molecule having a binding partner or target tissue. A further aspect of the present invention provides a vaccine composition comprising cells, 25 liposomes, vesicles or membranous material having engrafted thereto molecules capable of modifying an immunological response to a subject to which the vaccine is administered, said vaccine further comprising one or more pharmaceutical carriers and/or diluents.

WO 00/64471 PCT/AUOO/00397 - 11 BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic diagram showing anchoring of a hexa-histidine-tagged protein to a model membrane formed on the glass sensing surface of an lAsys biosensor cuvette. As 5 shown in Figure 1, to anchor hexa-histidine-tagged proteins onto a membrane for studying protein interactions with the IAsys biosensor, a lipid layer is first formed on the sensing surface of the biosensor cuvette using a known method which involves reacting the surface with a suspension of micelle (e.g. liposomes) made from 90% PC and 10% PE-NTA. The PE-NTA for these experiments was produced by first reacting PE with the bifunctional 10 cross-linker bis(sulfosuccinimidyl)suberate, and then reacting the mixture with NTA; the NTA-PE product was purified by thin layer chromatography (and its identity confirmed by mass spectroscopy). The layer is then washed to remove excess micelle (e.g. liposome) suspension and equilibrated in Tris-buffered saline (TBS). In accordance with the present invention, the inventors show that this membrane can be used to anchor 15 hexa-histidine-tagged molecules in a way that allows them to diffuse laterally and interact, thereby mimicking the cell surface. Figures 2(a) and 2(b) are traces showing the change in refractive index as a function of time due to binding of molecules as monitored with an lAsys biosensor. 20 Figure 3 is a biosensor trace showing changes in refractive index due to additions made to an lAsys biosensor cuvette after reacting the sensing surface of the cuvette with a suspension of micelles (e.g. liposomes) to form a layer. 25 Figure 4 is an illustration of the way hexa-histidine-tagged molecules can be engrafted onto the membranes of cells or other membranous structures. The illustration depicts how recombinant receptors bearing a hexa-histidine tag can be engrafted onto biological membranes such as the plasma membrane of cells or the membrane of sub-cellular membranous structures and onto the surface of artificial vesicles or liposomes. The 30 recombinant protein is engrafted onto the membrane structure through the binding of the hexa-histidine tag on the protein to the NTA metal chelating headgroup on the chelator lipid WO 00/64471 PCT/AUOO/00397 - 12 (denoted NTA-DTDA; this may also be referred to as di-C 14-NTA) which has been incorporated into the phospholipid membrane. Figure 5 is the fluorescence profile, as measured by fluorescence-activated cell sorting, of 5 P815 cells engrafted with biotinylated and hexa-histidine-tagged CD40 and B7.1 molecules and then stained with streptavidin-FITC. Figure 6 is a graphical representation showing an induction of tumor-specific cytoxicity in T lymphocytes isolated from mice vaccinated with tumor cells bearing engrafted co 10 stimulator molecules. Syngeneic DBA/2 mice were immunized subcutaneously with either PBS or 1 x 10' y-irradiated P815 cells engrafted with the recombinant proteins: EPOR-6H, B7.1-6H, and B7.1-6H plus CD40-6H, as indicated. Spleens were removed from the mice 14 days after immunization, and T lymphocytes (effector T cells) were isolated, suspended in incubation medium and aliquoted into 24-well flat-bottom plates at a concentration of 1 x 15 10' cells/well, and then co-cultured with 1 x 10' y-irradiated native P815 cells. After 5 days co-culture at 37'C in the presence of 5% CO2 the cells were incubated with '"Cr-labelled native P815 cell targets for 6 hrs at 37'C at the indicated E:T ratio, before harvesting the supernatants and determining the amount of 5 'Cr released through specific lysis. Results are expressed as the percentage specific lysis ± SEM, calculated as described in the Materials 20 and Methods. Figures 7(a) and (b) are graphical representations showing induction of tumor immunity by immunization with P815 tumor cells engrafted with recombinant co-stimulatory molecules. Mice were immunized by injection of either PBS or 1 x 105 y-irradiated P815 cells engrafted 25 with recombinant protein(s) including: EPOR-6H, B7.1-611, and B7.1-611 plus CD40-6H, as indicated. Two weeks after injection the mice in each group were challenged with 1 x 105 native P815 cells by subcutaneous injection, and then monitored for tumor growth and survival. Each point in (A) represents the mean tumor diameter for each group of mice as a function of time for the first five weeks. The data in (B) show the percentage survival of the 30 animals with time.

WO 00/64471 PCT/AUOO/00397 - 13 Figure 8 is a graphical representation showing that binding of fluorescently-labelled liposomes to D10 cells (murine CD4+ T cells) is significantly greater when the liposomes are engrafted with either of the co-stimulatory molecules CD40 and B7.1, than when engrafted with a control protein EPOR. (Note that the D10 cells express ligands for B7.1 and 5 CD40 but no ligand for EPOR.) The liposomes, composed of the lipids: PC:NTA DTDA:FITC-PE (10:1:0.1 molar ratio), were engrafted with one or more recombinant protein (as indicated, EPOR, B7.1 and CD40) each bearing a hexa-histidine tag. The fluorescence profile of the cells in each condition (which reflects the extent of binding of the liposomes to the cells) was determined by fluorescence-activated cell sorting; the 10 background fluorescence of cells (indicated "cells") is shown for comparison. The results indicate that the binding of liposomes engrafted with an appropriate recombinant protein is specific for the type of engrafted protein and, therefore, that liposomes bearing engrafted recombinant proteins can be targeted to cells expressing the appropriate cognate receptor. 15 Figure 9 is a graphical representation showing that synthetic liposomes engrafted with the co-stimulatory molecules B7.1 and CD40 can specifically stimulate the adherence of D10 cells (murine CD4+ T cells) to the culture dish. Cultured D10 cells were suspended in complete growth medium (RPMI 1640 plus 10% v/v FCS, 50 U/ml IL-2, antibiotics and 50 pM P-mercaptoethanol). The recombinant proteins EPOR, CD40 and B7.1 (each with a 20 hexa-histidine tag) were mixed with the cells either in soluble form (as indicated sEPOR, sB7.1 and sCD40) or engrafted onto liposomes composed of PC and NTA-DTDA (10:1) (as indicated NTA-DTDA-EPOR, NTA-DTDA-B7.1 and NTA-DTDA-CD40), before plating the cells into separate wells of a 12-well Linbro tissue culture plate and incubating in growth medium for 2 hrs at 37 0 C. After the incubation, the non-adherent cells were removed from 25 the wells by washing three times with PBS, and the remaining adherent cells were removed with a solution of 1 mM EDTA and counted microscopically. The data shows the proportion of adherent cells for each condition. The results demonstrate that liposomes bearing engrafted co-stimulatory molecules (i.e. B7.1 and/or CD40) can be used to modify immunological responses.

WO 00/64471 PCT/AUOO/00397 - 14 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS In one embodiment, the present invention contemplates a method of anchoring the extramembranous or transmembrane domains of receptors to overcome one or more of the 5 foregoing shortcomings of the prior art. In another embodiment, the subject invention provides a method of assaying interactions between membrane anchored molecules and between anchored molecules and molecules capable of interacting therewith. More particularly, the present invention is useful to study 10 interactions between extramembranous or transmembrane domains of receptors, and between receptor domains and a ligand(s), by anchoring the receptor domain(s) onto a fluid membrane system. Receptor domains may also be composed of proteins, glycoproteins or proteoglycans, oligosaccharides, or fragments or functional equivalents thereof. 15 An important application of the present invention, therefore, is in providing the technology for the screening of agents or drugs which affect either induced or spontaneous receptor aggregation and, hence, which influence receptor function. The present invention may also be used in the screening for drugs which disrupt pre-existing aggregates of receptor subunits, such as the normally associated a and P subunits of the MHC class II molecules described 20 below. The present invention is useful to monitor the spontaneous or ligand induced aggregation/ disaggregation processes of receptors or protein fragments which are not normally membrane associated, once they are engineered to have a hexa-histidine tag and anchored to 25 the membrane as described below for the cell surface derived molecules CD4 and MHC class II. Alternatively, a receptor domain or related protein fragment can be engineered to contain a suitably located strand which spontaneously inserts into the outermost leaflet of the membrane layer. The application of this system includes drug screening and surveying for new ligand species or biochemical agents (e.g. enzymes) which induce modifications that 30 affect aggregation processes.

WO 00/64471 PCT/AUOO/00397 - 15 Thus, the present invention is directed to a method of anchoring receptor domains (albeit extramembranous or transmembrane) onto a membrane such that said receptor domains are capable of lateral movement to facilitate interactions with biomolecules in the membrane. 5 Accordingly, the present invention provides a method of anchoring receptor domains onto a planar supported membrane comprising amphiphilic molecules arranged in a layer, wherein a proportion of the amphiphilic molecules have been modified by a covalent attachment of a metal chelating group such that some of the metal chelating groups are oriented toward the outside surface of said membrane; which method comprises the step of interacting a receptor 10 domain which is covalently attached to a polypeptide tag with said membrane for a time and under conditions sufficient for said polypeptide tag to attach to said membrane via the outwardly facing metal chelating residues of said membrane, such that the receptor domains are capable of lateral movement, dimerization or oligomerization, and interaction with a ligand molecule. 15 A preferred metal chelating group for use in the present invention is nitrilotriacetic acid (NTA). The membrane may be formed from a suspension of micelles (e.g. liposomes) from the 20 amphiphilic molecules, wherein a proportion of the amphiphilic molecules has been modified by covalent attachment of a metal chelating group. Alternatively, the membranes are formed by the addition of water or aqueous buffers to a solution of amphiphilic molecules in an organic solvent. 25 Another aspect of the invention provides a method of anchoring receptor domains onto a membrane such that the receptor domains are capable of lateral movement, said method comprising of: (i) forming a membrane on an appropriate planar support by either: 30 (a) contacting and incubating the support for a period of time with a solution in WO 00/64471 PCT/AUOO/00397 - 16 an organic solvent of amphiphilic molecules wherein a proportion of the molecules have been modified by covalent attachment of a metal chelating group, then forming a membrane by the addition of water or an aqueous buffer; or 5 (b) forming a suspension of micelles (e.g. liposomes) from amphiphilic molecules wherein a proportion of the amphiphilic molecules have been modified by a covalent attachment of a metal chelating group, then interacting said micelle (e.g. liposome) suspension with an appropriate planar 10 support for a time and under conditions sufficient to allow a membrane layer to form in which some metal chelating residues attached to the amphiphilic molecules of said micelle (e.g. liposome) suspension are oriented toward the outside surface of said membrane; and 15 (ii) interacting the receptor domain(s) to be anchored which is covalently attached to a polypeptide tag with said membrane for a time and under conditions sufficient for said polypeptide tag to attach via the metal chelating linkage to the outwardly-facing metal chelating residues of said membrane. 20 The amphiphilic molecules are normally surfactant molecules having a hydrophilic "head" portion and one or more hydrophobic "tails". Surfactants may be any of the known types, i.e. cationic (e.g. quaternary ammonium salts), anionic (e.g. organosulfonate salts), zwitterionic (e.g. the phospholipids: phosphatidylcholines and phosphatidylethanolamines), membrane spanning lipid, or non-ionic (e.g. polyether materials). 25 The membrane may be comprised of more than one type of amphiphilic molecule. In a preferred embodiment, the membrane is comprised of a first phospholipid and a second phospholipid. 30 Thus, in a preferred form, the present invention contemplates a method of anchoring receptor domains onto a lipid membrane such that said receptor domains are capable of WO 00/64471 PCT/AUOO/00397 - 17 lateral movement to facilitate interactions with biomolecules in the membrane, said method comprising of: (i) forming a membrane on an appropriate planar support by either: 5 (a) contacting and incubating the support for a period of time with a solution in an organic solvent of amphiphilic molecules wherein a proportion of the molecules have been modified by covalent attachment of a metal chelating group, then forming a membrane by the addition of water or an aqueous 10 buffer; or (b) forming a suspension of micelles (e.g. liposomes) from a first phospholipid and a second phospholipid wherein the second phospholipid has been modified by a covalent attachment of a nitrilotriacetic acid (NTA), and then 15 interacting said micelle (e.g. liposome) suspension with an appropriate planar support for a time and under conditions sufficient to allow a phospholipid membrane layer to form in which some NTA residues attached to the second phospholipid of said micelle (e.g. liposome) suspension are oriented toward the outside surface of said membrane; and 20 (ii) interacting the receptor domain(s) to be anchored which is covalently attached to a polypeptide tag with said membrane for a time and under conditions sufficient for said polypeptide tag to attach via the NTA-metal chelating linkage to the outwardly-facing NTA residues of said membrane. 25 The method of this aspect of the present invention is particularly useful for anchoring extramembranous or transmembrane receptor domains. In a further preferred form, the first phospholipid is phosphatidylcholine (PC) and the second 30 lipid is phosphatidyl-ethanolamine-NTA (PE-NTA) and the ratio of PC:PE-NTA (w/v) is about 10:1. However, the first phospholipid can be any phospholipid or hydrocarbon capable WO 00/64471 PCT/AUOO/00397 - 18 of forming a lipid layer; and the second phospholipid can be any lipid with a metal chelating headgroup which can be used to anchor receptor domains using a suitably engineered tag on the domain. In addition, the ratio of the first to the second phospholipid can be varied depending on the desired density of receptor domain molecules to be achieved on the 5 membrane layer. In one form of the present invention, the support is the glass-like sensing surface of an optical biosensor cuvette or a mica surface suitable for analysis by atomic force microscopy. Preferably, the support is a planar glass or mica surface, but could be any surface which can 10 form a substrate for a supported planar lipid membrane layer (6, 9, 10). In another form, the surface may be a layer of glass, gold or any material which can be reacted with a solution of a hydrocarbon-containing compound (e.g. octadecyltrichlorosilane and octadecanethiol), capable of attaching hydrocarbon chains to said surface (6, 11), such 15 that chains of hydrocarbon are oriented away from the surface of said support to form a lipid monolayer or a hydrophobic surface which is capable of reacting with a suspension of phospholipid micelles (e.g. liposomes) to form a planar membrane layer (6, 9, 10). In these forms the NTA residues attached to the second phospholipid of said micelle (e.g. liposome) suspension are oriented toward the outside surface of said membrane. Such surfaces may be 20 the sensing surface of optical biosensors that are capable of monitoring the interaction of receptor domains anchored on the membrane with ligand molecules in solution. Preferably, the polypeptide tag comprises a sequence of at least six amino acid residues such as a hexa-histidine molecule, but can be any sequence of amino acids that can bind strongly 25 through the formation of a complex with the metal chelating component of a lipid containing a metal chelating group such as NTA. In one application of the subject invention the molecule is a transcription factor molecule. In another form of the instant invention, the molecule is a receptor. More particularly, the receptor may be any cell surface receptor such as the human cell surface molecule CD4 or human MHC class II molecule, or domains of 30 such receptors.

WO 00/64471 PCT/AUOO/00397 - 19 Accordingly, in another aspect, the present invention provides a method of assaying interactions between a receptor and a ligand, comprising the steps of: (A) anchoring a receptor to a membrane by: 5 (i) forming a membrane on an appropriate planar support by either: (a) contacting and incubating the support for a period of time with a solution in an organic solvent of amphiphilic molecules wherein a 10 proportion of the molecules have been modified by covalent attachment of a metal chelating group, then forming a membrane by the addition of water or an aqueous buffer; or (b) forming a suspension of micelles (e.g. liposomes) from amphiphilic 15 molecules wherein a proportion of the amphiphilic molecules have been modified by a covalent attachment of a metal chelating group, and then interacting said micelle (e.g. liposome) suspension with an appropriate planar support for a time and under conditions sufficient to allow a membrane layer to form in which some metal chelating 20 residues attached to the amphiphilic molecules of said micelle (e.g. liposome) suspension are oriented toward the outside surface of said membrane; and (ii) interacting the receptor domain(s) to be anchored which is covalently 25 attached to a polypeptide tag with said membrane for a time and under conditions sufficient for said polypeptide tag to attach via the metal chelating linkage to the outwardly-facing metal chelating residues of said membrane; (B) allowing said receptor molecules to interact and/or oligomerize on the membrane; 30 and WO 00/64471 PCT/AUOO/00397 - 20 (C) contacting said anchored receptor with an effective concentration of ligand for a time and under conditions sufficient for binding to occur and detecting said binding. In one embodiment, the present invention contemplates a method of assaying interactions 5 between a receptor and a ligand comprising the steps of: (A) anchoring a receptor to a lipid layer by: (i) forming a membrane on an appropriate planar support by either: 10 (a) contacting and incubating the support for a period of time with a solution in an organic solvent of amphiphilic molecules wherein a proportion of the molecules have been modified by covalent attachment of a metal chelating group, then forming a membrane by 15 the addition of water or an aqueous buffer; or (b) forming a suspension of vesicles from a first phospholipid and a second phospholipid wherein said second phospholipid has been modified by covalent attachment of a metal chelating group such as 20 nitrilotriacetic acid (NTA), and then interacting said micelle (e.g. liposome) suspension with an appropriate planar support such as glass or mica for a time and under conditions sufficient to allow a hydrocarbon phospholipid membrane layer to form on the support in which some of the NTA residues attached to the second phospholipid 25 of said micelle (e.g. liposome) suspension are oriented toward the outside surface of said membrane; (ii) interacting the receptor domain to be anchored which is covalently attached to a polypeptide tag with said membrane for a time and under conditions 30 sufficient for said polypeptide tag to attach via the NTA-chelating linkage to the outwardly-facing NTA residues of said membrane; WO 00/64471 PCT/AUOO/00397 -21 (B) allowing said receptor molecules to interact and/or oligomerize on the membrane; and (C) contacting said anchored receptor with an effective concentration of ligand for a time 5 and under conditions sufficient for binding to occur and detecting said binding. In the case where the solid support (e.g. gold) may not be suitable for layer formation directly from the vesicle suspension, steps (i) to (iii) above are preceded by a step to form a monolayer or hydrophobic surface on the solid support. This is achieved by reacting the 10 solid support with a solution of a hydrocarbon-containing compound capable of attaching hydrocarbon chains to its surface, such that chains of hydrocarbon are oriented away from the surface of said support to form a lipid monolayer or a hydrophobic surface. The surface is then reacted with a suspension of phospholipid amphiphilic molecule micelles (e.g. liposomes), preferably micelles (e.g. liposomes) [as in steps (i)-(ii) above] for a time and 15 under the conditions necessary to form a membrane layer. The metal chelating, preferably NTA residues attached to the amphiphilic molecules such as a second phospholipid will be oriented toward the outside surface of said membrane, and may be used to anchor molecules with a hexa-histidine tag [as described in (iii) above]. Such surfaces may be the sensing surface of optical biosensors that are capable of monitoring the interaction of receptor 20 domains anchored on the membrane with ligand molecules in solution. Preferably, the oligomerization or self-association of the receptor and binding to the ligand is detected by atomic force microscopy or by an optical biosensor technique. 25 The present invention thus provides for the anchoring of receptor domains, proteins, glycoproteins and polysaccharides, onto lipid layers formed on mica, glass, gold or any other appropriate surface, that enable the molecules to diffuse laterally and interact. This technology is ideal in a preferred embodiment for studying the interaction between receptor domains and between receptor domains and ligands in a membrane system using optical 30 biosensor and atomic force microscope techniques.

WO 00/64471 PCT/AUOO/00397 - 22 Receptor domains can be engineered to have a hexa-histidine COOH-tail, or NH2-tail using standard recombinant DNA techniques. A hexa-histidine tag also may be covalently attached to polysaccharides and other molecules by chemical means. 5 Optical biosensors used in connection with this invention measure the change in refractive index due to the binding of a soluble macromolecule with a molecule immobilized onto the solid sensing surface and can be used to measure the binding affinity of the interacting molecules. The IAsys biosensor employs a sample cuvette which has a resonance mirror as the sensor surface. The glass-like sensing surface of the IAsys biosensor is ideal for reacting 10 with a suspension of micelles (e.g. liposomes) to form a membrane layer on the sensing surface. The present invention may also be used with a BlAcore surface plasmon resonance biosensor which utilizes a gold sensing surface. The gold is first reacted with 15 octadecanethiol to form an octadecane monolayer, and then reacted with a suspension of micelles (e.g. liposomes) to form the membrane layer. Atomic force microscopes can be used for mapping molecular structures on the model membrane layers containing a limited number of molecular species. Such layers can be 20 formed either directly on mica or glass surfaces by incubation with a suspension of micelles (e.g. liposomes). A hydrophobic monolayer also can be formed on glass or gold surface by treating with an appropriate reagent, and a hybrid membrane layer formed by reacting the surface of the monolayer with a suspension of micelles (e.g. liposomes) composed of the amphiphilic molecules, preferably an appropriate mixture of PC and PE-NTA to form the 25 layer. The anchoring of hexa-histidine tagged receptors onto model membrane systems of the present invention is also useful to anchor or engraft receptors and other molecules onto liposomes or vesicles which through the specificity of the engrafted molecules can target and 30 deliver drugs, DNA/RNA or any therapeutic agent that can be encapsulated into the liposomes, to specific cell types or tissues when the liposomes are administered in vivo.

WO 00/64471 PCT/AUOO/00397 - 23 Recombinant molecules also can be engrafted onto synthetic liposomes or vesicles for the purpose of developing vaccines to produce specific biological or therapeutic effects. According to this aspect of the present invention, there is provided a method of engrafting 5 molecules onto liposomes said method comprising: (i) preparing a suspension of liposomes with chelator lipid incorporated, and with or without an encapsulated drug or agent; 10 (ii) incubating the liposomes with a recombinant protein or target molecule bearing an appropriate metal affinity tag; and (iii) if necessary, removing excess protein by washing, filtering or other washing means and suspending them in an appropriate solution. 15 In a preferred embodiment, the molecules may be anchored or engrafted onto liposomes by the following method: (i) preparing a suspension of liposomes from an appropriate mixture of a chelator lipid 20 such as di-tetradecylamine nitrilotriacetic acid (NTA-DTDA) and 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) at a concentration of- 0.5 mM in aqueous solution such as PBS (phosphate buffered saline) containing a concentration of Ni2+ or Zn2+ approximately equal to that of the NTA-DTDA. The liposomes can be produced by sonicating the mixture for 5-10 mins at a temperature above the Tm. 25 Alternatively, the liposomes can also be produced by dissolving the lipids in an ethanolic solution and then dispersing in aqueous buffer, or by extruding an acqeous suspension of the lipids through polycarbonate or similar filter of suitable pore size. Typically, the ratio of NTA-DTDA:POPC can be 5:1, but can be different; 30 (ii) washing the liposomes by pelleting (by centrifuging at ~95,000 x g for 30 min at 4*C) and removing the supernatant, or by filtration techniques, and then suspending WO 00/64471 PCT/AUOO/00397 - 24 the liposomes in an appropriate volume of the buffering solution to facilitate incubation with the tagged protein(s); (iii) incubating the liposomes with a recombinant protein (e.g. human 5 hexa-histidine-tagged VEGF, vascular endothelial growth factor), or a combination of different recombinant proteins, each bearing a hexa-histidine or other suitable metal affinity tag to allow it to be anchored onto the liposomes; and (iv) removing excess soluble or unincorporated protein by washing the liposomes as in 10 step (ii) above, then suspending them in PBS or other buffer solution suitable for administration in vivo. Other combinations and different lipids can also be used in conjunction with the chelator lipid to give the liposomes specific properties. For example, the ganglioside GM1 or 15 derivatives polyethylene glycol can be included in the mixture (in step (i) above) to produce liposomes with "stealth" properties (12) to avoid them being taken up by macrophages or by the liver or spleen when used as vaccines in vivo. Also, step (i) can be carried out in the presence of a drug, DNA or other therapeutic agent for the purpose of encapsulating the material and permitting it, when administered in vivo, to be delivered to specific cells or 20 tissues defined by the specificity of the engrafted molecule(s). For example, liposomes with engrafted VEGF (vascular endothelial growth factor) can be used to target angiogenic epithelium which is known to express the VEGF receptor and is required for tumor growth. Liposomes with engrafted VEGF, therefore, can be used to deliver a cytotoxic drug or agent that can block the growth of new blood vessels needed for the growth of tumors. The 25 cytotoxic drug or agent is encapsulated within the liposome. Current methods of modifying the surfaces of cells to be used as vaccines for altering immunity to disease (e.g. the immune response to tumors - see below) generally require the transfection or genetic manipulation of the tumor cells, to induce them to express one or 30 more specific protein(s) on their surface (13-15). For example, in both animal and human tumor models evidence suggests that the transfection of tumor cells with genes inducing WO 00/64471 PCT/AUOO/00397 - 25 them to express T cell costimulator molecules like B7-1 (CD80), B7-2 (CD86), CD40 and ICAM- 1 on their surface, may be a useful approach to prepare the cells for use in vaccinations to enhance tumor immunity in the tumor bearing host (16-22). Unfortunately, in a clinical setting, such as in the treatment of cancer in humans, the transfection of tumor 5 cells with such genes can be time consuming and inconvenient. Thus, the frequency of transfection is generally low, and successful transfection with multiple genes (to induce expression of multiple proteins on the tumor cell surface) can be difficult to achieve. Furthermore, transfection techniques, even when carried out by the use of seemingly harmless viral vectors, can be associated with risks to the patient owing to the difficulty in 10 precisely controlling the expression of the gene or its integration into the genome. The present invention further provides a more convenient and safe method of engrafting co stimulatory and other molecules directly onto the surfaces of cells (such as tumor cells) and other membranous structures (either biological or synthetic), that can be used as vaccines to 15 enhance or modify immunity to tumors and other diseases in humans. The method of anchoring receptors onto model membrane systems to assay for inter molecular interactions and drug screening can be used to anchor or "engraft" molecules directly onto biological membranes (e.g. the membranes of cells or subcellular particles), 20 once a chelator lipid (e.g. NTA-DTDA) has been incorporated into the membranes, thereby providing a convenient way of engrafting recombinant molecules possessing a hexa-histidine or other suitable metal affinity tag, directly onto the membrane surface. In this aspect of the present invention, there is provided a method of anchoring or engrafting 25 recombinant molecules directly onto biological membranes said method comprising: (i) preparing a suspension of chelator lipid or liposomes containing the chelator lipid; (ii) incubating a suspension of cells or biological membranous structures with a 30 suspension of the chelator lipid; WO 00/64471 PCT/AUOO/00397 -26 (iii) washing away excess or unincorporated lipid; (iv) incubating the membranous structures with a solution of recombinant protein or target molecule possessing an appropriate metal affinity tag; and 5 (v) washing away excess or unbound soluble protein, and suspending the structures in a solution suitable for administration in vivo. In a preferred embodiment, the invention allows the engrafting of molecules onto cells and 10 other membranous structures using the following method: (i) washing a suspension of the cells or membranous structures with PBS or other aqueous buffer solution to remove excess soluble and/or loosely bound proteins. This can be carried out by pelleting the structures by appropriate centrifugation (e.g. 5 min 15 at 200-500 x g for murine and human cells), and then resuspending them in PBS; depending on the structures, excess soluble or loosely bound proteins may be removed by filtering or other washing means; (ii) preparing a suspension of chelator lipid (e.g. NTA-DTDA, at a concentration of ~0. 1 20 mM) in PBS containing an approximately equal concentration of either Zn2+ or Ni2+ by sonicating for 5-10 min an appropriate quantity of the lipid in the PBS solution. Other lipids or phospholipids (e.g. POPC) or other agents also can be included with the chelator lipid to promote the fusion and incorporation of the liposomes into the membrane structures; 25 (iii) incubating the cells or membranous structures with a suspension of the chelator lipid (e.g. 0.1 mM NTA-DTDA) in PBS for a suitable period of time and temperature (e.g. 30 min, at 37*C) to allow some of the lipid in the suspension to fuse and/or become incorporated into the structures. Note: the incubation conditions employed can be 30 altered to suit the nature of the chelator lipid used and the particular membrane structure into which the lipid is to be incorporated; also, incubations or wash steps in WO 00/64471 PCT/AUOO/00397 - 27 buffer containing additives such as polyethylene glycol can be used to promote lipid fusion and incorporation; (iv) removing unincorporated lipid from the mixture by washing the membranous 5 structures with PBS by pelleting and washing as in step (i) above; (v) incubating the washed structures containing incorporated chelator lipid with a solution of a recombinant protein, or a solution of a mixture of different recombinant proteins, each containing a hexa-histidine or any other appropriate metal affinity tag; 10 and (vi) washing the cells or structures with PBS (as in step (i) above) to remove excess or unbound soluble recombinant protein. 15 A similar procedure can be used to engraft tagged proteins to any cells, or subcellular membranous components. The structures so treated will contain a modified surface due to the engrafted protein, and can be used in vaccinations to alter immunological responses in vivo. The structures when administered in vivo also can be used to target a particular cell type or tissue within the body thereby altering the function of these cells. For example, the 20 engrafting of tumor cells with molecules known to bind receptors on dendritic cells can be employed to direct the engrafted tumor cells to the dendritic cells to enhance tumor antigen presentation and hence immunological responses against the tumor. In this form, the present invention contemplates a method of anchoring or engrafting 25 recombinant receptors and other molecules directly onto biological membranes, such as the membrane of cells, and, hence, of modifying the properties of such membranes. In particular, the instant invention provides the basis of a convenient strategy for modifying the surfaces of cells (e.g. tumor cells), any cellular or subcellular membranous component, infectious agent or particle (e.g. bacteria), as well as any biological or synthetic membrane including 30 synthetic vesicles or liposomes, into which the chelator lipid can be incorporated. In all these instances, the recombinant protein is engrafted by the formation of a metal chelating linkage WO 00/64471 PCT/AUOO/00397 - 28 between a peptide tag on the protein, and the NTA headgroup on the chelator lipid incorporated into the membranous structure. The biological membrane being modified by the anchoring of any recombinant protein, glycoprotein and any other molecular structure possessing an appropriate tag,.and designed to enhance immunity to diseases when used 5 either as a vaccine, or as an agent to target delivery of these biological membranes to specific cells and tissues when administered in vivo. Examples of suitable molecules in accordance with this aspect of the present invention includes therapeutic molecules, pharmaceutical compounds and nucleic acid molecules such 10 as RNA and DNA. A particularly useful molecule is VEGF or its homologue. VEGF and its homologues are also useful for targeting liposomes to cells carrying VEGF receptors. Accordingly, the molecules contemplated by this aspect of the present invention include molecules having binding partners on target tissue. Preferably, the molecules are engrafted, anchored or encapsulated within the liposome. 15 A further aspect of the present invention provides for altering the immunogenicity of a target cell. This may be readily accomplished by introducing foreign polypeptides, polysaccharides, glycoproteins, receptors, ligands and other molecules. Altering the immunogenicity of cells such as tumor cells is a useful way of enhancing an immune response against tumor cells. 20 Accordingly, another aspect of the present invention contemplates a method for altering the immunogenicity of a target cell or membranous component thereof, said method comprising anchoring a molecule to the membrane of said target cell by: 25 (i) preparing a suspension of chelator lipid or liposomes containing the chelator lipid; (ii) incubating a suspension of cells or membranous structures with a suspension of the chelator lipid; 30 (iii) washing away excess or unincorporated lipid; WO 00/64471 PCT/AUO0/00397 - 29 (iv) incubating the membranous structures with a solution of said molecule to be anchored; and (v) washing away excess or unbound soluble molecule, and suspending the structures in 5 a solution suitable for administration in vivo. In a more particular embodiment, the present invention contemplates a method of targeting liposomes or membranous structures to a particular cell type or tissue, said method comprising anchoring or engrafting a molecule having a binding partner on the particular 10 cells or tissue to be targeted by: (i) preparing a suspension of chelator lipid or liposomes containing the chelator lipid; (ii) incubating a suspension of cells or membranous structures with a suspension of the 15 chelator lipid; (iii) washing away excess or unincorporated lipid; (iv) incubating the liposomes or membranous structures with a solution of molecules to 20 be anchored; and (v) washing away excess or unbound soluble molecule, and suspending the structures in a solution suitable for administration in vivo. 25 As stated above, the methods of the present invention provides methods for altering the immunogenicity of cells. Accordingly, the present invention provides a method of treatment, said method comprising administering to a subject an effective amount of a liposome preparation or membranous material comprising an active material and optionally an anchored or engrafted molecule having a binding partner or target tissue. 30 Examples of active material include but are not limited to a recombinant polypeptide, co- WO 00/64471 PCT/AUOO/00397 - 30 stimulatory molecule, therapeutic drug or nucleic acid molecule. In one example, VEGF is incorporated into a liposome to target a cytotoxic drug to block the growth of new blood vessels needed for the growth of tumors. 5 Another aspect of the present invention provides a vaccine composition comprising cells or membranous material having engrafted thereto molecules capable of modifying an immunological response to a subject to which the vaccine is administered, said vaccine further comprising one or more pharmaceutical carriers and/or diluents. Preferably, the molecules engrafted to the cells or membranous material are co-stimulatory molecules. 10 Furthermore, the vaccine is preferably produced by the steps comprising: (i) incubating the cells or membranous material with a chelator lipid such as NTA DTDA to allow the lipid to incorporate in the cells or membranes; 15 (ii) washing off any unincorporated lipid by centrifugation or filtration and resuspension of the structures in the appropriate solution or buffer; (iii) incubating the membranous structures with incorporated chelator lipid with said molecules to be engrafted; and 20 (iv) washing off unincorporated molecular material. Accordingly, the present invention enables the incorporation of chelator lipids like NTA DTDA into tumor cell membranes, followed by the engraftment of recombinant co 25 stimulatory and/or other molecules (or combinations of molecules) with an appropriate tag, may be a convenient approach in the development of cell-based vaccines to enhance tumor immunity. Analogous to its demonstrated ability to alter tumor immunity therefore, the technique also can be expected to provide a convenient approach to engraft specific co stimulatory and/or other cell surface molecules (or combinations of such molecules) onto 30 other cell types including T cells, B-cells and dendritic cells, to see what role such molecules might play in regulating immune function. In addition to its potential use in cancer WO 00/64471 PCT/AUOO/00397 -31 immunotherapy, therefore, the technique described herein will have application to areas that could significantly enhance our understanding of immune function. The present invention further provides a use of membrane system comprising an agent 5 engrafted, encapsulated and/or anchored thereto in the manufacture of a medicament for modifying an immune response in an animal. Preferred animals in accordance with the present invention are humans, primates, livestock animals, laboratory test animals and captured wild animals. 10 Terms such as "anchoring" and "engrafting" may be used interchangedly throughout the subject specification. The term "engrafting" also encompasses the term "grafting". The terms "membrane" and "lipid membrane" include reference to biological and synthetic membranes as well as lipid layers. The term "layer" or its plural "layers" includes monolayers and 15 multilayers such as bilayers. A "chelator lipid" may be any suitable chelating lipid such as but not limited to NTA-DTDA.

WO 00/64471 PCT/AUOO/00397 - 32 The materials and methods below relate to some of the Examples which follow. Reagents 5 Analytical grade reagents were used in all experiments. Paraformaldehyde was obtained from BDH Chemicals. ZnSO 4 was used for all additions of Zn 2 to buffers and growth media. RPMI 1640 and EMEM (Eagles minimal essential medium) both were obtained from Gibco (Life Technologies, Melbourne, Australia). Fetal calf serum (FCS) was obtained from Trace Scientific (Noble Park, Vic. Australia). Sulfo-NHS-LC-Biotin was obtained from Pierce 10 (Rockford, IL). Na"CrO 4 , [ 3 H]-thymidine, and fluorescein isothiocyanate (FITC)-conjugated streptavidin (streptavidin-FITC) were obtained from Amersham (UK). Dioleoyl phosphatidylethanolamine (DOPE), a-palmitoyl- p-oleoyl-phosphatidylcholine (POPC), dimyristoyl-phosphatidylcholine (DMPC), Isopaque, ficoll, propyl gallate, and the polyethylene glycol (PEG) preparations PEG4 PEG 600 , PEG 900 , and PEG150, were all 15 obtained from Sigma-Aldrich Pty Ltd (Castle Hill, NSW, Australia). MicroScint scintillation fluid and other items such as filters and seals for 96-well plates for use with the Topcount NXT microplate scintillation counter were obtained from Canberra Packard (Canberra, ACT, Australia). 20 Mice and Cell lines Female or male DBA/2J mice (H-2d), were used for isolation of lymphoid tissue (spleen) for T cell proliferation, measurement of T cell cytotoxicity, and for vaccination and monitoring of tumor growth in vivo. C57BL/6J mice (H-2b) were used in experiments assessing 25 allogeneic stimulation of T cell proliferation. The mice were used at 6-8 weeks of age and were obtained from the Animal Breeding Establishment, John Curtin School of Medical Research (JCSMR), Australian National University (ANU), Canberra. The murine cell lines, P815 [murine DBA/2 (H-2) mastocytoma] and EL4 [murine C57BL/6 (H-2b)] T cell lymphoma, were obtained, respectively, from Drs P. Waring (Division of Immunology and 30 Cell Biology, JCSMR) and H. O'Neill (Division of Biochemistry and Molecular Biology), ANU. Both cell lines were cultured in complete medium consisting of EMEM containing WO 00/64471 PCT/AUOO/00397 - 33 10% v/v FCS. Synthesis of NTA-DTDA 5 The chelator-lipid nitrilotriacetic acid didtetradecylamine (NTA-DTDA), consisting of a nitrilotriacetic acid (NTA) head-group covalently linked to ditetradecylamine (DTDA) was synthesized by Dr C. Easton (Research School of Chemistry, ANU) following a procedure similar to that previously described (25). Briefly, the DTDA was synthesized from bromotetradecane and ammonia. DTDA was then N-succinylated with succinic anhydride to 10 produce N-succinyl-DTDA (DTDA-suc), which was reacted with N-hydroxysuccinimide (NHS) to produce N-[(hydroxysuccinimidyl)succinyl]-DTDA (DTDA-suc-NHS). The succinimidyl group of DTDA-suc-NHS was replaced with a N"-tert-butyloxycarbonyl-lysine (N-Boc-lys) group, and the butyloxycarbonyl (Boc) group was removed to produce N [(DTDA) succinyl]-L-lysine (DTDA-suc-Lys). DTDA-suc-Lys was finally reacted with 15 bromo-acetic acid to produce N,N-bis[carboxymethyl]-N-[(DTDA)suc]-L-lysine, which will be referred to as NTA-DTDA. The purity of each product was measured by thin layer chromatography, and the identity of the final product was confirmed by nuclear magnetic resonance spectroscopy, Fourier transformed infrared spectroscopy and mass spectroscopy. The purity of the final product was estimated to be in excess of 99%. 20 Preparation of NTA-DTDA liposome suspensions For NTA-DTDA incorporation into cells, dessicated NTA-DTDA was suspended to a concentration of 0.5 mM in PBS containing 0.5 mM Zn 2 , by sonication using a TOSCO 25 100W ultrasonic disintegrator at maximum amplitude for 2 min. The same procedure was used to produce suspensions of DMPC, and mixtures of NTA-DTDA and DMPC, POPC, or DOPE. Stock suspensions of lipids were stored at -20*C, and were always re-sonicated and diluted to the indicated concentration prior to use in experiments. 30 Monoclonal antibodies WO 00/64471 PCT/AUOO/00397 - 34 The monoclonal antibodies (mAb) and their sources were as follows: murine anti-CD40 (clone 3/23, Rat IgG 2 ,) and murine anti-CD3 (clone 145-2C 11, Armenian Hamster IgG) mAbs were both obtained from Pharmingen; and murine anti-B7.1 (clone 16-10Al, Armenian Hamster IgG) mAb was kindly provided by Dr. K. Shortman, WEHI, Melbourne. 5 Where indicated, mAbs were biotinylated by reacting with sulfo-LC-biotin (Pierce) as previously described (26). Recombinant proteins 10 Recombinant forms of the extracellular regions of the murine T cell co-stimulatory molecules B7.1 (CD80) and CD40, and the extracellular region of the human erythropoietin receptor (EPOR), each with a hexahistidine (6His) tag and denoted B7.1-6H, CD40-6H, and EPOR-6H, respectively, were produced using the baculovirus expression system. Briefly, genes encoding the extracellular domains of murine B7. 1, CD40 and EPOR were amplified 15 by polymerase chain reaction (PCR), and the sequences for 6His tags were incorporated into the end of each gene (corresponding to the carboxyl terminal of the protein) by PCR using primers containing the sequence of the tag. The constructs were then separately ligated into the pVL1393 plasmid baculovirus transfer vector and used to transform E. coli. Appropriate transformants were selected, and recombinant pVL1393 plasmids from these transformants 20 were co-transfected with the baculovirus AcMNPV into SF9 insect cells. Cells infected with virus which had the pVL1393 plasmid incorporated into the viral genome were selected by plaque assays, further amplified and these viral stocks were used to infect High-5 insect cells grown in Express-5 medium. Recombinant proteins were purified from the supernatants of recombinant virus infected High-5 cells by Ni 2 +-NTA affinity chromatography (using Ni 2

+

25 NTA Superflow, from QIAGEN Pty Ltd, Cifton Hill, Victoria, Australia) followed by size exclusion gel filtration on FPLC (Pharmacia Biotech, Upsalla, Sweden) using a Superdex-75 HR 10/30 column; the final purity of each protein was >95% as judged by SDS-PAGE analysis. For some experiments recombinant proteins were biotinylated by reacting with sulfo-LC-biotin (Pierce) as previously described (26). The proteins were routinely stored at 30 20*C in PBS at a concentration of 0.2-0.6 mg/ml, and then thawed at 37'C and vortexed gently prior to use in each experiment.

WO 00/64471 PCT/AUO0/00397 - 35 Incorporating and optimizing the incorporation of NTA-DTDA Cultured P815 tumor cells were washed twice in PBS to remove proteins from the culture media and suspended to 1 x 107 cells/ml in PBS. The cells were then aliquoted into 96-well 5 V-bottom Serocluster plates (Costar, Coming, NY) at 1.8 x 15 cells/well and incubated with 125 pM NTA-DTDA (alone or as a mixture with other lipids as indicated) or 125 gM DMPC (control) in PBS containing 125 pM Zn", for 40 min at 37 0 C. Following the incubation, unincorporated lipid was removed by washing three times with PBS containing 0.1% v/v BSA (PBS-0.1% v/v BSA). The relative level of NTA-DTDA incorporated was 10 routinely assessed by FACS analysis (see below) after incubating the cells with biotinylated 6His peptide (B-6His) (0.2 gg/ml) for 30 min at 4'C, washing twice with PBS-0.1% v/v BSA, and then staining with streptavidin-FITC. The cells were incubated with streptavidin FITC (33 pg/ml) in PBS containing 1% v/v BSA (PBS-l% v/v-BSA) for 30 min at 4*C, washed three times with PBS-I% v/v BSA, fixed with 2% v/v paraformaldehyde in PBS, 15 and then analyzed for FITC-fluorescence by FACS. To promote fusion of NTA-DTDA liposomes and hence incorporation of the NTA-DTDA into the membrane of cells, a number of agents previously reported to potentiate the fusion of cells and vesicles with lipid layers were tested. P815 cells aliquoted into 96-well V 20 bottom serocluster plates as described above were incubated with 125 PM NTA-DTDA, DMPC, POPC, or DOPE, or with 125 gM NTA-DTDA plus DMPC, POPC or DOPE (at the indicated molar ratio), in PBS containing 125 gM Zn", for 40 min at 37'C. For some experiments the cells were treated with PEG following the incubation: the cells were pelleted, suspended in 15% PEG 4 00 , mixed and diluted 1Ox with serum-free EMEM, and 25 then washed once with serum-containing EMEM and twice with PBS-0. 1% v/v BSA, before engrafting the cells with biotinylated recombinant protein (see below) and then staining with streptavidin-FITC as above for FACS analysis. Engrafting recombinant proteins onto cells 30 Cells with incorporated NTA-DTDA were incubated with purified B7.1-6H and CD40-6H WO 00/64471 PCT/AUOO/00397 - 36 (or biotinylated forms of these as indicated), either alone (each at 50 pig/ml) or in combination (100 ig/ml total protein, with a B7.1-6H:CD40-6H molar ratio of 4:1), in 96 well V-bottom Serocluster plates for 1 hr at 4'C. Unbound protein was then removed by washing twice with PBS-0. 1% v/v BSA, before using the cells bearing the engrafted 5 protein(s) either for immunizations or in assays of T cell proliferation. For experiments to determine the level of bound protein by FACS analysis the cells were stained with streptavidin-FITC (for cells bearing engrafted biotinylated protein), or were first incubated with the appropriate biotinylated mAb (B-mAb) (4'C for 30 min), washed twice with PBS 0.1% V/v BSA and then stained with streptavidin-FITC. 10 Time courses Cells with incorporated NTA-DTDA and DMPC, with or without engrafted CD40-6H, were suspended in EMEM containing 10% v/v FCS and 50 pM added Zn", and incubated in 12 15 well flat-bottom tissue culture plates (Linbro, ICN Biomedicals Inc, Aurora, OH) for approx. 2 min (time 0), or 4 or 24 hrs at 37 0 C. After the indicated incubation time, cells were collected from the 12-well flat-bottom plates, transferred to 96-well V-bottom Serocluster plates and washed twice in PBS-0. 1% v/v BSA, before either staining with streptavidin FITC (for cells with NTA-DTDA and engrafted B-CD40-6H), or first incubating with B 20 CD40-6H and then washing with PBS-0.1% v/v BSA and staining with streptavidin-FITC (for cells with only NTA-DTDA). Flow cytometry 25 Fluorescence-activated cell sorter (FACS) analysis was used to quantify the relative levels of NTA-DTDA incorporated into the membrane of cells following binding of B-6His, and the levels of biotinylated recombinant proteins anchored to the cell surface via the incorporated NTA-DTDA. Flow cytometric analyses were performed using a FACSort flow cytometer (Becton Dickinson, San Jose, CA) equipped with a 15 mW argon-ion laser. Cells were 30 analysed on the basis of forward light scatter (FSC), side light scatter (SSC) and FITC fluorescence; with the relative shift in fluorescence intensity above background providing a WO 00/64471 PCT/AUOO/00397 - 37 semi-quantitative measure of the level of NTA-DTDA incorporation and the level of peptide or recombinant protein on the surface of cells. Typically, fluorescence information for 10, 000 cells was collected for each condition using a log amplifier and the data processed using CELLQuest (Becton Dickinson) software. Data were analyzed by gating live cells, as 5 judged by FSC versus SSC dot plots, and plotting the fluorescence profile as a histogram. The fold increase in fluorescence intensity above background was determined by measuring the shift in fluorescence intensity, using the control sample as background, from peak to peak. The results of independent experiments were then represented as the mean + the standard error of the mean (SEM). 10 Confocal microscopy The distribution of the NTA-DTDA on the surface of P815 cells was studied by laser scanning confocal microscopy using cells bearing incorporated NTA-DTDA engrafted with 15 biotinylated CD40-6H, and stained with streptavidin-FITC. Briefly, the cells were suspended in embedding medium (2% propyl gallate in 87% v/v glycerol) and deposited into 0.05 mm deep chambers on microscope slides formed using perforated Scotch 465 adhesive transfer tape, and the chambers were then sealed with glass cover slips. The cells were examined for fluorescence at 520 nm with a MRC-500 Laser Scanning Confocal Imaging 20 System (BioRad), consisting of a Nikon confocal fluorescence microscope (x 60 Nikon objective), with a BioRad UV-laser scanner and an Ion Laser Technology laser head (model 5425, BioRad) with an argon ion laser. The image was acquired by Kalman averaging of 10 successive laser scans, and stored and analyzed using Image Processor PC (BioRad) and processed using NIH Image 1.61 software. 25 T cell proliferation assays Murine T cells for use in T cell proliferation assays were isolated and purified from the spleens of either allogeneic or syngeneic mice as described (27). Briefly, the spleens were 30 dissociated into single cell suspensions, and dead cells and red blood cells were removed by density gradient centrifugation using an Isopaque-Ficoll gradient. After centrifugation (20 WO 00/64471 PCT/AUOO/00397 - 38 min at 400 x g) the viable cells, mainly lymphocytes, were collected from the layer at the top of the gradient and suspended in RPMI 1640 containing 10% v/v FCS, 5 x 10- M 2-p mercaptoethanol, 100 i.u./ml penicillin, 100 ptg/ml neomycin, IL-2, and 10 mM HEPES. T cells were purified using an equilibrated nylon wool column (28). The purified T cells were 5 then suspended in growth medium at a concentration of 2 x 104 cells/50 pl/well in a 96-well flat bottom plate (Cell Wells, Coming, NY) for culture at 37'C in an atmosphere of 5% CO 2 . T cell proliferation assays were carried out as described (28). Syngeneic lymphocytes or responder cells were then co-cultured with y-irradiated (5000 rad) stimulator cells at a 10 concentration of 2 x 104 cells/50 pl/well. Stimulator cells included native P815 tumor cells, P815 cells with incorporated NTA-DTDA on their surface, and P815 cells with engrafted recombinant protein(s), as indicated. After 4 days co-culture at 37'C, the cells were pulsed with 1 pCi of [ 3 H]-thymidine per well for 6 hrs. The cells were then harvested using a Filtermate 196 cell harvester (Packard) and [ 3 H]-thymidine incorporation assessed using 15 MicroScint scintillant and a Topcount NXT microplate scintillation counter (Packard) using Topcount software. Cytotoxicity assays 20 Assays for in vivo tumor-specific CTL were performed by a procedure similar to that described by Chen et al. (29). Syngeneic DBA/2 mice were immunized subcutaneously with either PBS (control) or 1 x 10' y-irradiated (5000 rad) P815 cells engrafted with recombinant protein(s). Spleens were removed from mice 14 days after immunization, and T lymphocytes (effector T cells) were isolated by density gradient centrifugation using 25 Isopaque-Ficoll and nylon wool fractionation, as described above. Effector T cells were then suspended in incubation medium and aliquoted into 24-well flat-bottom plates at a concentration of 1 x 10' cells/well and co-cultured with 1 x 105 y-irradiated (5000 rad) native P815 cells. After 5 days of co-culture, the cytolytic activity of the effector cells was assessed in a standard 5 'Cr-release assay, as described (29). Briefly, 2 x 106 native P815 cells 30 were labelled with 250 gCi 51 Cr (Na"CrO 4 ) for 90 min. Labelled target cells were washed three times and resuspended in culture medium. Effector and target cells were co-incubated WO 00/64471 PCT/AUOO/00397 - 39 with effector cells at different effector to target ratios, as indicated, for 6 hrs at 37 0 C. Supernatants were harvested and "Cr release assessed with a Topcount NXT microplate scintillation counter (Packard) using Topcount software (Packard). Percent specific lysis was calculated as follows: WO 00/64471 PCT/AUOO/00397 - 40 % ID x(pbwti qm - pntns qm) Mid qm - zpnnas qm) Immunization of animals and tumor challenge in vivo Mice were immunized using a protocol similar to that described (29). Briefly, either PBS (control) or 1 x 10' y-irradiated P815 cells with the engrafted recombinant protein(s) as 5 indicated, were suspended in a 0.2 ml volume of PBS and injected into the shaved right back of syngeneic DBA/2 mice using a 25-gauge needle and 1 ml syringe. After 14 days the mice were either used in cytotoxicity assays using T cells isolated from the spleens of the mice, or were challenged with 1 x 10' native P815 cells by subcutaneous injection in the shaved left back. For monitoring tumor growth, the mice were scored for tumor size once a week by 10 measuring two perpendicular diameters in millimeters using a caliper (29). Survival data represent animals that were still alive when scored; animals that were near death were euthanized after scoring and were deemed to have died of the tumor. Data for a total of 10 or 12 mice in the group for each experimental condition is presented. 15 EXAMPLE 1 The IAsys biosensor can be used to study the interaction of hexa-histidine-tagged proteins anchored onto the lipid layer. 20 A planar membrane layer consisting of PC and PE-NTA was formed on the sensing surface of the IAsys biosensor cuvette. Figure 2(a) shows the refractive index change due to formation of the layer (indicated Memb); there is no signal following two lots of washings with TBS (as marked), indicating that a stable layer is formed. Subsequently, the biosensor showed no change in refractive index on addition of BSA (see Figure 2(b)), indicating a low 25 level of non-specific binding. However, the addition of a 13 kDa transcription factor with hexa-histidine tag (denoted by HH in Figure 2(b)) resulted in a strong signal indicating binding of this protein to the NTA-PE in the membrane via the NTA chelating linkage. The anchored protein was not removed after washing and incubating in TBS, but was removed WO 00/64471 PCT/AUOO/00397 -41 with 200 mM imidazole known to break the chelating linkage (denoted Imid+TBS). Moreover, the interaction of other molecules with this protein also could be readily detected. The results show that hexa-histidine-tagged proteins can be stably and effectively anchored onto the layer to study molecular interactions with the biosensor. 5 EXAMPLE 2 Molecules anchored onto the lipid layer can diffuse laterally and interact. 10 A microscope slide was reacted with octadecyltrichlorosilane to form an octadecane monolayer (as described above). A phospholipid layer was then formed on the monolayer by placing a drop of the micelle (e.g. liposome) suspension (PC and biot-PE, 10:1) and incubating for 2 hours. After washing, the surface of the layer was stained with fluoresceinated streptavidin, incubated with Biot-BSA at 4'C, and then examined for 15 fluorescein fluorescence. Preliminary studies show that under these conditions the fluorescence is seen uniformly over the surface. However, after a 30-60 minute incubation of the slide at 37'C the fluorescence is seen in discrete patches, indicating that the fluoresceinated streptavidin forms aggregates with the biot-BSA. This shows that, depending on the temperature, molecules on the layer can diffuse laterally and interact. 20 EXAMPLE 3 Using the technique of this invention to resolve current difficulties in understanding of the CD4-MHC Class II interaction. 25 One application of the invention described herein is to study the interaction of CD4 with Major Histocompatibility Complex (MHC) Class II molecules, two key molecules involved in initiating an immune response by white blood cells. Crucial for the initiation of an antigen-specific immune response is the interaction of CD4 with invariant regions on the 30 MHC Class II molecule, which provides adhesion and stabilizes the interaction between the T cell receptor and the presented antigen. Although cellular immunology experiments WO 00/64471 PCT/AUOO/00397 - 42 strongly suggest that such an interaction is crucial for efficient T cell activation and the triggering of an immune response, to date no direct evidence of this interaction or how it takes place, has been obtained. 5 Recent X-ray crystallographic studies on the three-dimensional structure of the extracellular portion of the human MHC Class II (HLA-DR1) molecule have shown that this molecule crystallizes as a dimer of non-covalently associated heterodimers. This suggests a capacity of the MHC Class II to dimerize, perhaps leading to an increased avidity for the CD4 receptor. The crystal structure of human soluble CD4 has shown that the Dl-D2 and the D3-D4 10 regions of CD4 crystallize in multimeric form; but the affinity of self-association must be low because multimers do not form in solution. It has been suggested, that the extreme concentration of soluble CD4 in the crystal may reveal a natural propensity for oligomerization. Interestingly, biochemical evidence indicates that some CD4 immunoprecipitates as a disulfide-linked homodimer from lysates of sheep lymphocytes. 15 Recently, evidence that human CD4 can oligomerize also was obtained using CD4 chimeras in transfectants, and by chemically cross-linking adjacent CD4 molecules on the cell surface. However, these findings do not clarify whether oligomerization is essential to increase the avidity of the CD4-MHC Class II interaction. Application of the technique as described below will provide the means to redress this deficiency. 20 EXAMPLE 4 The aim of this example is to determine whether the extracellular regions of mouse and human CD4 can oligomerize when anchored onto membrane layers that allow lateral CD4 25 mobility. The atomic force microscope can be used to study the oligomerization of CD4 when anchored onto a lipid layer. A phospholipid layer consisting of 90% PC and 10% PE-NTA can be formed on a freshly cleaved and atomically flat surface of mica to permit high 30 resolution atomic force microscopy. The procedure for this is the same as that used for forming layer on the biosensor surface (see above). Alternatively, the surfaces of glass-like WO 00/64471 PCT/AUOO/00397 -43 materials, or surfaces coated with a uniform layer of gold (400-1000 A thick) using sputtered techniques, can be suitably treated with compounds like octadecyltrichlorosilane and octadecanethiol, respectively, to form a hydrophobic monolayer of hydrocarbon chains covalently attached to the surface and capable of reacting with a suspension of micelles (e.g. 5 liposomes) to produce a hybrid layer consisting of the attached monolayer of octadecane hydrocarbon chains and the phospholipid layer comprised of PC/PE-NTA. The recombinant extracellular region of CD4 with hexa-histidine tail can be anchored using the NTA chelating linkage onto layers formed on the glass or mica surface suitable for use in 10 the atomic force microscope. The particular PC used to produce the PC/PE-NTA membrane layer can be chosen with chain lengths such that the membrane transition temperature is about 23*C (eg. dimyristoylphosphatidylcholine). Atomic force measurements can then initially be carried out with the system maintained at 10*C to prevent CD4 lateral mobility to map the force structure with CD4 in the monomeric state. The temperature can then be 15 increased to 37'C for 30-60 minutes to allow CD4 to diffuse and interact laterally, before again cooling the system to 10*C to map the structure. Comparison of three-dimensional force maps of the CD4 molecules under the different conditions should allow determination of whether murine CD4 can oligomerize. 20 The IAsys biosensor can be used to measure the binding affinities (association and dissociation constants) for the CD4-MHC Class II interaction at 1 0 0 C and 37*C, using CD4 anchored onto layers formed on the sensing surface of the biosensor cuvette. The finding of a higher binding affinity for the interaction of CD4 with soluble MHC Class II after subjecting to temperatures above the membrane transition temperature would provide further evidence 25 that the extracellular region of CD4 has oligomerized. These experiments initially can be carried out using murine CD4 and MHC Class II with and without bound peptide. Similar experiments can be carried out using anchored human CD4 and soluble human MHC Class II. The force structure of CD4 anchored onto layers under conditions expected to exist in oligomeric form also can be mapped after binding soluble MHC Class II, if the interaction is 30 stable to permit analysis, to see if MHC Class II binding can induce changes in the structure.

WO 00/64471 PCT/AUO0/00397 - 44 EXAMPLE 5 The aim of this Example is to determine whether the extracellular regions of mouse and human MHC Class II oligomerize when anchored onto layers that allow lateral MHC Class 5 II mobility. Analogous to the studies described above for CD4, the atomic force structure of murine MHC Class II when anchored onto the layers can be mapped with the system maintained at 10*C, and compared with that observed after exposure to 37'C, to see if oligomerization 10 occurs. The studies also can be carried out with and without peptide bound to the MHC Class II groove (for the mouse system), to determine if bound peptide has an effect on MHC Class II oligomerization. The atomic force structure also can be mapped after binding soluble murine CD4 to see if this can induce changes in the force structure and/or promote MHC Class II oligomerization. The biosensor can be used with membrane-anchored MHC 15 Class II to measure the binding affinities of the interaction with soluble CD4, under conditions in which the MHC Class II is expected to exist in either monomeric or oligomeric form. The finding of a higher binding affinity of the MHC Class II for soluble CD4 after subjecting the system to temperatures above the membrane melting temperature, may provide additional evidence that MHC Class II can oligomerize. 20 EXAMPLE 6 The aim of this Example is to test the hypothesis that oligomerization of CD4, and oligomerization of MHC Class II, are both required for CD4-MHC Class II interaction. 25 Failure to detect binding of either monomeric or oligomeric CD4 to soluble MHC Class II, and either monomeric or oligomeric MHC Class II to soluble CD4 in the experiments outlined above could indicate that oligomerization of CD4 and MHC Class II are both necessary for stable interaction. If both CD4 and MHC Class II can be shown to oligomerize 30 in the membrane system, this possibility can be tested by examining whether CD4 and MHC Class II are able to interact under conditions where both are expected to exist in oligomeric WO 00/64471 PCT/AUOO/00397 - 45 form. This can be done by anchoring hexa-histidine-tagged MHC Class II onto unilamellar vesicles using the NTA chelating linkage, expose the vesicles to 37'C to permit MHC Class II oligomerization, and study the ability of these vesicles to interact with oligomeric CD4 using the IAsys biosensor. This can be done in both the mouse and human systems, but 5 since the mouse MHC Class II with and without peptide in the groove is available, the mouse MHC in the presence of bound peptide may be required to achieve the correct molecular confirmation required for the interaction. The feasibility of this approach has been demonstrated recently by studies showing the 10 interaction of biotin-PE and PC-containing vesicles with strepavidin anchored onto a lipid layer formed on the IAsys biosensor surface. Figure 3 is a biosensor trace showing changes in refractive index due to additions made to an lAsys biosensor cuvette after reacting the sensing surface of the cuvette with a suspension of vesicles consisting of PC and biot-PE. The initial change in refractive index is due to the formation of the layer. Subsequently, the 15 cuvette was washed with Tris buffer. After equilibration, the addition of BSA (indicated BSA) resulted in no change in refractive index, indicating a low level of non-specific binding. The addition of streptavidin (indicated Strep) resulted in a change in refractive index, which did not alter upon washing with Tris buffer (indicated Tris). The addition of a suspension of vesicles made of PC (indicated PC) did not bind to the membrane, but the 20 addition of vesicles made of PC and biot-PC did interact with the membrane, reflecting the binding of the vesicles containing the biot-PE with the streptavidin anchored onto the membrane. EXAMPLE 7 25 Since the Examples above use recombinant forms of the extracellular regions of receptor molecules oligomerization would be detected only if it arises from interactions via the extracellular region of the molecules. For many receptors this is unlikely to present difficulties as both the transmembrane and intracellular regions are short and are unlikely to 30 be involved in oligomerization. Certainly, for the CD4 and MHC Class II described above, this notion also is supported by X-ray crystallographic studies which show that the WO 00/64471 PCT/AUOO/00397 -46 extracellular domains of the molecules each can dimerize/oligomerize spontaneously in the absence of other molecules (e.g. those in the cytoplasm). However, other applications of the technique also include the anchoring of the cytoplasmic or transmembrane domain of receptor molecules onto the model membranes to study their involvement in oligomerization 5 and/or in interactions with other membrane associated or cytoplasmic molecules involved in signal transduction. Another useful application of the instant technology is to study the interaction of CD2 with its ligands. Thus, the use of multimeric binding techniques have identified the glycosyl 10 phosphatidylinositol (GPI) anchored molecule CD59 as an alternative ligand for human CD2, but biosensor studies have failed to confirm the interaction of CD2 with monomeric soluble CD59. This indicates that this interaction is of low affinity and that oligomerization of CD59 may be required to form a stable interaction with CD2. Clearly, analogous to the technique used above for studying CD4-MHC interactions, the anchoring of CD59 onto lipid 15 layers coupled with biosensor and atomic force analysis could be extremely useful in determining whether oligomerization of CD59 is required for interaction with CD2. This technique also is particularly suited in applications for studying the interactions of other GPI-anchored receptor molecules. Such receptor molecules could be anchored to the membrane either directly through their native GPI anchor, or by engineering a hexa-histidine 20 tag to replace the native GPI moiety, and using the hexa-histidine-NTA chelating linkage to anchor the receptors onto the membrane. The Examples described herein establish that the technique in conjunction with the technologies outlined is very useful for the analysis of other low affinity molecular 25 interactions. The subject technology can also be used to assay for interactions or aggregation events between receptor domains, proteins, glycoproteins, polysaccharides, or fragments thereof, after attachment of an appropriate hexa-histidine tag and the anchoring of the molecules onto the layer membrane system. This provides a method of screening for agents or drugs that may influence such receptor interactions. The invention therefore, provides the 30 basis of a novel method of screening for drugs or other agents that influence receptor interactions, and consequently, the ability of the receptors to transduce transmembrane WO 00/64471 PCT/AUOO/00397 - 47 signals and/or to elicit responses in biological systems. EXAMPLE 8 5 Using the instant invention to anchor or engraft hexa-histidine tagged molecules onto the surfaces of cells and other biological and synthetic membranes (see Figure 4), for the development of vaccines and for drug targeting. The histograms in Figure 5 show fluorescence-activated cell sorting (FACS) profiles of 10 murine mastocytoma P815 cells carrying engrafted recombinant hexa-histidine-tagged murine B7.1 and CD40. P815 cells were pre-incubated for 30 min at 37'C with a suspension (0.1 mM) of control lipid di-myristoyl-phosphatidylcholine (DMPC; also referred to as di-C 14-PC; control), or the chelator lipid NTA-DTDA, before being washed in PBS and incubated with a mixture of hexa-histidine-tagged B7.1 and CD40 (each at -20 mg/ml). The 15 cells were then washed again in PBS and stained by an incubation (30 min at 4"C) with either biotinylated 16-1OA1 or biotinylated B-3/23 monoclonal antibody (ie. biotinylated anti-B7.1 or anti-CD40), as indicated, followed by an incubation with FITC-conjugated streptavidin. Cells incubated with DMPC and recombinant proteins show a low level of fluorescence after staining with either monoclonal antibody (Control). The fluorescence of 20 P815 cells pre-incubated with NTA-DTDA is 10-100-fold higher than that of cells pre-incubated with DMPC (Control). Each result is a representative of two experiments performed in duplicate. The results show that chelator lipids (in this instance NTA-DTDA) can be incorporated into the membrane of these cells, and that the incorporated lipid can be used to anchor or engraft hexa-histidine tagged B7.1 and CD40 directly onto the P815 cell 25 surface via the NTA-DTDA. In other studies we showed that recombinant murine B7.1 and CD40 bearing a hexa-histidine tag can be engrafted onto the surface of all the different cell lines tested; these included murine P815 and EL4 tumor cells, human leukemic Jurkat cells and yeast cells. 30 EXAMPLE 9 WO 00/64471 PCT/AUOO/00397 -48 The Example relates to modifying the surface of tumor cells to enhance tumor immunity. Recent work indicates that the transmembrane and cytoplasmic regions of B7-1 and B7-2 are not required for T cell co-stimulation (23), and that T cell co-stimulation also occurs when 5 the B7-1 is expressed on tumor cell surfaces in a GPI-anchored form (24). Also, the extracellular regions of any cell surface receptor molecules (e.g. the murine T cell co stimulator molecules B7.1 and CD40) can be produced to contain a hexa-histidine or other appropriate peptide tag on the carboxyl terminal. In this form the present invention provides a method of anchoring these co-stimulator molecules directly onto the cell surface in the 10 correct orientation, thereby mimicking the co-stimulatory function of these molecules on the surface of antigen presenting cells. The instant invention, therefore, has implications for tumor vaccine development, by providing a more convenient and safe alternative to transfection for putting co-stimulator and/or other relevant molecules onto tumor cells for use in immunizations to enhance immunity to tumors. 15 The viability of using engrafted molecules can be tested by assaying for functional responses dependent on the engrafted molecules. Thus, the ability of murine P815 mastocytoma (DBA/2, H-2d) cells carrying engrafted hexa-histidine tagged B7-1 and/or CD40 to stimulate a T cell proliferative response in an allogenic system was examined using splenocytes 20 isolated from C57B1/6 (H-2b) mice co-cultured with an appropriate number of y-irradiated P815 cells (as control), or P815 cells engrafted with hexa-histidine-tagged B7-1 and/or CD40. Preliminary experiments in which the incorporation of 3H-thymidine was used to measure T cell proliferation, show that the P815 cells bearing engrafted hexa-histidine tagged B7-1 and/or CD40 are able to stimulate an increased level of T cell proliferation in 25 this mixed cell reaction. These results are consistent with the invention being useful to modify cells for use in vaccinations to enhance anti-tumor responses. EXAMPLE 10 30 To test the ability of P815 cells bearing engrafted co-stimulatory molecules to induce anti tumor responses in vivo, mice were immunized with P815 cells bearing the engrafted WO 00/64471 PCT/AUOO/00397 - 49 molecules to see if this could stimulate CTL activity and/or affect tumor growth in syngeneic animals. Separate groups of DBA/2 mice were immunized with either PBS, or with y irradiated P815 cells bearing engrafted EPOR-6H, B7.1-6H, or B7.1-6H plus CD40-6H. Two weeks after immunization, spleens were removed from the mice, and splenic T cells 5 were isolated and assessed for their ability to kill native P815 cells in a standard "Cr release assay. The data in Figure 6 show that at all the effector target cell ratios indicated (0.5:1, 1:1, 5:1), only a low level (2-5%) of lysis was induced by T cells from mice immunized with PBS (as control). The lytic activity of T cells from mice immunized with P815-EPOR (as control protein) was also low ranging from 7-16%. Interestingly, at all effector:target cell 10 ratios tested, the level of tumor cell-specific lysis was higher for conditions where the effector T cells were derived from mice immunized with P815 cells bearing one or more engrafted co-stimulatory molecule(s) (see Figure 6). The highest cytolytic activity was observed at the effector:target cell ratio of 5:1, for which the specific lysis induced by T cells obtained from mice immunized with P815 cells bearing engrafted B7. 1, and P815 cells with 15 engrafted B7.1 and CD40, was 3- and 5-fold higher, respectively, than that for T cells obtained from mice immunized with P815 cells engrafted with control protein (see Figure 6). Parallel experiments using native EL4 cells instead of P815 cells showed only background levels of lysis, indicating that the cytolytic response was specific for P815 cells as targets. The results indicate that CTL responses against P815 cells can be generated in 20 mice immunized with P815 cells bearing engrafted B7/CD40. To determine whether the immunization of mice with P815 cells bearing engrafted co stimulatory molecules could induce tumor immunity, groups of mice immunized with y irradiated cells bearing the engrafted proteins, also were monitored for tumor growth and 25 survival after a challenge with native P815 cells. These studies indicated a slower rate of tumor growth in mice immunized with P815 cells bearing engrafted co-stimulatory molecule(s), compared to mice immunized with cells bearing control protein. Thus, at 5 weeks after tumor challenge the mean tumor diameter was 3.36 ± 1.0 mm and 1.1 ± 0.9 mm, for mice immunized with P815 cells bearing engrafted B7.1-6H and B7.1-6H plus CD40 30 6H, respectively; and 10.7 ± 2.5 mm and 8.3 ± 2.7 mm for mice immunized with PBS and P815 cells engrafted with EPOR-6H, respectively. Tumor growth data as reflected by the WO 00/64471 PCT/AUOO/00397 -50 mean tumor diameter for only the first 5 weeks after challenge is presented since from this time some animals died from the tumor. At 14 weeks after tumor challenge survival was -17% for control mice, -30% for mice immunized with P815 cells engrafted with B7.1, and -60% for mice immunized with P815 cells engrafted with both B7.1 and CD40 (see Figures 5 7(a) and (b)). Consistent with the observed increase in CTL activity, the results indicate that the immunization of syngeneic animals with P815 cells bearing engrafted co-stimulatory molecules can inhibit tumor growth and prolong survival of the animals after a challenge with the native P815 tumor. 10 EXAMPLE 11 Vascular endothelial growth factor (VEGF) is a homodimeric glyocoprotein hormone of -40 kDa that is among the most potent of angiogenic mitogens, and a major regulator of angiogenesis (30). Considerable evidence suggests that VEGF is secreted by tumor cells and 15 other cells exposed to hypoxia, and that VEGF is a major angiogenic factor in solid tumor development (31). In addition to its potent angiogenic activity, VEGF increases vascular permeability and favours the migration of endothelial cells through the extravascular matrix, processes that are essential for tumor angiogenesis, tumor spread, and metastasis (32). Human VEGF is known to stimulate endothelial cell growth and differentiation by binding 20 to high affinity VEGF receptors such as the kinase domain receptor (human KDR, or murine flk-1), and the Fms-like tyrosine kinase-1 (Flt-1). These receptors are expressed exclusively on proliferating vascular endothelial cells, and their expression is known to be increased by a number of factors often produced by tumors (30-33). That VEGF and its receptors are important for tumor growth has been demonstrated by the fact that the neutralization of 25 VEGF by the use of antibodies (34) or recombinant soluble receptor domains (35) exhibit therapeutic potential as agents that can suppress tumor growth and metastasis in vivo. The substantially exclusive expression of VEGF receptors on proliferating endothelial cells, suggests the VEGF receptor can be used as a targeting molecule in therapeutic strategies that 30 target neovascularization. The development of sterically stabilized or "stealth" liposomes (SLs) which can evade elimination by the phagocytic cells of the immune system (e.g. the WO 00/64471 PCT/AUOO/00397 -51 reticulo-endothelial system in the liver and spleen), recently has provided a major advance for liposomal drug delivery in cancer chemotherapy (36-38). In contrast to conventional liposomes which are cleared rapidly from the blood (often within minutes) after their administration in vivo, SLs remain in the blood circulation for several days. A number of 5 studies have demonstrated therapeutic benefit of SLs containing an encapsulated cytotoxic drug like doxorubicin, in the treatment of AIDS-related Kaposi sarcomas and other lesions characterized by leaky vasculature (38). Recent studies also describe the targeting of SLs to specific tumors, with the targeting being achieved primarily by the use of "immuno liposomes", or liposomes with specific antibody (or F(ab') 2 fragments) covalently attached to 10 the liposome surface (39). The immobilization of targeting proteins such as antibodies onto SLs encapsulated with doxorubicin apparently does not alter their stealth-like characteristics, but can endow the liposomes with specific targeting properties (40-43). Until the advent of the present invention, the coupling of target molecules to liposomes has 15 been difficult and there is potential to induce unwanted anti-idiotypic responses to the antibody used. In accordance with an aspect of the present invention, two chelator lipids, nitrilotriacetic acid di-tetra-decylamine (NTA-DTDA) and nitrilotriacetic acid polyethyleneglycol (2000) phosphatidylethanolamine (NTA-PEG2000-PE), are used with a recombinant form of VEGF, to develop SLs containing encapsulated doxorubicin that will 20 target and specifically destroy proliferating vascular endothelial cells in vivo, thereby blocking neovascularization and tumor growth. VEGF is a particularly attractive targeting molecule as VEGF receptors are expressed substantially exclusively on angiogenic endothelium. In accordance with the present 25 invention, it is proposed to produce a "stealth" liposome containing encapsulated doxorubicin and surface VEGF anchored through NTA-lipids like NTA-DTDA. It is proposed in accordance with the present invention that when administered in vivo, this agent will target and destroy proliferating endothelial cells, and will inhibit tumorigenesis and tumor metastasis. 30 Liposomes composed largely of conventional lipids like egg-yolk phosphatidylcholine (PC) WO 00/64471 PCT/AUOO/00397 - 52 and cholesterol (Chol), and a small proportion (-10%) of a sterically "stabilizing" lipid such as ganglioside GM1 (35), or a phosphatidyl-ethanolamine conjugated to polyethyleneglycol (2000) (PEG2000-PE) (36, 37), are reported to exhibit increased stability and prolonged circulation times in blood, largely escaping elimination by the reticulo-endothelial system. 5 These properties of SLs have been attributed to the presence of lipids possessing uncharged headgroups which increase interaction with water, but inhibit interaction with either charged or hydrophobic structures likely to be encountered on proteins and cells in plasma (35, 36). Evidence suggests that SLs (or immunoliposomes) with antibody molecules attached to the distal end of the PEG chains on the SL surface, interact more effectively with their target 10 than liposomes with the antibody attached directly onto the SL surface. This has been explained by the PEG chains sterically interfering with the ability of the antibody to interact with antigen under these conditions (40). SLs made using ganglioside GM 1, or a PEG-lipid with a shorter PEG chain length (eg. PEG750-PE, rather than PEG2000-PE), therefore, are likely to be more suited for the binding of 6His-VEGF directly to the NTA-DTDA on the 15 liposome surface, and for optimal binding of the engrafted VEGF to VEGF receptors on target cells. Ganglioside GM1 and PEG750-PE are both commercially available (from Avanti Polar Lipids), and each will be tested with liposomes made from PC, Chol and NTA DTDA. To further reduce possible steric effects the inventors produce a novel lipid, namely NTA-PEG2000-PE, which consists of the NTA group attached to the distal end of the PEG 20 chain on the PE. This compound, is used in combination with PEG2000-PE to produce SLs which allow convenient engraftment of targeting 6His-proteins (such as 6His-VEGF), while eliminating the possibility of steric hindrance. This is approach greatly facilitates the use of SLs in therapeutic applications requiring their targeting to specific cells and/or tissues. 25 Recombinant 6His-VEGF is produced. SLs are produced from a mixture of lipids including PC, Chol, NTA-DTDA, and "stealth" lipids such as ganglioside GM 1, PEG2000-PE and NTA-PEG2000-PE. The SLs are engrafted with 6His-VEGF (VEGF-SLs) and then assessed for their ability to target endothelial cells. Conditions for specific binding of the liposomes to proliferating endothelial cells in culture are optimized, relative to their binding to cells that 30 lack the VEGF receptor. The specific cytoxicity of VEGF-SLs encapsulated with doxorubicin will be assessed using human vascular endothelial cells in vitro. VEGF-SLs WO 00/64471 PCT/AUOO/00397 - 53 intrinsically labelled with fluorescent dyes and/or radioactive tracers are administered intravenously into mice to determine their distribution in various tissues with time. The proportion of each stabilizing lipid used for producing SLs is altered to optimize the "stealth" properties of the liposomes, as judged by a reduction in the proportion of the 5 liposomes taken up by the liver, spleen and other major organs, relative to vascularizing tumors. As the VEGF receptor is endocytosed upon binding its ligand, VEGF-SLs made to contain encapsulated doxorubicin, are taken up by proliferating vascular endothelial cells, resulting in their destruction. The viability of this method is tested by examining the ability of the liposomes to inhibit tumor growth and/or to eradicate established tumors in vivo. This 10 work provides therefore a novel approach to anti-angiogenic cancer therapy. EXAMPLE 12 The method used to anchor proteins onto membranes is also used to engraft recombinant 15 receptors onto cells which, when used, vaccines can modify immunological responses in vivo. This is demonstrated by the fact that P815 cells engrafted with the co-stimulator molecules B7.1 and CD40 can be used as a vaccine to enhance tumor immunity. Analogously, these or 20 any other recombinant protein or molecules (possessing the appropriate tag) may be engrafted onto any other biological membranous structure(s) (e.g. a membranous structure derived from cells and/or sub-cellular components, such as plasma membranes vesicles etc.). The engrafted structures can then be used as a vaccine to enhance tumor immunity and/or modify immunological responses in vivo for therapeutic purposes. The preferred method 25 comprises: (i) incubating the cells or membranous material with a chelator lipid such as NTA DTDA to allow the lipid to incorporate in the cells or membranes; 30 (ii) washing off any unincorporated lipid by centrifugation or filtration and resuspension of the structures in the appropriate solution or buffer; WO 00/64471 PCT/AUOO/00397 - 54 (iii) incubating the membranous structures containing incorporated chelator lipid with an appropriate recombinant protein(s) possessing an appropriate affinity tag; and 5 (iv) washing off unincorporated protein material; and then using the modified structure as a vaccine administrable in vivo for therapeutic purposes such as to modify immunological responses in vivo. 10 The subject approach can also be used with synthetic membrane structures (i.e. synthetic liposomes or vesicles composed of a mixture of any phospholipid (e.g. PC or PE) and the NTA-DTDA. The synthetic structures can be made to incorporate the NTA-DTDA, therefore, only steps (iii) to (v) above are required. 15 EXAMPLE 13 Preliminary experiments indicate that synthetic liposomes (composed of say PC and NTA DTDA in 10:1 ratio) engrafted with an appropriate recombinant receptor protein can be used to specifically target cells bearing the cognate receptor or ligand (see Figure 8). Such 20 liposomes also can be used to modify biological response (see Figure 9). The present invention provides, therefore, a method of engrafting recombinant proteins onto liposomes for use in therapeutic applications to deliver an encapsulated drug or other therapeutic agent to cells or tissues within the body. Such liposomes are used to modify a biological response(s) for the treatment of disease, or for targeting the delivery of cytotoxic drugs or 25 agents to specific cells (e.g. tumor cells) in order to destroy such cells for therapeutic purposes. Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood 30 that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in WO 00/64471 PCT/AUOO/00397 - 55 this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

WO 00/64471 PCT/AUOO/00397 - 56 BIBLIOGRAPHY 1. Shao, Z. (1995) Quart. Revs Biophys. 28: 195-25 1. 2. Garland, P.B. (1996) Quart. Revs Biophys. 29: 91-117. 3. Khilko et al. (1995) J. mmunol. Meth. 183: 77-94. 4. Velge-Rousel et al. (1995) J. Immunol. Meth. 183: 141-148. 5. Gershon, P.D. and Khilko, S. (1995) J Immunol. Meth. 183: 65-67. 6. Plant et al. (1995) Analyt. Biochem. 226: 342-348. 7. Dietrich et al. (1995) Proc. Nat. Acad. Sci. (USA) 92: 9014-9018. 8. Dietrich et al. (1996) Biochemistry 35: 1100-1105. 9. Kalb et al. (1992) Biochim. Biophys. Act. 1103: 307-316. 10. McConnel et al. (1986) Biochim. Biophys. Act. 864: 95-106. 11. Kalury et al. (1992) Langmuir 8: 947-954. 12. Du et al. (1997) Biochim Biophys Acta, 1326(2), 236-248. 13. Owens, T. (1996) Current Biology 6: 32-35. 14. Guinan et al. (1994) Blood 84: 3261-3282. 15. Hellstrom et al. (1995) Immunol. Rev. 145: 123-145. 16. La Motte et al. (1996) Cancer Immunol. Immunother. 42: 161-169. 17. Chaux et al. (1996) Int. J. Cancer 66: 244-248. 18. Fujii et al. (1996) Int. J. Cancer 66: 219-224. 19. Martin-Fontecha et al. (1996) Eur. J. Immunol. 26: 1851-1859. 20. Yang et al. (1995) J. Immunol. 154:2794-2800. 21. Shinde et al. (1996) J. Immunol. 157: 2764-2768. 22. Cavallo et al. (1995) Eur. J. Lmmunol. 25:1154-162. 23. Brunschwig et al. (1995) J. Immunol. 155: 5498-5505. 24. McHugh et. al. (1995) Proc. Nat. Acad. Sci. 92:8059-8063. 25. Schmitt et al. (1994) J. Am. Chem. Soc. 116:8485-8491. 26. Altin et al. (1994) Eur. J. Immunol. 24:450-457. 27. Parish et al. (1974) Eur. J Immunol. 4:808-815. 28. Greenfield et al. (1997) J Immunol. 158:2025-2034. 29. Chen et al. (1994) Cancer Res. 54:5420-5423.

WO 00/64471 PCT/AUOO/00397 - 57 30. Achen M.G. and Stacker, S.A. (1988) Int. J. Exp. Pathol. 79:255-265. 31. Maxwell et al. (1997) Proc. Nat. Acad. Sci. (USA) 94:8104-8109. 32. Brown, J.M. and Giaccia, A.J. (1998) Cancer Research 58:1408-1416. 33. Weismann et al. (1997) Cell 91:695-704. 34. Wang et al. (1998) J Cancer Res. Clin. Oncol. 124:615-620. 35. Lin et al. (1998) Cell Growth Differ. 9:49-58. 36. Papahadjopoulos et al. (1991) Proc. Natl. Acad. Sci. (USA) 88:11460-11464. 37. Allen et al. (199) Biochim. Biophys. Act. 1066:29-36. 38. Lasic, D.D. and Papahadjopoulos D. (1995) Science 267:1275-1276. 39. Ahmad et al. (1993) Cancer Research 53:1484-1488. 40. Hansen et al. (1995) Biochim. Biophys. Act. 1239:133-144. 41. DeMenezes et al. (1998) Cancer Research 58:3320-3330. 42. Kirpotin et al. (1997) Biochem. 36:66-75. 43. Vaage et al. (1999) Int. J. Cancer. 80:134-137.

Claims (38)

1. A method of anchoring receptor domains onto a planar supported membrane comprising amphiphilic molecules arranged in a layer, wherein a proportion of the amphiphilic molecules have been modified by a covalent attachment of a metal chelating group such that some of the metal chelating groups are oriented toward the outside surface of said membrane; which method comprises the step of interacting a receptor domain which is covalently attached to a polypeptide tag with said membrane for a time and under conditions sufficient for said polypeptide tag to attach to said membrane via the outwardly facing metal chelating residues of said membrane, such that the receptor domains are capable of lateral movement, dimerization or oligomerization, and interaction with a ligand molecule.

2. A method according to Claim 1 wherein the metal chelating group is nitrilotriacetic (NTA).

3. A method according to Claim 2 wherein the membrane is formed from a suspension of micelles or liposomes from the amphiphilic molecules.

4. A method according to Claim 2 wherein the membrane is formed by the addition of water or aqueous buffers to a solution of amphiphilic molecules in an organic solvent.

5. A method according to Claims 3 or 4 wherein a proportion of the amphiphilic molecules have been modified by a covalent attachment of a metal chelating group.

6. A method of anchoring receptor domains onto a lipid membrane such that the receptor domains are capable of lateral movement, said method comprising of: (i) forming a membrane on an appropriate planar support by either: (a) contacting and incubating the support for a period of time with a solution in an organic solvent of amphiphilic molecules wherein a proportion of the WO 00/64471 PCT/AUOO/00397 - 59 molecules have been modified by covalent attachment of a metal chelating group, then forming a membrane by the addition of water or an aqueous buffer; or (b) forming a suspension of micelles (e.g. liposomes) from amphiphilic molecules wherein a proportion of the amphiphilic molecules have been modified by a covalent attachment of a metal chelating group, then interacting said micelle (e.g. liposome) suspension with an appropriate planar support for a time and under conditions sufficient to allow a membrane layer to form in which some metal chelating residues attached to the amphiphilic molecules of said micelle (e.g. liposome) suspension are oriented toward the outside surface of said membrane; and (ii) interacting the receptor domain(s) to be anchored which is covalently attached to a polypeptide tag with said membrane for a time and under conditions sufficient for said polypeptide tag to attach via the metal chelating linkage to the outwardly-facing metal chelating residues of said membrane.

7. A method according to Claims 1 or 6 wherein the amphiphilic molecules are surfactant molecules having a hydrophilic "head" portion and one or more hydrophobic "tails".

8. A method of anchoring receptor domains onto a lipid membrane such that said receptor domains are capable of lateral movement to facilitate interactions with biomolecules in the membrane, said method comprising of: (i) forming a membrane on an appropriate planar support by either: (a) contacting and incubating the support for a period of time with a solution in an organic solvent of amphiphilic molecules wherein a proportion of the molecules have been modified by covalent attachment of a metal chelating WO 00/64471 PCT/AUOO/00397 - 60 group, then forming a membrane by the addition of water or an aqueous buffer; or (b) forming a suspension of micelles (e.g. liposomes) from a first phospholipid and a second phospholipid wherein the second phospholipid has been modified by a covalent attachment of a nitrilotriacetic acid (NTA), and then interacting said micelle (e.g. liposome) suspension with an appropriate planar support for a time and under conditions sufficient to allow a phospholipid membrane layer to form in which some NTA residues attached to the second phospholipid of said micelle (e.g. liposome) suspension are oriented toward the outside surface of said membrane; and (ii) interacting the receptor domain(s) to be anchored which is covalently attached to a polypeptide tag with said membrane for a time and under conditions sufficient for said polypeptide tag to attach via the NTA-metal chelating linkage to the outwardly-facing NTA residues of said membrane.

9. A method according to Claim 8 wherein the first phospholipid is phosphatidylcholine (PC) and the second lipid is phosphatidyl-ethanolamine-NTA (PE-NTA) and the ratio of PC:PE-NTA (w/v) is about 10:1.

10. A method according to any one of the preceding claims wherein the support is the sensing surface of an optical biosensor or a mica surface suitable for analysis by atomic force microscopy.

11. A method according to any one of the preceding claims wherein the polypeptide tag comprises a sequence of at least six amino acid residues.

12. A method according to Claim 11 wherein the six amino acid residues is hexa histidine. WO 00/64471 PCT/AUOO/00397 - 61

13. A method of assaying interactions between a receptor and a ligand, comprising the steps of: (A) anchoring a receptor to a membrane by: (i) forming a membrane on an appropriate planar support by either: (a) contacting and incubating the support for a period of time with a solution in an organic solvent of amphiphilic molecules wherein a proportion of the molecules have been modified by covalent attachment of a metal chelating group, then forming a membrane by the addition of water or an aqueous buffer; or (b) forming a suspension of micelles (e.g. liposomes) from amphiphilic molecules wherein a proportion of the amphiphilic molecules have been modified by a covalent attachment of a metal chelating group, and then interacting said micelle (e.g. liposome) suspension with an appropriate planar support for a time and under conditions sufficient to allow a membrane layer to form in which some metal chelating residues attached to the amphiphilic molecules of said micelle (e.g. liposome) suspension are oriented toward the outside surface of said membrane; and (ii) interacting the receptor domain(s) to be anchored which is covalently attached to a polypeptide tag with said membrane for a time and under conditions sufficient for said polypeptide tag to attach via the metal chelating linkage to the outwardly-facing metal chelating residues of said membrane; (B) allowing said receptor molecules to interact and/or oligomerize on the membrane; and WO 00/64471 PCT/AUOO/00397 - 62 (C) contacting said anchored receptor with an effective concentration of ligand for a time and under conditions sufficient for binding to occur and detecting said binding.

14. A method according to Claim 13 wherein the interaction and/or oligomerization of the receptor and/or binding to the ligand is detected by atomic force microscopy or by an optical biosensor technique, for use in assays of intermolecular interactions, receptor dimerization/oligomerization, or in devices for the screening of drugs which affect these processes.

15. A method of engrafting or anchoring molecules onto liposomes, said method comprising: (i) preparing a suspension of liposomes with chelator lipid incorporated, and with or without an encapsulated drug or agent; (ii) incubating the liposomes with a recombinant protein or target molecule bearing an appropriate metal affinity tag; and (iii) if necessary, removing excess protein by washing, filtering or other washing means and suspending them in an appropriate solution.

16. A method according to Claim 15 wherein the molecules engrafted, anchored or encapsulated within the liposome are therapeutic molecules, pharmaceutical compounds, DNA and/or RNA.

17. A method according to Claim 16 wherein the molecules engrafted or anchored onto the liposome surface is VEGF or its homologue.

18. A method according to Claim 17 wherein the liposome is encapsulated with a cytotoxic drug or agent together with the engrafted VEGF or its homologue to block the growth of new blood vessels needed for the growth of tumors. WO 00/64471 PCT/AUOO/00397 - 63

19. A method according to Claim 16 wherein the liposome comprises an immunogenic agent and together with an agent which targets the liposome to different cell types in the body including immune cells and tumor cells to alter immunogenicity or immunological responses.

20. A method of anchoring a recombinant molecule directly onto biological membranes, said method comprising: (i) preparing a suspension of chelator lipid or liposomes containing the chelator lipid; (ii) incubating a suspension of cells or biological membranous structures with a suspension of the chelator lipid to allow the chelator lipid to incorporate into the structures; (iii) washing away excess or unincorporated lipid; (iv) incubating the membranous structures with a solution of recombinant protein or target molecule possessing an appropriate metal affinity tag; and (v) washing away excess or unbound soluble protein, and suspending the structures in a solution suitable for administration in vivo.

21. A method according to Claim 20 wherein the recombinant molecule is a co stimulatory molecule.

22. A method according to Claim 20 or 21 wherein the biological membrane is from a tumor cell.

23. A method according to Claim 20 or 21 or 22 for use in enhancing or WO 00/64471 PCT/AUOO/00397 - 64 modifying immunity to tumors or other disease conditions.

24. A method according to Claim 20 wherein the recombinant molecule is a receptor or ligand.

25. A method according to Claim 24 wherein the recombinant molecule is a ligand for a receptor on specific cell types within the body or on cells such as tumor cells that arise as a consequence of disease.

26. A method for altering the immunogenicity of a target cell or membranous component thereof, said method comprising anchoring a molecule to the membrane of said target cell by: (i) preparing a suspension of chelator lipid or liposomes containing the chelator lipid; (ii) incubating a suspension of cells or membranous structures with a suspension of the chelator lipid; (iii) washing away excess or unincorporated lipid; (iv) incubating the membranous structures with a solution of said molecule to be anchored; and (v) washing away excess or unbound soluble molecule, and suspending the structures in a solution suitable for administration in vivo.

27. A method according to Claim 26 wherein the target cell is a tumor cell.

28. A method according to Claim 26 or 27 wherein the molecule is a ligand, receptor, recombinant protein, polysaccharide, glycoprotein or antigen. WO 00/64471 PCT/AUOO/00397 - 65

29. A method of targeting a liposome or a biological membranous structure to a particular cell or tissue, said method comprising anchoring or engrafting a molecule having a binding partner on the particular cell or tissue to be targeted by: (i) preparing a suspension of chelator lipid or liposomes containing the chelator lipid; (ii) incubating a suspension of cells or biological membranous structures with a suspension of the chelator lipid; (iii) washing away excess or unincorporated lipid; (iv) incubating the liposomes or membranous structures with a solution of molecules to be anchored; and (v) washing away excess or unbound soluble molecule, and suspending the structures in a solution suitable for administration in vivo.

30. A method of treatment, said method comprising administering to a subject an effective amount of a liposome preparation or membranous material comprising an active material and optionally an anchored or engrafted molecule having a binding partner or target tissue.

31. A method according to Claim 30 wherein the active material is a recombinant polypeptide, co-stimulatory molecule, therapeutic drug or nucleic acid molecule, either engrafted onto the surface or encapsulated within the liposome or membranous material.

32. A method according to Claim 30 or 31 wherein the anchored or engrafted molecule is a receptor, ligand, glycoprotein, polysaccharide or recombinant polypeptide.

33. A method according to Claim 32 wherein the anchored molecule is VEGF. WO 00/64471 PCT/AUOO/00397 - 66

34. A method according to any one of Claims 30 to 33 when used to enhance immunity to a specific tumor or disease.

35. A method according to Claim 31 wherein the co-stimulatory molecule is CD40 or B7.1.

36. A vaccine composition comprising cells or membranous material having engrafted thereto molecules capable of modifying an immunological response to a subject to which the vaccine is administered, said vaccine further comprising one or more pharmaceutical carriers and/or diluents.

37. A vaccine according to Claim 36 wherein the molecules engrafted to the cells or membranous material are co-stimulatory molecules.

38. A vaccine according to Claim 36 or 37 prepared by the steps of: (i) incubating the liposomes, cells or membranous material with a chelator lipid such as NTA-DTDA to allow the lipid to incorporate in the cells or membranes; (ii) washing off any unincorporated lipid by centrifugation or filtration and resuspension of the liposomes, cells or membranous structures in the appropriate solution or buffer; (iii) incubating the liposomes, cells or membranous structures with incorporated chelator lipid with said molecules to be engrafted; and (iv) washing off unincorporated molecular material. WO 00/64471 PCT/AUOO/00397 67 AMENDED CLAIMS [received by the International Bureau on 25 August 2000 (25.08.00); original claims 1-14 replaced by; new claims 1-14; amended claims 15-38 (8 pages)] CLAIMS 1. A method of targeting a membranous structure such as a biological or synthetic membrane, cell or organelle, to a specific cell type or tissue in vivo, said method comprising incorporating into said membranous structure, a chelator lipid and/or amphiphilic molecule which facilitates anchoring targeting molecules such as receptors, to allow the membranous structures to be targeted and be used to modify a biological or physiological response. 2. A method according to Claim 1 wherein the amphiphilic molecule is modified by a covalent attachment of a metal chelating group, with metal chelating groups orientated toward the outside surface of said membranous structure. 3. A method according to Claim 2 comprising the steps of: (i) incorporating the chelator lipid into the membranous structure by mixing and/or co-incubation, or by production of the membrane from a composite mixture of lipids comprising a chelator lipid and one or more other lipids or phospholipids; and (ii) interacting a targeting molecule with said membranous structure for a sufficient time and under suitable conditions to attach to said membranous structure via the outwardly facing metal chelating residues of said membranous structure, such that the receptor domains or targetable molecules are capable of lateral movement, dimerization/oligomerization and/or interaction with a ligand. WO 00/64471 PCT/AUOO/00397 68 4. A method according to Claim 3 wherein the targeting molecule is a receptor domain and/or other targetable molecule engineered to possess a metal binding polypeptide tag. 5. A method according to Claim 4 wherein the receptor domain or targetable molecule is interacted with a membranous structure for a time and under conditions sufficient for the polypeptide tag to bind to the chelator lipid incorporated into the membranous structure. 6. A method according to Claim 1 or 2 or 3 or 4 or 5 wherein the membranous structure is a suspension of micelles or liposomes formed from the amphiphilic molecules by sonication, or extrusion/filtration techniques. 7. A method according to Claim 2 wherein the metal chelating group is nitrilotriacetic (NTA). 8. A method according to Claim I wherein a proportion of the amphiphilic molecules have been modified by a covalent attachment of a metal chelating group. 9. A method according to Claims 1 or 8 wherein the amphiphilic molecules are surfactant molecules having a hydrophilic head portion and one or more hydrophobic tails. 10. A method according to any one of the preceding claims wherein the polypeptide tag comprises a sequence of amino acid residues. 11. A method according to claim 10 wherein the amino acid residues are histidine residues. WO 00/64471 PCT/AUOO/00397 69 12. A method to claim 10 or 11 wherein the polypeptide tag comprises at least five amino acid residues. 13. A method according to claim 12 wherein the polypeptide tag comprises at least six amino acid residues. 14. A method according to claim 13 wherein the polypeptide tag comprises hexa histidine. 15. A method of modifying biological or synthetic membranes for: (i) vaccine development; (ii) for modification of biological response(s); and/or (iii) for the targeting of drugs or agents to specific tissue or cell types within the body to achieve a therapeutic effect, said method comprising: (i) preparing a suspension of liposomes with chelator lipid incorporated and with or without an encapsulated drug or agent; (ii) incubating the liposomes with a recombinant protein or target molecule bearing an appropriate metal affinity tag; and (iii) if necessary, removing excess protein or molecule by washing, filtering or other washing means and suspending them in an appropriate solution. WO 00/64471 PCT/AUOO/00397 70 16. A method according to Claim 15 wherein the molecules engrafted, anchored or encapsulated within the liposome are therapeutic molecules, pharmaceutical compounds, DNA and/or RNA. 17. A method according to Claim 16 wherein the molecules engrafted or anchored onto the liposome surface is VEGF or its homologue. 18. A method according to Claim 17 wherein the liposome is encapsulated with a cytotoxic drug or agent together with the engrafted VEGF or its homologue to block the growth of new blood vessels for the growth of tumours. 19. A method according to Claim 15 wherein the liposome comprises an immunogenic agent and together with an agent which targets the liposome to different cell types in the body including immune cells and tumor cells to alter immunogenicity or immunological responses. 20. A method of anchoring a recombinant molecule directly onto biological membranes, said method comprising: (i) preparing a suspension of chelator lipid or liposomes containing the chelator lipid; (ii) incubating a suspension of cells or biological membranous structures with a suspension of the chelator lipid to allow the chelator lipid to incorporate into the structures; (iii) washing away excess or unincorporated lipid; WO 00/64471 PCT/AUO0/00397 71 (iv) incubating the membranous structures with a solution of recombinant protein or target molecule possessing an appropriate metal affinity tag; and (v) washing away excess or unbound soluble protein, and suspending the structures in a solution suitable for administration in vivo. 21. A method according to Claim 20 wherein the recombinant molecule is a co stimulatory molecule. 22. A method according to Claim 20 or 21 wherein the biological membrane is from a tumor cell. 23. A method according to Claim 20 or 21 or 22 for use in enhancing or modifying immunity to tumors or other disease conditions. 24. A method according to Claim 20 wherein the recombinant molecule is a receptor or ligand. 25. A method according to Claim 24 wherein the recombinant molecule is a ligand for a receptor on specific cell types within the body or on cells such as tumor cells that arise as a consequence of disease. 26. A method for altering the immunogenicity of a target cell or membranous component thereof, said method comprising anchoring a molecule to the membrane of said target cell by:- WO 00/64471 PCT/AUOO/00397 72 (i) preparing a suspension of chelator lipid or liposomes containing the chelator lipid; (ii) incubating a suspension of cells or membranous structures with a suspension of the chelator lipid; (iii) washing away excess or unincorporated lipid; (iv) incubating the membranous structures with a solution of said molecule to be anchored; and (v) washing away excess or unbound soluble molecule, and suspending the structures in a solution suitable for administration in vivo. 27. A method according to Claim 26 wherein the target cell is a tumor cell. 28. A method according to Claim 26 or 27 wherein the molecule is a ligand, receptor, recombinant protein, polysaccharide, glycoprotein or antigen. 29. A method of targeting a liposome or a biological membranous structure to a particular cell or tissue, said method comprising anchoring or engrafting a molecule having a binding partner on the particular cell or tissue to be targeted by: (i) preparing a suspension of chelator lipid or liposomes containing the chelator lipid; WO 00/64471 PCT/AUOO/00397 73 (ii) incubating a suspension of cells or biological membranous structures with a suspension of the chelator lipid; (iii) washing away excess or unincorporated lipid; (iv) incubating the liposomes or membranous structures with a solution of molecules to be anchored; and (v) washing away excess or unbound soluble molecule, and suspending the structures in a solution suitable for administration in vivo. 30. A method of treatment, said method comprising administering to a subject an effective amount of a liposome preparation or membranous material comprising an active material and optionally an anchored or engrafted molecule having a binding partner or target tissue. 31. A method according to Claim 30 wherein the active material is a recombinant polypeptide, co-stimulatory molecule, therapeutic drug or nucleic acid molecule, either engrafted onto the surface or encapsulated within the liposome or membranous material. 32. A method according to Claim 30 or 31 wherein the anchored or engrafted molecule is a receptor, ligand, glycoprotein, polysaccharide or recombinant polypeptide. 33. A method according to Claim 32 wherein the anchored molecule is VEGF. 34. A method according to any one of Claims 26 to 33 when used to enhance immunity to a specific tumor or disease. WO 00/64471 PCT/AUOO/00397 74 35. A method according to Claim 31 wherein the co-stimulatory molecule is CD40 or B7.1. 36. A vaccine composition comprising cells or membranous material having engrafted thereto molecules capable of modifying an immunological response to a subject to which the vaccine is administered, said vaccine further comprising one or more pharmaceutical carriers and/or diluents. 37. A vaccine according to Claim 36 wherein the molecules engrafted to the cells or membranous material are co-stimulatory molecules. 38. A vaccine according to Claim 36 or 37 prepared by the steps of: (i) incubating the liposomes, cells or membranous material with a chelator lipid such as NTA-DTDA to allow the lipid to incorporate in the cells or membranes; (ii) washing off any unincorporated lipid by centrifugation or filtration and resuspension of the liposomes, cells or membranous structures in the appropriate solution or buffer; (iii) incubating the liposomes, cells or membranous structures with incorporated chelator lipid with said molecules to be engrafted; and (iv) washing off unincorporated molecular material.