WO2021116712A1 - Functionalised microbubble mediated cell tagging - Google Patents

Functionalised microbubble mediated cell tagging Download PDF

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Publication number
WO2021116712A1
WO2021116712A1 PCT/GB2020/053198 GB2020053198W WO2021116712A1 WO 2021116712 A1 WO2021116712 A1 WO 2021116712A1 GB 2020053198 W GB2020053198 W GB 2020053198W WO 2021116712 A1 WO2021116712 A1 WO 2021116712A1
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microbubble
macromolecule
lipid
cell
cells
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PCT/GB2020/053198
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French (fr)
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Eleanor Stride
Veerla BRANS
Erdinc SEZGIN
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Oxford University Innovation Limited
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Publication of WO2021116712A1 publication Critical patent/WO2021116712A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0023—Agression treatment or altering
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6925—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a microcapsule, nanocapsule, microbubble or nanobubble
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers

Definitions

  • the present invention provides a method for presenting macromolecules of interest at the outer surface of the plasma membrane of eukaryotic cells. More specifically, the method uses microbubbles carrying the macromolecule of interest bound to a functionalised lipid component of the microbubble shell together with focused low intensity ultrasound to transfer the macromolecule from the microbubble to a site in or on the outer surface of the plasma membrane and/or to induce the uptake of the lipid-macromolecule complex by the plasma membrane of eukaryotic cells to which the microbubbles have been exposed.
  • the method may be used to incorporate macromolecules of interest into the plasma membrane of cells in vivo, ex vivo or in vitro and thus therapeutic and non-therapeutic uses are envisaged, in particular the combat of hyperproliferative disorders.
  • the invention further provides said functionalised microbubbles, with or without the macromolecule of interest bound thereto, and kits for the preparation of the same.
  • Gas microbubbles coated with a surfactant or polymer shell, have become established as an effective type of contrast agent available for diagnostic ultrasound imaging.
  • the gas core of the microbubbles scatters ultrasound both more efficiently and over a wider range of frequencies than biological cells, producing strong contrast between the vasculature and the surrounding tissue.
  • microbubbles may be used in the targeted delivery of a drug or therapeutic agent to a specific organ or tissue, or cell thereof.
  • Therapeutic agents may be incorporated into the encapsulating shell of the microbubbles.
  • the microbubbles can then be introduced into the body at their intended site of action or introduced intravenously and their passage through the bloodstream can be traced using low intensity imaging ultrasound.
  • the therapeutic agent can be released by applying focused low intensity therapeutic ultrasound (typically longer pulses and higher frequencies than imagining ultrasound) to rupture the microbubbles selectively.
  • focused low intensity therapeutic ultrasound typically longer pulses and higher frequencies than imagining ultrasound
  • the rupture of the microbubbles may also enhance the cellular uptake of the therapeutic agent or drug, e.g. macromolecules such as proteins and nucleic acids, by temporarily increasing the membrane permeability of nearby cells (Rahim et al; Ultrasound Med. Biol., 2006; 32(8): 1269-79). This effect is known as sonoporation.
  • the cells of an animal subject may be tagged selectively with a macromolecule which is recognised by the subject's immune system and which induces the immune system to eliminate the cells displaying such a label.
  • target cells of an animal subject may be tagged selectively with a macromolecule which is part of a specific and selective binding pair and which is not found naturally in the subject or the local region in which such target cells are found and administration of a drug conjugated to a specific binding partner of the macromolecule will direct that drug to those cells that display the label.
  • delivery of macromolecules to the cell surface will allow the study of cell surface biology. Many other applications of such a technique would be readily apparent to the skilled person.
  • microbubbles of a particular composition may be engineered to carry macromolecules in a manner which permits the presentation of said macromolecules at the outer surface of the plasma membrane of eukaryotic cells when said microbubbles are exposed to particular ultrasound conditions.
  • the invention provides a method for presenting a macromolecule of interest at the outer surface of the plasma membrane of a eukaryotic cell, said method comprising
  • the invention also provides a microbubble comprising a gas core and a shell, wherein said shell comprises a lipid-modified chelator and wherein a metal cation is immobilised by said chelator and a macromolecule of interest is bound to the metal cation via one or more coordinate bonds for use in a method for presenting said macromolecule at the outer surface of the plasma membrane of a eukaryotic cell in or on a subject, said method comprising
  • the invention also provides the use of a microbubble comprising a gas core and a shell, wherein said shell comprises a lipid-modified chelator and wherein a metal cation is immobilised by said chelator and a macromolecule of interest is bound to the metal cation via one or more coordinate bonds in the manufacture of a medicament for presenting said macromolecule at the outer surface of the plasma membrane of a eukaryotic cell in or on a subject, said method comprising
  • the macromolecule may be therapeutically or diagnostically effective in that its presentation at the outer surface of the plasma membrane of a eukaryotic cell can result in a therapeutic effect, e.g. an immunotherapeutic effect, either directly or indirectly, or can provide information useful in the diagnosis or monitoring of disease.
  • a therapeutic effect e.g. an immunotherapeutic effect, either directly or indirectly, or can provide information useful in the diagnosis or monitoring of disease.
  • the above methods may be defined as therapeutic and/or diagnostic methods.
  • Presentation of a macromolecule at the outer surface of the plasma membrane may be expressed alternatively as the display or showing of said macromolecule at this location.
  • the act of using the microbubbles of the invention to present a macromolecule at the outer surface of the plasma membrane may be described as incorporating a macromolecule into the plasma membrane, or introducing, delivering or transferring a macromolecule to the plasma membrane, more specifically from the microbubble.
  • the act of using the microbubbles of the invention to present a macromolecule at the outer surface of the plasma membrane may be described as tagging, labelling or marking the plasma membrane with the macromolecule.
  • “presentation of the macromolecule at the outer surface of the plasma membrane of a eukaryotic cell” may be a result of the transfer of the macromolecule from the microbubble to a site in or on the outer surface of the plasma membrane of a eukaryotic cell and/or a result of the introduction of the macromolecule-lipid complex of the microbubble into the outer surface of the plasma membrane of a eukaryotic cell upon exposure to suitable ultrasound conditions. This latter process may be due to fusion of the microbubble shell with the plasma membrane or by selective transfer of the macromolecule-lipid complex from the microbubble shell to the plasma membrane or by any other means.
  • contact therefore includes sufficiently close proximity between the eukaryotic cell and the microbubble to permit the transfer of the macromolecule from the microbubble to a site in or on the outer surface of the plasma membrane of a eukaryotic cell and/or the introduction of the macromolecule-lipid complex of the microbubble into the outer surface of the plasma membrane of the eukaryotic cell upon exposure to suitable ultrasound conditions.
  • Microbubbles of use in the invention comprise a gas core and a shell, wherein said shell comprises a lipid-modified chelator and wherein a metal cation is immobilised by said chelator.
  • a chelator is an organic compound which carries one or more polar groups capable of forming at least one, preferably at least two, stable coordinate bonds with a metal cation. Such compounds may be referred to as chelants, chelating agents, or sequestering agents.
  • the chelator may be nitrilotriacetic acid (NTA), iminodiacetic acid (IDA), diethylenetriaminepentaacetic acid (DTPA), 1,4,7,10-tetraazacyclododecane;
  • DOTA 1,4, 8,11-tetraazacyclotetradecane (cyclam);
  • the chelator is not a lipophilic chelator.
  • the chelators described above are considered not to be lipophilic chelators. However, in other embodiments lipophilic chelators may be used so long as the chelator selected does not interfere with the capacity of the microbubble to present macromolecules of interest at the outer surface of the plasma membrane of eukaryotic cells under suitable ultrasound conditions.
  • Lipophilic chelators include, but are not limited to, 2-hydroxyquinoline-4-carboxylic acid; 6-chloro-2-hydroxyquinoline; 8-chloro-2-hydroxyquinoline; carbostyril 124; carbostyril 165; 4,6-dimethyl-2-hydroxyquinoline; 4,8-dimethyl-2-hydroxyquinoline; or other 2-quinolinol compounds; 8- hydroxyquinoline (oxine); 8-hydroxyquinoline b- D-galactopyranoside; 8- hydroxyquinoline b-D-glucopyranoside; 8-hydroxyquinoline glucuronide; 8- hydroxyquinoline-5-sulfonic acid; 8-hydroxyquinoline ⁇ -D- glucuronide sodium salt; 8-quinolinol hemisulfate salt; 8-quinolinol N-oxide; 2- amino-8-quinolinol; 5,7-dibromo-8-hydroxyquinoline; 5,7-dichloro-8- hydroxyquinoline; 5,7-d
  • the lipid of the lipid-modified chelator may be any hydrophobic or amphiphilic hydrocarbon-containing compound which, when linked to a chelator with an immobilised metal ion carrying, bound to, or associated with a macromolecule in accordance with the invention, is capable of stable incorporation into the shell of the microbubble and the outer layer of the lipid bilayer of the plasma membrane of a eukaryotic cell.
  • the lipid may be saturated or unsaturated. Typically, these are hydrophobic or amphiphilic hydrocarbon-containing compounds of at least 10 carbon residues. This may include representatives from the cationic lipids, zwitterionic lipids, neutral lipids, or anionic lipids.
  • the lipid may be a fatty acid, a monoacylglycerol, a diacylgylcerol, a triacylglycerol, a sterol, a glycolipid, or a phospholipid (e.g. a glycerophospholipids or a sphingolipid).
  • the lipid may be an amphiphilic lipid, i.e. any lipid composed of a hydrophilic portion and a hydrophobic portion (typically a hydrophilic head and a hydrophobic tail).
  • the hydrophilic portion of useful amphiphilic lipids may comprise polar or charged groups such as carbohydrates, phosphate, carboxylic, sulfato, amino, sulfhydryl, nitro, hydroxy and other like groups.
  • the hydrophobic portion may comprise apolar groups that include without limitation long chain saturated and unsaturated aliphatic hydrocarbon groups and groups substituted by one or more aromatic, cyclo-aliphatic or heterocyclic group(s).
  • amphipathic lipid compounds include, but are not limited to, phospholipids, aminolipids, glycolipids and sphingolipids.
  • lipids with acyl groups e.g. monoacylglycerol, diacylgylcerol, or triacylglycerol lipids
  • acyl groups e.g. monoacylglycerol, diacylgylcerol, or triacylglycerol lipids
  • the lipids may be anionic or neutral (including zwitterionic and polar) lipids, e.g. anionic or neutral phospholipids.
  • Neutral lipids exist in an uncharged or neutral zwitterionic form at a selected pH.
  • such lipids include, for example, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides and diacyl glycerols (e.g. 1,2-dioleoyl-sn- glycerol (DG)).
  • Suitable zwitterionic lipids include, without limitation, diacylphosphatidylcholine (e.g. dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC)).
  • DOPC dioleoylphosphatidylcholine
  • DMPC dimyristoylphosphatidylcholine
  • An anionic lipid is a lipid that is negatively charged at physiological pH.
  • lipids include, without limitation, phosphatidylglycerol (e.g. dioleoylphosphatidylglycerol (DOPG)), diacylphosphatidylethanolamine, cardiolipin, diacylphosphatidylserine (e.g.
  • DOPG dioleoylphosphatidylglycerol
  • diacylphosphatidylethanolamine e.g. cardiolipin
  • diacylphosphatidylserine e.g.
  • dioleoylphosphatidylserine diacylphosphatidic acid
  • N-dode-canoyl phosphatidylethanolamines N-succinyl phosphatidylethanolamines
  • N- glutarylphosphatidylethanolamines N- glutarylphosphatidylethanolamines
  • lysylphosphatidylglycerols palmitoyloleyolphosphatidylglycerol (POPG)
  • POPG palmitoyloleyolphosphatidylglycerol
  • non-cationic lipids may contain phosphorus.
  • non-cationic lipids of use in the invention include lecithin, lysolecithin, phosphatidylethanolamine, lysophosphatidylethanolamine, dioleoylphosphatidylethanolamine (DOPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DM PE), distearoylphosphatidy 1-ethanolamine (DSPE), palmitoyloleoylphosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoy
  • the lipid of the lipid-modified chelator of the present invention can also be a cationic lipid.
  • Cationic lipids contain positively charged functional groups under physiological conditions.
  • Cationic lipids include, but are not limited to, N,N-dioleyl- N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), 2,3-dioleoyloxy trimethylammonium propane (DOTAP), 2,3-di- (oleyloxy)propyl trimethyl ammonium (DOTMA), N-[1-(2,3,-ditetradecyloxy)propyl]- N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N-[1- (2,3,dioleyloxy)propyl]-N,N-dimethyl-N-hydroxy ethylammonium bromide (DORIE), 3b-
  • amphiphilic lipid may be selected from phosphatidylcholines, e.g. 1,2-dioleoyl-phosphatidylcholine, 1,2-dipalmitoyl- phosphatidylcholine, 1 ,2-dimyristoyl-phosphatidylcholine, 1 ,2-distearoyl- phosphatidylcholine, 1-oleoyl-2-palmitoyl-phosphatidylcholine, 1-oleoyl-2-stearoyl- phosphatidylcholine, 1-palmitoyl-2-oleoyl-phosphatidylcholine and 1-stearoyl-2- oleoyl-phosphatidylcholine; phosphatidylethanolamines, e.g.
  • 1,2-dioleoyl- phosphatidylethanolamine 1,2- dipalmitoylphosphatidylethanolamine, 1,2- dimyristoylphosphatidylethanolamine, 1,2- distearoylphosphatidylethanolamine, 1- oleoyl-2-palmitoyl-phosphatidylethanolamine, 1-oleoyl-2-stearoyl- phosphatidylethanolamine, 1-palmitoyl-2-oleoyl-phosphatidylethanolamine, 1- stearoyl-2-oleoyl-phosphatidylethanolamine and N-succinyl-dioleoyl- phosphatidylethanolamine; phosphatidylserines, e.g.
  • ganglioside GM1 glucolipids; sulphatides; phosphatidic acids, e.g. di-palmitoyl-glycerophosphatidic acid; palmitic fatty acids; stearic fatty acids; arachidonic fatty acids; lauric fatty acids; myristic fatty acids; lauroleic fatty acids; physeteric fatty acids; myristoleic fatty acids; palmitoleic fatty acids; petroselinic fatty acids; oleic fatty acids; isolauric fatty acids; isomyristic fatty acids; isostearic fatty acids; sterol and sterol derivatives, e.g.
  • phosphatidic acids e.g. di-palmitoyl-glycerophosphatidic acid
  • palmitic fatty acids palmitic fatty acids
  • stearic fatty acids arachidonic fatty acids
  • lauric fatty acids myristic fatty acids
  • polyoxyethylene fatty acids esters and polyoxyethylene fatty acids alcohols polyoxyethylene fatty acids alcohol ethers; polyoxyethylated sorbitan fatty acid esters; glycerol polyethylene glycol oxy- stearate; glycerol polyethylene glycol ricinoleate; ethoxylated soybean sterols; ethoxylated castor oil; polyoxyethylene polyoxypropylene fatty acid polymers; polyoxyethylene fatty acid stearates; di-oleoyl-sn-glycerol; 1,2-dioleoyl-sn-glycero- 3-succinate; dipalmitoyl-succinylglycerol; 1,3-dipalmitoyl-2- succinylglycerol; 1-alkyl
  • 1-hexadecyl-2-palmitoyl-phosphatidylcholine 1- alkyl-2-acyl-phosphatidylethanolamines, e.g. as 1-hexadecyl-2- palmitoyl- phosphatidylethanolamine; 1-alkyl-2-acyl-phosphatidylserines, e.g. 1- hexadecyl-2- palmitoyl-phosphatidylserine; 1-alkyl-2-acyl-phosphatidylglycerols, e.g.
  • the lipid may be a diglyceride, e.g. 1,2-dioleoyl-sn-glycerol or 1,2-dioleoyl-sn-glycero-3-succinate or 1,2-distearoyl-sn-glycerol (18:0 DG).
  • diglyceride e.g. 1,2-dioleoyl-sn-glycerol or 1,2-dioleoyl-sn-glycero-3-succinate or 1,2-distearoyl-sn-glycerol (18:0 DG).
  • a lipid-modified chelator is a chelator which is linked to a lipid via a direct covalent bond or a covalent molecular linker.
  • a direct covalent bond between the lipid and the chelating agent is a covalent bond formed by an atom of the lipid and an atom of the chelating agent.
  • the atoms contributing to the bond may together or independently be carbon, oxygen, sulphur, nitrogen and/or phosphorous.
  • the bond may be single, double or triple.
  • the bond is part of an organic functional group. The skilled person would be entirely familiar with the options available for suitable organic functional groups which could act as linkers between the lipid and the chelating agent.
  • Non-limiting examples thereof may include ester, carbonate ester, orthoester, ketone, ketal, hemiketal, ketene, ether, acetal, hemiacteal, peroxy, methylenedioxy, carbamate, amide, amine, amine oxide, hydroxamic acid, imine, imide, imidate, azide, azo, oxime, carbodiimide, carbazone, hydrozone, sulfide, disulfide, sulfinyl, sulfonyl, carbonothioyl, thioamide, thioester, thioether, thioketone, thioketal, sulphonate ester, dithiocarbamate, semicarbazone, phosphine or phosphodiester functional groups. Ester and amide bonds may be used in particular.
  • the covalent molecular linker may be any molecule, typically an organic molecule, or part thereof, which has a structure formed from covalently bonded atoms which is capable of bonding covalently with the lipid and the chelating agent. Within the lipid-chelator conjugate there will be a continuous series of covalently bonded atoms from the lipid to chelating agent via the molecular linker. In preferred embodiments, at least one of the covalent bonds in said series is as defined above.
  • the molecular linker may however further comprise non-covalent, e.g. ionic bonds, in parts of the molecule which are not contributing to the covalent linkage between the chelating agent and the lipid.
  • the covalent molecular linker may be linear, circular or branched. In certain embodiments, the molecular linker will have a molecular weight of equal to or less than 1500 Daltons, e.g. equal to or less than 1250, 1000, 900, 800, 700, 600, 500, 400, 300, 200 or 100 Daltons. In certain embodiments, at least one direct covalent bond between the lipid and the covalent molecular linker is as defined above. In certain embodiments, at least one direct covalent bond between the chelating agent and the covalent molecular linker is as defined above. Each bond may be the same or different.
  • the covalent linker molecule may comprise at least one covalent bond as defined above, preferably in the part of that molecule which contributes to the continuous series of covalently bonded atoms from the lipid to the chelating agent via the molecular linker.
  • the covalent molecular linker may be or comprise an amino acid or a peptide, e.g. of equal to or fewer than 15 amino acid residues, e.g. of equal to or fewer than 12, 10, 8, 6, 5, 4, 3 or 2 amino acid residues.
  • Specific examples of peptide linkers which may be used include but are not limited to peptides of Gly and/or Ser residues (e.g. (Gly)2-8, (Ser)2-8, (GGGGS)1-3); (EAAAK)1-3; A(EAAAK)1-3A; Leu-Glu; (Xaa-Pro)1-6 (e.g. (Glu-Pro)1-6, (Lys-Pro)1-6, (Ala-Pro)1-6).
  • the covalent molecular linker may be or comprise a monosaccharide or an oligosaccharide or a polymer formed therefrom, e.g. a saccharide of equal to or fewer than 12 amino acid residues, e.g. equal to or fewer than 10, 8, 6, 5, 4, 3 or 2 amino acid residues.
  • the covalent molecular linker may be a monosaccharide, disaccharide or trisaccharide or sugar derivatives thereof, e.g. aldonic and uronic acids, deoxy or amino sugars, sulfated sugars, and sugar alcohols.
  • the monosaccharide or one or more of the monosaccharide residues of the disaccharide or trisaccharide may be a those, a tetrose, a pentose, a hexose, a heptose, an octose, a nonose or a decose in pyranose or furanose form and/or L- or D- form where appropriate and/or sugar derivatives thereof.
  • Pentose or hexose saccharides/residues are preferred, e.g. mannose (e.g. D-mannose), galactose (e.g. D- galactose), glucose (e.g.
  • D-glucose D-glucose
  • fructose e.g. L-fucose
  • N- acetyl-glucosamine N-acetylgalactosamine
  • rhamnose galactosamine
  • glucosamine e.g.
  • D-glucosamine D-glucosamine
  • galacturonic acid glucuronic acid
  • mannuronate mannuronate
  • guluronate N-acetylneuraminic acid
  • methyl D-mannopyranoside mannoside
  • a-methyl-glucoside galactoside
  • ribose xylose, arabinose, saccharate
  • mannitol sorbitol
  • inositol glycerol and derivatives of these monomers.
  • the disaccharide may be exemplified by acarviosin, allolactose, cellobiose, chitobiose, galactose-alpha-1, 3-galactose, dentiobiose, isomalt, isomaltose, isomaltulose, kojibiose, lactitol, lactobionic acid, lactose, lactulose, laminaribiose, maltitol, maltose, mannobiose, melibiose, melibiulose, neohesperidose, nigerose, robinose, rutinose, sambubiose, sophorose, sucralfate, sucralose, sucrose, sucrose acetate isobutyrate, sucrose octaacetate, trehalose, truranose, xylobiose or derivatives of these disaccharides.
  • the covalent molecular linker may be or comprise a nucleotide or an oligonucleotide, i.e. a nucleic acid, e.g. a ribonucleotide or a deoxyribonucleotide.
  • the linker may also be or comprise a straight chain, branched or cyclic, substituted or unsubstituted, alkyl, alkenyl or alkynl group (typically C2-8) or derivative thereof, e.g. aminohexanoic acid or one of a range of commercially available PEG (polyethylene glycol) linkers.
  • suitable covalent linker molecules include but are not limited to acetyl, glycol, succinyl, aconityl (c/s or trans), glutaryl, methylsuccinyl, trimellityl cysteamine, penicillamine, N-(2- mercaptopropionyl)glycine, 2-mercaptopropionic acid, homocysteine, 3- mercaptopropionic acid and deamino-penicillamine groups.
  • the covalent linker molecule may be a plurality of the molecules and/or groups described above.
  • the covalent linker may have one or more polar groups capable of forming one or more coordinate bonds with a metal cation and said groups may form a stable coordinate bond with the metal cation of the microbubble of use in the invention.
  • the metal cation may be any metal cation which when immobilised by the lipid modified chelator is capable of binding a macromolecule carrying a metal ion binding motif, e.g. a metal ion binding amino acid sequence as defined herein.
  • the metal ion may be a monovalent, diavalent, trivalent or tetravalent metal cation.
  • the metal cation is a transition metal cation.
  • the cation should be non-toxic in the amounts used.
  • the metal cation is not radioactive.
  • the metal cation may be Zn 2+ , Cu 2+ , Cd 2+ , Hg 2+ , Co 2+ , Ni 2+ , Fe 2+ , Fe 3+ , Pd 3+ , Ga 3+ , Al 3+ .
  • Zn 2+ , Cu 2+ , Ni 2+ , and Fe 2+ are preferred. Ni 2+ and Zn 2+ is most preferred.
  • a reference to a metal cation “immobilised” by a chelator is a reference to the arrangement of a metal cation and the chelator group of the lipid- modified chelator of use in the invention such that there exists at least one, preferably at least two, stable coordinate bonds between one or more polar groups of the chelator and the metal ion.
  • Notable lipid-modified chelators of use in the invention are 18:1 and 16:0 DGS- NTA(Ni).
  • Microbubbles comprise a shell which surrounds an internal void comprising a gas. Generally, these are approximately spherical in shape, although the shape of the microbubble is not essential in carrying out the invention and is therefore not to be considered limiting.
  • the size of the microbubble may vary depending on its intended application. For in vivo applications the microbubble may be of a size such as to permit its passage through the vascular system following administration, e.g. by intravenous injection, but in embodiments in which the microbubbles are administered directly to a target site such constraints are not so limiting. Likewise, in vitro and/or ex vivo applications the size of the microbubble need not be constrained in this way.
  • microbubbles typically have a diameter of less than about 200 pm and greater than about 0.1 pm, preferably in the range from about 0.5 to about 100 pm.
  • Particularly suitable for use in the invention are microbubbles having a diameter of less than about 10 pm and greater than about 0.5 pm, more preferably 1 to 8 pm, particularly preferably up to 5 pm, e.g. about 2 pm.
  • the shell of the microbubble will vary in thickness and will typically range from about 10 to about 200 nm. The precise thickness is not essential provided that the shell performs the desired function of retaining the gas core and carrying the lipid modified chelator and the microbubble is functional in the methods of the invention described herein.
  • Size selection and size reduction techniques may be employed to select/reduce the size of microbubbles (e.g. sonication, size exclusion chromatography, filtration).
  • Individual microbubble diameter may be measured by microscopy.
  • the size of a plurality of microbubbles e.g. the weight-average particle size, may be determined according to a dynamic light scattering method (e.g., quasi-elastic light scattering method).
  • microbubble particle sizes can be measured using dynamic light scattering instruments (e.g. Zetasizer Nano ZS model manufactured by Malvern Instruments Ltd. and ELS-8000 manufactured by Otsuka Electronics Co., Ltd.). The instruments measure Brownian motion of the particles and particle size is determined based on established dynamic light scattering methodological theory.
  • the shell of the microbubbles of the invention should be biocompatible and suitable materials are well known in the art.
  • the shell of the microbubble will comprise a surfactant or a polymer.
  • Surfactants which may be used include any material which is capable of forming and maintaining a microbubble by forming a layer at the interface between the gas within the core and an external medium, e.g. an aqueous solution which contains the microbubble.
  • a surfactant or combination of surfactants may be used.
  • Those which are suitable include lipids, in particular phospholipids. Such lipids are present in the microbubbles in addition to the lipid-modified chealtor.
  • Suitable microbubble shell forming lipids include phospholipids and/or glycolipids.
  • suitable lipids include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidic acid, phosphatidylinositol, dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dimyristoyiphosphatidylcholine (DMPC), dioleylphosphatidylcholine (DOPE), dibehenoylphosphatidylcholine (dibehenoyl-sn-glycero-3-phosphocholine; DBPC), dimyristoylphosphatidylethanolamine, dipalmitolphosphatidylethanolamine, distearoylphosphatidylethanolamine, DSPE-PEG-2000, cardiolipin, sphingomyelin, glycosphin
  • the lipids used may be of either natural or synthetic origin.
  • phospholipids derived from plants and animals e.g. egg yolk or soybeans (e.g. egg yolk lecithin or soya bean lecithin) and their hydrogenation products or hydroxide derivatives, so- called semi-synthetic phospholipids.
  • Fatty acids constituting a phospholipid are not specifically limited, and saturated and unsaturated fatty acids are usable.
  • Suitable lipids may include any of those described above in the context of the lipid modified chelator component.
  • the microbubble of use in the invention comprises a phosphatidylcholine or a phosphatidylethanolamine, e.g. distearoylphosphatidylcholine (DSPC), dimyristoyiphosphatidylcholine (DMPC), dibehenoylphosphatidylcholine (DBPC) or 1,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (DSPE-PEG-2000)
  • DSPC distearoylphosphatidylcholine
  • DMPC dimyristoyiphosphatidylcholine
  • DBPC dibehenoylphosphatidylcholine
  • Polymer materials which are suitable for use in forming the shell of the microbubble include proteins, in particular albumin, particularly human serum albumin.
  • Other biocompatible polymers which may be used include poly(vinyl alcohol) (PVA), poly(D,L-lactide-co-glycolide) (PLGA), cyanoacrylate, poloxamers (Pluronics) or combinations thereof.
  • the shell materials may also be functionalised microbubble shell forming compounds.
  • Representative, non-limiting examples of the above mentioned materials include sialic acid derivatives, glucuronic acid derivatives; glutaminic acid derivatives; polyglycerin derivatives; polyethylene glycol derivatives (including methoxypolyethylene glycol condensates, etc.), e.g.
  • the microbubble of use in the invention comprises a polyethylene glycol modified lipid, polyethylene glycol (40) stearate or DSPE-PEG-2000.
  • the amounts of shell components and the ratio between the amounts of shell components may vary depending on the number and identity of those selected.
  • the microbubble of use in the invention comprises DSPC, polyethylene glycol (40) stearate (PEG40S), and DGS-NTA (18:1 and/or 16:0).
  • At least a portion of said DGS- NTA of said microbubble has an immobilised Ni 2+ or Zn 2+ ion.
  • the ratio of DSPC, PEG40S and DGS-NTA is about 9 : about 0.5 : about 0.2, respectively, e.g. 5-14 : 0.25-0.75 : 0.1-0.3 or 7-11 : 0.4-0.6 ; 0.15-0.25.
  • PEG40S will be present in the shell at about 2 to about 10 % w/w, e.g. about 3, 4,
  • DGS- NTA will be present in the shell at less than 5% w/w, e.g. at about 0.1 to about 5 % w/w, e.g. about 0.5, 1 , 2, 3 or 4 to about 5%, or about 0.1 to about 0.5, 1 , 2, 3 or about 4 % w/w.
  • the remainder of the shell mass may be DSPC.
  • % w/w (or “percentage weight by weight”) is a commonly used expression of the amount of a compound in a solid. 1% w/w equates to 1 gram of compound per 100g of solid, 2% w/w equates to 2g of compound per 100g of solid, and so on. Accordingly, % w/w may be expressed as g/1 OOg, grams per 100 grams and g 100g _1 . 1% w/w also equates to 10 gram of compound per kilogram of solid. In the present context, the term refers to the amount of shell component component in question in grams per 100g of microbubble shell.
  • the microbubble shells may comprise single or multiple layers of the same or different materials. Multiple layers may, for example, be formed in cases where the basic shell material (e.g. a lipid) bears one or more polymers or polysaccharides. Examples of such polymers include polyethylene glycol and polyvinylpyrrolidone.
  • the microbubble shell may also be coated with polymers, e.g. poly-L-lysine and PLGA, and/or polysaccharides, e.g. alginate, dextran, diethylamino-ethyl- dextran hydrochloride (DEAE) or chitosan. Methods for attaching these coating materials may involve electrostatic or covalent interactions. Different coating materials (polymers, polysaccharides, proteins, etc.) may be used in order to improve the properties of the microbubble, for example by increasing the rigidity, stability in circulation and/or tissue permeation capability of the microbubble-based reagents, by manipulating the net surface charge of the microbubble and by increasing its payload capacity.
  • polymers e.g. poly-L-lysine and PLGA
  • polysaccharides e.g. alginate, dextran, diethylamino-ethyl- dextran hydrochloride (DEAE) or chitosan.
  • the gas within the core of the microbubble should be biocompatible.
  • gas encompasses not only substances which are gaseous at ambient temperature and pressure, but also those which are in liquid form under these conditions. Where the "gas" is liquid at ambient temperature this will generally undergo a phase change to a gas at a temperature of 30°C or above, more preferably 35°C or above. For any gas which is a liquid at ambient temperature, it is generally preferred that this will undergo a phase change to a gas at a temperature between about 30 and 37°C, preferably at around normal body temperature. Any reference herein to "gas” should thus be considered to encompass not only gases and liquids, but also liquid vapours and any combination thereof, e.g. a mixture of a liquid vapour in a gas.
  • Gases which are suitable for incorporation within the microbubbles for use according to the invention include air, nitrogen, oxygen, carbon dioxide, hydrogen; inert gases, e.g. helium, argon, xenon or krypton; sulphur fluorides (e.g. sulphur hexafluoride, disulphur decafluoride); low molecular weight hydrocarbons e.g. alkanes (e.g. methane, ethane, propane, butane), cycloalkanes (e.g. cyclopropane, cyclobutane, cyclopentane), alkenes (e.g. ethylene, propene); and alkynes (e.g.
  • halogenated hydrocarbons are those which contain one or more fluorine atoms and include, for example, bromochlorodifluoromethane, chlorodifluoromethane, dichlorodifluoromethane, bromotrifluoromethane, chlorotrifluoromethane, chloropentafluoroethane, dichlorotetrafluoroethane, chlorotrifluoroethylene, fluoroethylene, ethyl fluoride, 1,1-difluoroethane and perfluorocarbons.
  • Perfluorocarbons include perfluoroalkanes (e.g. perfluoromethane, perfluoroethane, perfluoropropanes, perfluorobutanes, perfluoropentanes, perfluorohexanes and peril uoroheptanes); perfluoroalkenes (e.g. perfluoropropene, perfluorobutenes); and perfluorocycloalkanes (e.g. perfluorocyclobutane).
  • Microbubbles containing perfluorinated gases, in particular, perfluorocarbons e.g. perfluoropropanes, perfluorobutanes, perfluoropentanes and peril uorohexanes
  • perfluoroalkanes e.g. perfluoromethane, perfluoroethane, perfluoropropanes, perfluorobutanes, perfluoropentanes, perfluoro
  • the microbubble of use in the invention comprises a high molecular weight gas, e.g. a perfluorocarbon or a sulphur fluoride.
  • the microbubble of the use in the invention further comprises a macromolecule bound to (associated with) the metal cation immobilised by the chelator via one or more coordinate bonds.
  • the number and/or strength of the one or more coordinate bonds are sufficient to ensure a sufficiently persistent relationship exists between the macromolecule and the lipid-modified chelator such that macromolecule-lipid complex is capable of stable incorporation into both the shell of the microbubble and the outer layer of the lipid bilayer of the plasma membrane of a eukaryotic cell following appropriate ultrasound exposure.
  • the number and/or strength of the one or more coordinate bonds are sufficient to ensure a sufficiently persistent relationship exists between the macromolecule and the lipid-modified chelator such that macromolecule-lipid complex is capable of stable incorporation into the shell of the microbubble but that the macromolecule may still be transferred from the microbubble to a site in or on the outer surface of the plasma membrane of a cell following appropriate ultrasound exposure.
  • the relationship between the macromolecule and the lipid-modified chelator need not be permanent however, and some embodiments may be designed to permit a gradual release of the macromolecule from the lipid-modified chelator.
  • the macromolecule may be a protein (including peptides), a carbohydrate, a nucleic acid or a combination or complex thereof.
  • the macromolecule may be therapeutically or diagnostically effective in that its presentation at the outer surface of the plasma membrane of a eukaryotic cell can result in a therapeutic effect, e.g. an immunotherapeutic effect, either directly or indirectly, or can provide information useful in the diagnosis or monitoring of disease. If the macromolecule does not have a molecular motif capable of forming one or more coordinate bonds with the immobilised metal cation of sufficient stability, the macromolecule must be engineered to comprise or carry such a metal cation binding motif.
  • a motif examples include, but are not limited to a histidine residue, a cysteine residue, a tryptophan residue, an amino acid analogue comprising an imidazole side chain or a combination and/or a plurality thereof, i.e. a contiguous sequence of subunits selected from the aforementioned amino acid residues and analogues.
  • the metal cation binding motif is a contiguous sequence of subunits selected from histidine residues and amino acid analogues comprising an imidazole side chain.
  • the contiguous sequence may consist of 2-10 of such subunits, e.g. 3-10, 4-10, 5-10, 6-10, 7-10, 8-10 or 9-10 of such subunits.
  • the contiguous sequence may consist of 2, 3, 4, 5, 6, 7, 8, 9, or 10 of such subunits.
  • the sequence will be entirely histidine residues and in this context the motif may be described as a His tag.
  • the macromolecule of use in the invention will comprise a metal cation binding motif comprising a contiguous sequence of 3-10 amino acids selected from histidine, cysteine, tryptophan or an amino acid analogue comprising an imidazole side chain or a combination thereof, preferably a contiguous sequence of 5-10 histidine residues.
  • the macromolecule may be a fusion protein comprising, e.g. consisting of, at least one protein of interest and at least one heterologous sequence of amino acids which is capable of forming one or more coordinate bonds with the immobilised metal cation of sufficient stability, e.g. a metal cation binding motif as described above.
  • the amino acids of the protein of interest may be contiguous with the amino acids of the metal cation binding motif, but in other embodiments at least one amino acid spacer sequence may be included.
  • spacer sequences may be up to 10, e.g. up to 9, 8, 7, 6, 5, 4, 3, 2 or 1 , amino acids in length.
  • the spacer amino acids may be any of the proteinogenic amino acids or structurally similar analogues.
  • the metal cation binding motif of the fusion protein is “heterologous” in the sense that it is a sequence of amino acids which is not present in the consensus wild type sequence of the at least one protein of interest of the fusion protein.
  • the macromolecule including any heterologous metal cation binding motif, will typically be water-soluble. Water-soluble macromolecules can be considered to be macromolecules for which less than 1000 parts pure water are required to solubilise 1 part of the macromolecule, e.g. less than 500, 250, 100, 50, 40, 30, 20, 10, 9, 8,
  • the identity of the macromolecule is limited only insofar as the skilled person is interested in presenting that macromolecule, or a part thereof, at the outer surface of the plasma membrane of a eukaryotic cell.
  • the macromolecule may be, or comprise, a tumour antigen, e.g. those listed in the Cancer Antigenic Peptide Database hosted by the Cancer Immunity Journal (https://caped.icp.ucl.ac.be/Peptide/list).
  • the macromolecule may be, or comprise, a hydrophilic portion of a macromolecule with hydrophilic and hydrophobic regions, or one or more hydrophilic portions of a macromolecule complex with hydrophilic and hydrophobic regions.
  • the macromolecule may be selected from hydrophilic portions, e.g. extracellular domains or a portion thereof, of a cluster of differentiation (CD) protein (e.g. CD58 (lymphocyte function-associated antigen 3), CD106 (VCAM-1), CD54 (ICAM-1), CD2, CD4, CD19), an interleukin receptor protein (e.g. IL-4 receptor, IL-2 receptor or IL-12 receptor), a MUC protein (e.g.
  • MUC16/CA-125, MUC5AC and MUC1 a major histocompatibility complex (MHC), preferably an peptide loaded MHC capable of inducing, maintaining or propagating an immune response thereto, a T cell receptor or a B cell receptor.
  • MHC major histocompatibility complex
  • the macromolecule may be, or comprise, a portion, or all, of a caspase, carcinoembryonic antigen (CEA), alphafetoprotein (AFP), tyrosinase, epithelial tumour antigen (ETA), melanoma-associated antigen (MAGE), an antibody (including fragments thereof, e.g. Fab, Fab', F(ab')2, Fv, scFv, dAb, minibody), preferably wherein said antibody can induce, maintain or propagate an immune response when bound to its target antigen, or Protein A/G (preferably bound to an antibody, e.g. those mentioned above).
  • the antibody specifically binds any of the above-mentioned macromolecules, e.g. a tumour antigen, or an extracellular portion of CD58, CD106, CD54, CD2, CD4, or CD19.
  • references to the above named proteins extends to mutations thereof, e.g. mutations that result in the formation of a tumour antigen.
  • microbubbles of the invention are specifically designed to be used in ultrasound-mediated tagging of eukaryotic cell plasma membranes.
  • the ultrasound conditions which may be used, i.e. applied to the target cell and the microbubble when each are in contact, will be appropriate to result in the presentation of the macromolecule of interest at the outer surface of the plasma membrane of the target eukaryotic cell in the relevant context.
  • Conditions for use in vitro or ex vivo may vary from those required in vivo. The skilled person would be able to adjust the various conditions to achieve successful results. Adjusting the frequency and/or the peak rarefaction pressure and/or the pulse length may give the most effective control.
  • the ultrasound may be provided in continuous or pulsed mode. Pulsed mode may be helpful in in vivo contexts where there may be concerns over prolonged exposures.
  • the frequency of the ultrasound will be about 0.25 to about
  • 2.5 MHz e.g. any of about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, or 2.0 to about
  • the peak rarefaction pressure will be about 0.10 to about
  • 1.5 MPa e.g. any of about 0.15, 0.2, 0.25, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, or 1.25 to about 1.5 MPa or about 0.10 to any of about 0.15, 0.2, 0.25, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, or 1.25 MPa. Any and all ranges which may be formed from any of these range endpoints are specifically contemplated. A range of about 0.1 to about 0.3 MPa, e.g. about 0.15 to about 0.25 or about 0.18 MPa, may be advantageous. In other embodiments, a range of about 0.22 to about 0.28 MPa, or about 0.25 MPa, may be advantageous.
  • the number of cycles per pulse may be about 1 to about 10 million, e.g. about 1 to about 2, 3, 4, 5, 6, 7, 8, 9 million or about 1, 2, 3, 4, 5, 6, 7,
  • the cycles per pulse may however be different and the skilled person would be able to determine appropriate values for the specific needs in question.
  • the duty cycle will be about 0.5 to about 100%, e.g. any of about 1, 10, 20, 30, 40, 50, 60, 70, 80 or 90 to about 100% or about 0.5 to about 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100%. Any and all ranges which may be formed from any of these range endpoints are specifically contemplated. A range of about 70 to about 100%, or about 100% may be advantageous.
  • the duration of exposure will be about 10 to about 300 seconds, e.g. any of about 20, 40, 60, 80, 100, 120, 150, 180, 210, 240, 270 to about 300 seconds or about 10 to about 20, 40, 60, 80, 100, 120, 150, 180, 210, 240, or 270 seconds. Any and all ranges which may be formed from any of these range endpoints are specifically contemplated. A range of about 10 to about 120 seconds, e.g. about 30 to about 90 seconds, or about 60 seconds may be advantageous.
  • any source capable of producing ultrasound may be used in the methods herein described.
  • the source should be capable of directing the energy to the target site and may include, for example, a probe or device capable of directing energy to the target tissue from the surface of the body.
  • the equipment used may comprise an focused ultrasound transducer, a transformer, an amplifier, a waveform generator, a digitiser, a pre-amplifier, a high-pass filter, and a passive cavitation detector (PCD).
  • PCD passive cavitation detector
  • the method may be performed in vitro, e.g. on cultured cells or tissue.
  • Such cell cultures may be planktonic cultures or a solid support bound culture.
  • the contacting step may take place on a suspension of cells (which may include cells which have previously been cultured on a solid support and are in suspension transiently) or on cells associated with a solid support (which may include cells which have previously been cultured in suspension and are associated with a solid support transiently).
  • the cells/tissue will typically be maintained in a suitable buffered aqueous medium and then be exposed to microbubbles provided in an aqueous medium.
  • the microbubble may be applied to the cells/tissue or vice versa.
  • the cells/tissue and microbubbles will be exposed to ultrasound as described above. Following ultrasound treatment, the cells/tissue may be washed to remove excess microbubbles and/or tested to assess, e.g. quantify, the presentation of the macromolecule of interest at the outer surface of the plasma membrane of the target eukary
  • Such methods are expected to represent a highly advantageous investigational tool for use to investigate the role or cell membrane components and their impact upon interactions with other cells, pathogens, drug molecules, and so on.
  • the method may also be performed on cells or tissue which are ex vivo, i.e. cells or tissue which have been removed from a subject in order to have the method of the invention performed thereon and then to be returned to the subject or be administered to a different subject.
  • immune cells in particular cell-eliminating immune cells, e.g. T cells (in particular Tc cells), natural killer cells, monocytes and macrophages
  • T cells in particular Tc cells
  • macrophages may be tagged with macromolecules which function to modulate their (cell-eliminating, e.g. cytotoxic and/or cytophagic) activities and/or target such activities towards a target cell type, e.g. a hyperproliferative cell.
  • B cells and dendritic cells may be tagged with macromolecules which function to modulate their immunogenic activities, in particular towards a target cell type, e.g. a hyperproliferative cell.
  • immune cells involved in immunotolerance e.g. T cells (in particular Th and Treg cells), dendritic cells, mast cells and B cells may be tagged with macromolecules which function to modulate their immunotolerance activities towards a target cell type, e.g. those cells associated with autoimmune disease, inflammatory disease or allergy.
  • aberrant cells from a subject may be tagged with immunostimulatory macromolecules which are designed to induce a targeted immune response to those aberrant cells and others like them within the subject, thereby promoting their elimination from the subject undergoing treatment.
  • particular cells of interest may be tagged ex vivo with a detectable macromolecule, e.g. radioactive or fluorescent macromolecule, and their distribution within the subject may be monitored upon their return to the subject.
  • a detectable macromolecule e.g. radioactive or fluorescent macromolecule
  • aberrant cells in or on a subject may be tagged with immunostimulatory macromolecules which are designed to induce a targeted immune response to those aberrant cells and others like them within the subject and promote their elimination.
  • target cells may be tagged with macromolecules which are part of a specific and selective binding pair and which are not found naturally in the subject or the local region in which such target cells are found.
  • a suitable drug may then be targeted to those cells if administered as a conjugate with the relevant specific and selective binding partner.
  • the invention further provides a microbubble of the invention for use in such methods and the use of a microbubble of the invention in the manufacture of a medicament for use in such methods.
  • a pharmaceutically acceptable suspension of the microbubbles of the invention is generally introduced directly into the region comprising the target cells (e.g. intratumourally), or intradermally, subcutaneously, intraperitoneally or intravenously, or into lymph vessels at or near the area to be treated.
  • the suspension of microbubbles is introduced intravenously or directly.
  • the target area is then exposed to ultrasound to image the area and to determine the location of the microbubbles. Once the microbubbles are at the intended target, they are exposed to the ultrasound conditions described above.
  • the optional step of monitoring the microbubbles using ultrasound is generally performed with ultrasound of a lower intensity (typically corresponding to a lower frequency) than that used to introduce the macromolecule-lipid complex of the microbubble into the outer surface of the plasma membrane of a eukaryotic cell and/or to transfer of the macromolecule from the microbubble to a site in or on the outer surface of the plasma membrane of a eukaryotic cell.
  • ultrasound of a lower intensity typically corresponding to a lower frequency
  • the microbubbles may be derivatised with receptor affinity molecules specific to receptors on the target aberrant cells.
  • affinity molecules include receptor specific polypeptides, nucleic acids and carbohydrates, e.g. antibodies, antibody fragments, peptide growth factors, DNA and RNA.
  • Said receptors may be polypeptides, nucleic acids or carbohydrates.
  • a plethora of receptor-ligand pairs are known to the skilled person and may be identified without undue burden and any of these pair could provide the basis of the choice of receptor affinity molecule for use in the invention.
  • microbubbles of the invention may be rendered magnetic, e.g. by further comprising magnetic nanoparticles (e.g. as described in US 9427396), and magnetic fields may be applied at the region containing the target aberrant cells prior to or during exposure to the ultrasound conditions recited described above.
  • microbubbles that may be administered to the subject will depend on the specific microbubble formulation used and the macromolecule that is carried by the microbubble. Nevertheless, it is, in general, preferred that a dose of the microbubble formulation should not exceed 1 cc/kg, preferably 0.3 cc/kg, and more preferably 0.15 cc/kg.
  • the invention provides a method of immunotherapy of a human or non-human animal subject, said method comprising presenting a macromolecule at the outer surface of the plasma membrane of a target cell on or in the subject, wherein said macromolecule promotes elimination of cells displaying said macromolecule on their surface by the immune system of the subject, wherein said method comprises contacting said target cell whilst on or in the subject with a microbubble of the invention carrying the macromolecule as defined herein and, whilst in contact with one another, exposing the cell and the microbubble to ultrasound conditions effective to result in presentation of said macromolecule at the outer surface of the plasma membrane of the cell.
  • the invention provides a microbubble of the invention carrying a macromolecule as defined herein, wherein said macromolecule promotes elimination of cells displaying said macromolecule on their surface by the immune system of a human or non-human animal subject for use in a method of immunotherapy of a human or non-human animal subject, said method comprising presenting said macromolecule at the outer surface of the plasma membrane of a target cell on or in the subject by contacting said target cell whilst on or in the subject with the microbubble and, whilst in contact with one another, exposing the cell and the microbubble to ultrasound conditions effective to result in presentation of said macromolecule at the outer surface of the plasma membrane of the cell.
  • the invention provides for the use of a microbubble of the invention carrying a macromolecule as defined herein, wherein said macromolecule promotes elimination of cells displaying said macromolecule on their surface by the immune system of a human or non-human animal subject in the manufacture of a medicament for use in a method of immunotherapy of a human or non-human animal subject, said method comprising presenting said macromolecule at the outer surface of the plasma membrane of a target cell on or in the subject by contacting said target cell whilst on or in the subject with the microbubble and, whilst in contact with one another, exposing the cell and the microbubble to ultrasound conditions effective to result in presentation of said macromolecule at the outer surface of the plasma membrane of the cell.
  • Elimination of cells by the immune system refers to any mechanism by which the subject’s immune system recognises cells presenting the macromolecule in question and then acts to reduce the number of viable forms of such cells in the subject. This may be via a cytotoxic and/or a cytophagic mechanism, e.g. involving T cells (in particular Tc cells), natural killer cells, phagocytes and/or macrophages.
  • T cells in particular Tc cells
  • phagocytes e.g. involving T cells (in particular Tc cells), natural killer cells, phagocytes and/or macrophages.
  • T cells in particular Tc cells
  • the term encompasses complete eradication of viable forms of such cells from the subject but also the eradication of a portion of viable forms of such cells.
  • the elimination may be transient or permanent.
  • the term “cell-eliminating” should be construed accordingly.
  • Examples of macromolecules which may be presented on the target cells in this embodiment may be, or comprise, full length or functional portions, e.g. extracellular domains of portions thereof, of CD58, CD106, CD54, an MHC (preferably an peptide loaded MHC capable of inducing, maintaining or propagating an immune response thereto), a MUC protein (e.g. MUC16/CA-125, MUC5AC and MUC1), a caspase, carcinoembryonic antigen (CEA), alphafetoprotein (AFP), tyrosinase, epithelial tumour antigen (ETA), melanoma-associated antigen (MAGE).
  • MUC16/CA-125, MUC5AC and MUC1 e.g. MUC16/CA-125, MUC5AC and MUC1
  • a caspase e.g. MUC16/CA-125, MUC5AC and MUC1
  • CEA carcinoembryonic antigen
  • AFP alphafetoprotein
  • the step of exposing the target cell on or in a subject to ultrasound conditions which are effective to result in presentation of said macromolecule at the outer surface of the plasma membrane of said cell may be as described above.
  • the invention provides a method of immunotherapy of a human or non-human animal subject, said method comprising providing an ex vivo sample of immune cells, preferably immune cells of the subject; contacting said immune cells with a microbubble of the invention carrying a macromolecule as defined herein, wherein said macromolecule functions to target the cell-eliminating activity of the immune cell towards a target cell type in or on the subject or to modulate the cell-eliminating activity of the immune cell; and, whilst in contact with one another, exposing the cells and the microbubble to ultrasound conditions effective to result in presentation of said macromolecule at the outer surface of the plasma membrane of one or more of the immune cells; and administering at least a portion of the immune cells presenting the macromolecule to the subject.
  • the invention provides a microbubble of the invention carrying a macromolecule as defined herein, wherein said macromolecule functions to target the cell-eliminating activity of an immune cell towards a target cell type in or on a human or non-human animal subject or to modulate the cell-eliminating activity of an immune cell for use in a method of immunotherapy of a human or non- human animal subject, said method comprising providing an ex vivo sample of said immune cells, preferably immune cells of the subject; contacting said immune cells with the microbubble and, whilst in contact with one another, exposing the cells and the microbubble to ultrasound conditions effective to result in presentation of said macromolecule at the outer surface of the plasma membrane of one or more of the immune cells; and administering at least a portion of the immune cells presenting the macromolecule to the subject.
  • the invention provides for the use of a microbubble of the invention carrying a macromolecule as defined herein, wherein said macromolecule functions to target the cell-eliminating activity of an immune cell towards a target cell type in or on a human or non-human animal subject or to modulate the cell-eliminating activity of an immune cell in the manufacture of a medicament for use in a method of immunotherapy of a human or non-human animal subject, said method comprising providing an ex vivo sample of said immune cells, preferably immune cells of the subject; contacting said immune cells with the microbubble and, whilst in contact with one another, exposing the cells and the microbubble to ultrasound conditions effective to result in presentation of said macromolecule at the outer surface of the plasma membrane of one or more of the immune cells; and administering at least a portion of the immune cells presenting the macromolecule to the subject.
  • the invention also provides an ex vivo method useful in the immunotherapy of a human or non-human animal subject said method comprising providing an ex vivo sample of immune cells, preferably immune cells of the subject; contacting said immune cells with a microbubble of the invention carrying a macromolecule as defined herein, wherein said macromolecule functions to target the cell-eliminating activity of the immune cell towards a target cell type in or on the subject or to modulate the cell-eliminating activity of the immune cell; and, whilst in contact with one another, and exposing the cells and the microbubble to ultrasound conditions effective to result in presentation of said macromolecule at the outer surface of the plasma membrane of one or more of the immune cells.
  • the immune cells of use in this aspect of the invention are typically cytotoxic and/or cytophagic immune cells, e.g. T cells (in particular Tc cells), natural killer cells, monocytes and macrophages, but in other embodiments, B cells and dendritic cells may be used.
  • Modulation of the cell-eliminating activity of an immune cell refers to any effect which stimulates, maintains, prolongs or enhances an immune cell’s role in promoting the elimination of a target cell from the body of a subject, e.g. by cytotoxic or cytophagic mechanisms. It may also refer to any effect which inhibits, reduces or prevents an immune cell’s role in suppressing the elimination of a target cell from the body of a subject, e.g. by cytotoxic or cytophagic means.
  • These roles of an immune cell in cell elimination mechanisms may be direct and/or indirect.
  • the sample of immune cells of use in this aspect of the invention may be provided by any of the convenient cell separation/isolation techniques known in the art or which may be developed in the future. Such techniques may involve one or more steps of blood fractionation, centrifugation, precipitation, filtration, cell sorting. In other embodiments, the immune cells may be provided from cell culture.
  • macromolecules which may be presented on the immune cells in this embodiment may be, or comprise, full length or functional portions, e.g. extracellular domains of portions thereof, of CD2, CD4, CD19, IL-4 receptor, IL-2 receptor, IL-12 receptor, an antibody (including fragments thereof, e.g. Fab, Fab', F(ab')2, Fv, scFv, dAb, minibody) directed to CD106, CD58 or CD54, Protein A/G (preferably bound to an antibody, e.g. those mentioned above), a T cell receptor or a B cell receptor.
  • the step of exposing ex vivo samples of immune cells to ultrasound conditions which are effective to result in presentation of said macromolecules at the outer surface of the plasma membrane of one or more of the immune cells may be as described above.
  • the immune cells may be administered to the subject by any convenient means.
  • Many approaches are available and are performed routinely, but may, for instance, include parental administration of a liquid or gel composition comprising the cells or implantation of a solid support, e.g. a natural or artificial polymer scaffold, seeded with the cells.
  • Administration may be to a particular site at which the immune cells are designed to exert their effects or could be via systemic delivery to the blood and/or lymph circulatory systems.
  • Transfusion based approaches may be used.
  • the immune cells may be mixed with blood or plasma and then transfused to the patient. Delivery may take the form of a bone marrow transplant or injection into the bone marrow.
  • the invention provides a method of immunotherapy of a human or non-human animal subject, said method comprising
  • step (c) contacting a target cell on or in the subject with a microbubble of the invention carrying the second macromolecule and, whilst in contact with one another, exposing the target cell and the second microbubble to ultrasound conditions effective to result in presentation of said second macromolecule at the outer surface of the plasma membrane of the target cell, wherein step (c) may be performed before, after or simultaneously with step
  • step (a) and/or step (b).
  • the invention further provides a microbubble of the invention for use in one or other part of such methods (i.e. a microbubble carrying the first or the second macromolecule) and the use of a microbubble of the invention in the manufacture of a medicament for use in one or other part of said methods (i.e. a microbubble carrying the first or the second macromolecule).
  • the invention still further provides a microbubble of the invention carrying the first macromolecule and a microbubble of the invention carrying the second macromolecule for use in such methods and the use of a microbubble of the invention carrying the first macromolecule and a microbubble of the invention carrying the second macromolecule in the manufacture of a medicament for use in said methods.
  • binding pair may be a specific antibody (or antibody fragment, e.g. Fab, Fab', F(ab')2, Fv, scFv, dAb, minibody) and antigen pair.
  • the pair may be selected from following pairs, preferably in the arrangement target cell : immune cell; CD58:CD2,
  • the target cell/cell type is the cell/cell type the immunotherapy method is designed to eliminate.
  • Such cells will be aberrant, abnormal or dysfunctional in some way and thus give rise to or drive a clinical disease or condition which would benefit from treatment.
  • the target cell type is a hyperproliferative or neoplastic cell, e.g. a cancer cell, in particular a tumour cell.
  • it is a cell from the hyperproliferative or neoplastic diseases or conditions mentioned herein.
  • immunotherapy methods of the invention may further comprise the administration of further immunostimulatory therapeutic agents, e.g. agents which stimulate or enhance the subject’s immune system directly or those which do so indirectly by inhibiting, reducing or preventing immunosuppression pathways in the subject’s immune system.
  • further immunostimulatory therapeutic agents e.g. agents which stimulate or enhance the subject’s immune system directly or those which do so indirectly by inhibiting, reducing or preventing immunosuppression pathways in the subject’s immune system.
  • immunostimulatory therapeutic agents e.g. agents which stimulate or enhance the subject’s immune system directly or those which do so indirectly by inhibiting, reducing or preventing immunosuppression pathways in the subject’s immune system.
  • the immune checkpoint inhibitor may be selected from ipilimumab (anti-CTLA4), tremelimumab (anti-CTLA4), MDX-1106 (also known as BMS-936558 and nivolumab; anti-PD1 antibody), MK3475 (also known as pembrolizumab; anti-PD1 antibody), CT-011 (anti-PD1 antibody), AMP-224 (anti- PD1 fusion protein - PDL2-lg fusion protein), MDX-1105 (anti-PDL1 antibody), RG7446 (anti-PDL1 antibody), BMS-936559 (anti-PDL1 antibody), MGA271 (anti- B7-H3 antibody), atezolizumab (also known as MPDL3280A; anti-PDL1 antibody), avelumab (also known as MSB0010718C; anti-PDL1 antibody) and durvalumab (anti-PDL1 antibody).
  • anti-CTLA4 ipilimumab
  • the immunostimulatory cytokine may be selected from cytokines e.g. TNF, IL-1, IL-2, IL-4, IL-6, IL-8, IL-12 and IFNa,
  • the disease or condition treatable by the above immunotherapy methods of the invention may be any hyperproliferative or neoplastic disease or condition, which terms include diseases or conditions caused by any malignant, pre-malignant or non-malignant (benign) neoplastic entities.
  • the term therefore encompasses, inter alia, the treatment of cancers, tumours, malignancies, sarcomas, carcinomas, germinomas, lymphomas, leukaemias, blastomas, papillomas and adenomas.
  • the hyperproliferative or neoplastic disease or condition may be selected from colorectal cancer (also known as colon cancer, rectal cancer or bowel cancer), prostate cancer, kidney (renal) cancer (e.g.
  • pancreatic cancer pancreatic cancer
  • testicular cancer skin cancer (e.g. melanoma and non melanoma (e.g. basal-cell cancer, squamous-cell cancer)), breast cancer, ovarian cancer, vulvar cancer, cervical cancer, endometrial cancer, bladder cancer, stomach (gastric) cancer, intestinal cancer (e.g. duodenal cancer, ileal cancer, jejunal cancer, small intestine cancer), liver (hepatic) cancer, lung (pulmonary) cancer, oesophageal cancer, oral cancer, throat cancer, brain cancer (e.g. glioblastoma, medulloblastoma), adrenal cancer (e.g.
  • skin cancer e.g. melanoma and non melanoma (e.g. basal-cell cancer, squamous-cell cancer)
  • breast cancer e.g. melanoma and non melanoma (e.g. basal-cell cancer, squamous-
  • adrenocortical cancer thyroid cancer
  • thyroid cancer e.g. anaplastic thyroid carcinoma
  • uterine cancer e.g. uterine carcinosarcoma
  • haematological cancer also known as the haematological malignancies
  • haematopoietic and lymphoid cancer malignancies e.g. leukemia, lymphoma and myeloma
  • non- malignant neoplasm or tumour in these anatomical sites e.g.
  • colorectal polyps pilomatrixoma, hemangioma, osteoma, chondroma, lipoma, fibroma, lymphangioma, leiomyoma, rhabdomyoma, astrocytoma, meningioma, ganglioneuroma, papilloma, adenoma).
  • the immunotherapy methods of the invention may be used to treat autoimmune disease.
  • the invention provides a method of immunotherapy of a human or non-human animal subject, said method comprising providing an ex vivo sample of immune cells, preferably immune cells of the subject; contacting said immune cells with a microbubble of the invention carrying a macromolecule as defined herein, wherein said macromolecule functions to modulate the immunotolerance activity of the immune cell; and, whilst in contact with one another, exposing the cell and the microbubble to ultrasound conditions effective to result in presentation of said macromolecule at the outer surface of the plasma membrane of one or more of the immune cells; and administering at least a portion of the immune cells presenting the macromolecule to the subject.
  • the invention further provides a microbubble of the invention for use in such methods and the use of a microbubble of the invention in the manufacture of a medicament for use in said methods.
  • the immune cells of use in this embodiment of the invention are immune cells involved in immunotolerance, e.g. T cells (in particular Th and Treg cells), dendritic cells, mast cells and B cells.
  • Modulation of the immunotolerance activity of an immune cell refers to any effect which stimulates, maintains, prolongs or enhances an immune cell’s role in promoting immunotolerance towards a target cell from the body of a subject. It may also refer to any effect which inhibits, reduces or prevents an immune cell’s role in promoting immunotolerance towards a target cell from the body of a subject.
  • These roles of an immune cell in immunotolerance mechanisms may be direct and/or indirect.
  • the macromolecule is, or comprises, a functional portion, e.g. extracellular domains of portions thereof, of the IL-2 receptor, IL-4 receptor, IL-12 receptor, CD19 or CD4.
  • the disease or condition treatable by the above immunotherapy method of the invention may rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease (including Crohn’s disease and ulcerative colitis), multiple sclerosis, type 1 diabetes mellitus, Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, psoriasis, Graves' disease, Hashimoto's thyroiditis, myasthenia gravis, autoimmune vasculitis, autoimmune hepatitis, alopecia areata, celiac disease, eczema, atopic dermatitis, urticara, asthma, allergic conjunctivitis, allergic rhinitis, food allergies and other allergies.
  • the invention provides a method of targeted drug delivery to a human or non-human animal subject, said method comprising presenting a macromolecule at the outer surface of the plasma membrane of a target cell of the subject, wherein said macromolecule is part of a specific binding pair and is not found in the subject or a region or tissue of the subject in which the target cells are found, wherein said method comprises contacting said target cell with a microbubble of the invention carrying the macromolecule as defined herein and, whilst in contact with one another, exposing the cell and the microbubble to ultrasound conditions effective to result in presentation of said macromolecule at the outer surface of the plasma membrane of the cell.
  • binding pair may be a specific antibody (or antibody fragment, e.g. Fab, Fab', F(ab')2, Fv, scFv, dAb, minibody) and antigen pair.
  • the method may further comprise administering to the subject a conjugate of a drug and the binding partner of said macromolecule.
  • drug conjugates will typically be administered, e.g. orally, by inhalation or by injection (e.g. intravenous, intratumoural, intradermal, subcutaneous, intraperitoneal or intramuscular) in the form of a pharmaceutically acceptable composition together with at least one pharmaceutically acceptable carrier or excipient. Examples of suitable carriers or excipients are disclosed below.
  • the drug conjugate will be directly administered to the region or the tissue of the subject in which the target cells are found.
  • the drug may be a therapeutic agent selected from: anti-allergic agents (e.g. amelexanox); anti-anginals (e.g. diltiazem, nifedipine, verapamil, erythrityl tetranitrate, isosorbide dinitrate, nitroglycerin (glyceryl trinitrate) and pentaerythritol tetranitrate); anticoagulants (e.g. phenprocoumon, heparin); antibodies, (e.g. a polyclonal antibody or a monoclonal antibody, including ranibizumab, pembrolizumab); antibiotics (e.g.
  • anti-allergic agents e.g. amelexanox
  • anti-anginals e.g. diltiazem, nifedipine, verapamil, erythrityl tetranitrate, isosorbide dinitrate, nitroglycerin (g
  • dapsone chloramphenicol, neomycin, cefaclor, cefadroxil, cephalexin, cephradine erythromycin, clindamycin, lincomycin, amoxicillin, ampicillin, bacampicillin, carbenicillin, dicloxacillin, cyclacillin, picloxacillin, hetacillin, methicillin, nafcillin, oxacillin, penicillin G, penicillin V, ticarcillin, rifampin and tetracycline); anti-cancer agents (e.g.
  • platinum compounds (spiroplatin, cisplatin and carboplatin) methotrexate, adriamycin, taxol, mitomycin, ansamitocin, bleomycin, cytosine arabinoside, arabinosyl adenine, mercaptopolylysine, vincristine, busulfan, chlorambucil, melphalan and phenylalanine mustard (PAM)), mercaptopurine, mitotane, procarbazine hydrochloride, dactinomycin (actinomycin D), daunorubicin hydrochloride, doxorubicin hydrochloride, mitomycin, plicamycin (mithramycin), paclitaxel aminoglutethimide, estramustine phosphate sodium, flutamide, leuprolide acetate, megestrol acetate, tamoxifen citrate, testolactone, trilostane, amsacrine (m
  • anti-fungal agents e.g. ketoconazole, nystatin, griseofulvin, flucytosine, miconazole, amphotericin B
  • anti-fungal agents e.g. ketoconazole, nystatin, griseofulvin, flucytosine, miconazole, amphotericin B
  • anti- parasitics e.g. anti-protozoans (e.g. chloroquine, hydroxychloroquine, metronidazole, quinine and meglumine antimonate); anti-rheumatics (e.g. penicillamine); antituberculars (e.g.
  • para-aminosalicylic acid isoniazid, capreomycin sulfate cycloserine, ethambutol hydrochloride ethionamide, pyrazinamide, rifampin, and streptomycin sulfate); anti-virals (e.g. acyclovir, amantadine, azidothymidine, e.g. as AZT or Zidovudine, ribavirin, amantadine, vidarabine and vidarabine monohydrate); anti-inflammatories (e.g.
  • vitamins e.g. cyanocobalamin constituoic acid, retinoids and derivatives, e.g. retinol palmitate, a-tocopherol, naphthoquinone, cholecalciferol, folic acid, and tetrahydrofolate
  • blood products e.g.
  • parenteral iron, hemin, hematoporphyrins and their derivatives cardiac glycosides (e.g. deslanoside, digitoxin, digoxin, digitalin and digitalis); circulatory drugs (e.g. propranolol); hormones or steroids (e.g.
  • betamethasone dipropionate betamethasone, betamethasone acetate and betamethasone sodium phosphate
  • vetamethasone disodium phosphate vetamethasone sodium phosphate
  • cortisone acetate dexamethasone, dexamethasone acetate, dexamethasone sodium phosphate, flunsolide, hydrocortisone, hydrocortisone acetate, hydrocortisone cypionate, hydrocortisone sodium phosphate, hydrocortisone sodium succinate, methylprednisolone, methylprednisolone acetate, methylprednisolone sodium succinate, paramethasone acetate, prednisolone, prednisolone acetate, prednisolone sodium phosphate, prednisolone tebutate, prednisone, triamcinolone, triamcinolone acet
  • paregoric and opiates e.g. codeine, heroin, methadone, morphine and opium
  • neuromuscular blockers e.g. atracurium besylate, gallamine triethiodide, hexafluorenium bromide, metocurine iodide, pancuronium bromide, succinylcholine chloride, tubocurarine chloride and vecuronium bromide
  • peptides e.g.
  • angiostatin manganese super oxide dismutase, tissue plasminogen activator, glutathione, insulin, dopamine, human chorionic gonadotropin, corticotropin release factor, cholecystokinins, bradykinins, elastins, vasopressins, pepsins, glucagon, integrins, adrenocorticotropic hormone, oxytocin, calcitonins, IgG, IgA, IgM, thrombin, streptokinase, urokinase, Protein Kinase C, interferon, colony stimulating factors, granulocyte colony stimulating factors, granulocyte-macrophage colony stimulating factors, tumour necrosis factors, nerve growth factors, platelet derived growth factors, lymphotoxin, epidermal growth factors, fibroblast growth factors, vascular endothelial cell growth factors, erythropoietin, transforming growth
  • the method of targeted drug delivery of the invention may be considered a method for the treatment of a disease or condition in a human or non-human animal which is responsive to a drug, e.g. those disclosed above, in which said drug undergoes targeted drug delivery to target cells, said method comprising performing the above described method of targeted drug delivery and administering to the subject a conjugate of said drug and the binding partner of said macromolecule.
  • this aspect of the invention may be expressed as method of targeted drug delivery in the treatment of a disease or condition in a human or non-human animal which is responsive to a drug, e.g. those disclosed above, said method comprising performing the above described method of targeted drug delivery and administering to the subject a conjugate of said drug and the binding partner of said macromolecule
  • the invention further provides a microbubble of the invention for use in such methods and the use of a microbubble of the invention in the manufacture of a medicament for use in said methods.
  • the invention provides a microbubble of the invention, or a suspension thereof, for use in the medical (e.g. therapeutic, diagnostic or surgical) treatment of a human or non-human animal subject.
  • the invention also relates to methods of medical treatment involving the use of a microbubble of the invention, or a suspension thereof, and also the use of a microbubble of the invention, or a suspension thereof, in the manufacture of a medicament for use in the medical treatment of a human or non-human animal subject.
  • the subject may be any human or non-human animal subject, but more particularly may be a human or a non-human vertebrate, e.g. a non-human mammal, bird, amphibian, fish or reptile.
  • the subject is a mammalian subject.
  • the animal may be a livestock or a domestic animal or an animal of commercial value, including laboratory animals or an animal in a zoo or game park. Representative animals therefore include dogs, cats, rabbits, mice, guinea pigs, hamsters, horses, pigs, sheep, goats and cows. Veterinary uses of the invention are thus covered.
  • the subject may be viewed as a patient.
  • the subject is a human.
  • Treatment when used in relation to the treatment of a disease or medical condition in a subject in accordance with the invention is used broadly herein to include any therapeutic effect, i.e. any beneficial effect in relation to the disease or the condition.
  • any therapeutic effect i.e. any beneficial effect in relation to the disease or the condition.
  • eradication or elimination of the disease or the condition, or cure of the subject but also an improvement in the disease or the condition of the subject.
  • an improvement in any symptom or sign of the disease or the condition or in any clinically accepted indicator of the disease or the condition (for example a decrease in tumour size (volume, area and/or cell number), a decrease in tumour invasion, a reduction in general discomfort or pain in the surrounding tissue, or a reduction in inflammation).
  • Treatment thus includes both curative and palliative therapy, e.g. of a pre-existing or diagnosed disease or condition, i.e. a reactionary treatment.
  • the invention provides a method for the diagnosis or monitoring of a disease or condition in a human or non-human animal subject said method comprising providing an ex vivo sample of a cell of interest, preferably immune cells, preferably cells of the subject; contacting said cells with a microbubble of the invention carrying a detectable macromolecule as defined herein (e.g.
  • a fluorescent or radioactive macromolecule and, whilst in contact with one another, exposing the cells and the microbubble to ultrasound conditions effective to result in presentation of said macromolecule at the outer surface of the plasma membrane of one or more of the cells of interest; administering at least a portion of the cells presenting the macromolecule to the subject and detecting said macromolecule in the body of said subject and/or assessing the distribution of said macromolecule in the body of said subject.
  • the detection of said macromolecule in the body of said subject and/or assessing the distribution of said macromolecule in the body of said subject allows the skilled person to monitor or diagnose a disease or condition in the subject. Methods for the formation of microbubbles are known in the art.
  • Such methods include the formation of a suspension of the gas in an aqueous medium, e.g. PBS, in the presence of the selected shell materials.
  • the macromolecule-lipid complex described herein is present as part of the selected shell materials.
  • the macromolecule is added to preformed microbubbles of the invention, i.e. microbubbles comprising a gas core and a shell, wherein said shell comprises the lipid-modified chelator and wherein a metal cation is immobilised by said chelator.
  • microbubbles include sonication, shaking, high speed mixing (mechanical agitation), coaxial electrohydrodynamic atomisation and microfluidic processing using a T-junction (see e.g. Stride & Edirisinghe, Med. Biol. Eng. Comput., 47: 883-892, 2009). Sonication is widely used and generally preferred. This technique may be carried out using an ultrasound transmitting probe. Sonication or shaking may be performed until formation of a suspension of bubbles.
  • these techniques are typically carried out under an atmosphere of the gas that is to be trapped as the gas core of the microbubbles, e.g. sulfur hexafluoride.
  • the gas for the gas core of the microbubbles may be bubbled through the aqueous solution used to prepare the suspension in addition to, or as an alternative to, carrying out sonication and/or shaking under an atmosphere of the desired gas.
  • the microbubble suspension so formed is allowed to settle before a lower part of the suspension is isolated.
  • nanodroplet core in a nanoemulsion examples include vaporisation of a nanodroplet core in a nanoemulsion (see e.g. Rapoport et al, Bubble Sci. Eng. Technol. 1 : 31 -39, 2009).
  • the core of such nanodroplets will typically be formed by an organic perfluorocompound which is encased by walls of a biodegradable amphiphilic block copolymer such as poly(ethylene oxide)-co-poly(L-lactide) or poly(ethylene oxide)-co-caprolactone.
  • nanoemulsions may be prepared by extrusion through sizing membranes, for example using albumin as the shell material.
  • the droplet-to-bubble transition may be induced by physical and/or mechanical means which include heat, ultrasound and injection through a fine- gauge needle.
  • Such microbubbles may be formed at the point of administration to the subject (e.g. during the step of administration using a fine-gauge needle) or in vivo at the desired target cells or tissues (e.g. by subjecting the nanoemulsion to ultrasound).
  • the step of administering a microbubble of the invention to a human or non-human animal subject includes administration of a nanoemulsion of the microbubble shell materials and the gas under conditions suitable for the formation of the microbubbles of the invention.
  • a suspension of microbubbles which may have been prepared according to the sonication/shaking method outlined above, or according to a method known in the art, may be filtered to obtain a suspension having a desired distribution of optimised microbubbles.
  • a suspension of the microbubbles of the invention in an aqueous phase will contain a distribution of microbubbles having different particle sizes. It is preferred that the distribution is centred on the desired weight average particle size.
  • the standard deviation of microbubble diameters in a distribution representing a suspension of the microbubbles is preferably ⁇ 3 pm, more preferably ⁇ 1 pm.
  • aqueous is used herein to describe a liquid composition which is comprised mainly, e.g. substantially, predominantly or essentially of water, e.g. at least 80%, 90%, 95%, 99% or 100% of the solvent in the composition is water. In other embodiments less than 20%, 10%, 5%, or 1% of the solvent in the composition is a non-polar solvent, or at least the amount of non-polar solvent is insufficient to interfere with vesicle formation and integrity.
  • the “aqueous suspension” can be considered to comprise, in addition to any microbubbles of the invention or the non-assembled components thereof, less than 20%, 10%, 5%, or 1% of a non-polar organic phase, or at least an amount of non polar organic phase that is insufficient to interfere with microbubble integrity.
  • the aqueous phase is devoid of non-polar solvent/phase. Consistent with this the presence of other molecules dissolved or suspended in the aqueous phase is not excluded, again as long as they are present in amounts that are insufficient to interfere with microbubble integrity.
  • the aqueous suspension of the microbubbles of the invention is a suspension in water or a saline solution, e.g. phosphate-buffered saline.
  • the macromolecule may be loaded onto preformed microbubbles of the invention by any convenient means. Typically this may by combining an aqueous suspension of microboubbles of the invention comprising a gas core and a shell, wherein said shell comprises a lipid-modified chelator and wherein a metal cation is immobilised by said chelator, with an aqueous solution of the macromolecule and incubating the mixture under conditions suitable for the metal ion binding motif of the macromolecule to form at least one stable coordinate bond with the immobilised metal ion. Suitable washing and/or isolation steps may follow, e.g. as described above.
  • the microbubbles of the invention will generally be provided in a pharmaceutical composition, e.g. together with at least one pharmaceutically acceptable carrier or excipient.
  • a pharmaceutical composition e.g. together with at least one pharmaceutically acceptable carrier or excipient.
  • compositions comprising microbubbles for use according to the invention may be formulated using techniques well known in the art.
  • the route of administration will depend on the intended use. Typically, these will be administered systemically or directly to an internal body location and may thus be provided in a form adapted for parenteral administration, e.g. by intratumoural, intradermal, subcutaneous, intraperitoneal or intravenous injection.
  • Suitable pharmaceutical forms include suspensions and solutions which contain the microbubbles of the invention, or the aqueous suspension thereof, together with one or more inert carriers or excipients.
  • Suitable carriers include saline, sterile water, phosphate buffered saline and mixtures thereof.
  • compositions may additionally include other agents, e.g. emulsifiers, suspending agents, viscosity modifiers, dispersing agents, solubilisers, stabilisers, buffering agents, preserving agents, etc.
  • agents e.g. emulsifiers, suspending agents, viscosity modifiers, dispersing agents, solubilisers, stabilisers, buffering agents, preserving agents, etc.
  • the compositions may be sterilised by conventional sterilisation techniques.
  • the non-gaseous components of the microbubbles of the invention may be supplied in the form of a dry mixture, e.g. a lyophilised powder or dehydrated film, for reconstitution together with the gaseous component at the point of use, e.g. for reconstitution in water, saline or PBS.
  • a dry mixture e.g. a lyophilised powder or dehydrated film
  • the invention provides a dry mixture of a lipid-modified chelator, wherein a metal cation is immobilised by said chelator and one or more additional microbubble shell forming components.
  • these components of the mixture may be any of the examples described herein.
  • the dry mixtures of the invention may further comprise a macromolecule as defined herein.
  • the dry mixtures of the invention may be in the form of a powder or a film.
  • the mixture is a lyophilised mixture.
  • the dry mixtures of the invention are substantially, e.g. essentially, water-free (moisture-free). This may be expressed as a water content of less than 10% w/w, e.g. less than 9%, 8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5% or 1% w/w as measured by weight loss on drying or chemically by the Karl Fischer method (United States Pharmacopeia; European Pharmacopoeia).
  • kits comprising the dry mixtures of the invention and a gas suitable for forming a microbubble, e.g. any of the examples described herein.
  • the kit comprises, in separate containers, (i) a dry mixture of a lipid-modified chelator, wherein a metal cation is immobilised by said chelator and one or more additional microbubble shell forming components; (ii) a macromolecule as defined herein and (iii) a gas suitable for forming a microbubble.
  • the kit comprises, in separate containers, (i) a dry mixture of a lipid-modified chelator, wherein a metal cation is immobilised by said chelator and one or more additional microbubble shell forming components; and (ii) a gas suitable for forming a microbubble.
  • the kit comprises, in separate containers, (i) a dry mixture of a lipid-modified chelator, wherein a metal cation is immobilised by said chelator, one or more additional microbubble shell forming components and a macromolecule as defined herein and (ii) a gas suitable for forming a microbubble.
  • kits comprising the components of the microbubbles of the invention, or combinations thereof, in separate containers. These components of the kit may be any of the examples described herein.
  • the kit comprises, in separate containers, (i) a lipid- modified chelator, wherein a metal cation is immobilised by said chelator, and (ii) a gas suitable for forming a microbubble, and optionally (iii) a macromolecule as defined herein and optionally (iv) one or more additional microbubble shell forming components.
  • the kit comprises, in separate containers, (i) a lipid- modified chelator, wherein a metal cation is immobilised by said chelator and (ii) a macromolecule as defined herein, and optionally (iii) a gas suitable for forming a microbubble and optionally (iv) one or more additional microbubble shell forming components.
  • the kit comprises, in separate containers, (i) a lipid- modified chelator, wherein a metal cation is immobilised by said chelator and one or more additional microbubble shell forming components and (ii) a macromolecule as defined herein, and optionally (iii) a gas suitable for forming a microbubble.
  • the kit comprises, in separate containers, (i) a lipid- modified chelator, wherein a metal cation is immobilised by said chelator and one or more additional microbubble shell forming components and (ii) a gas suitable for forming a microbubble, and optionally (iii) a macromolecule as defined herein.
  • Figure 3 shows representative confocal fluorescence microscopy images of DSPC- PEG40S-DGS-NTA(Ni) MB in (A) DPBS and (B) DPBS supplemented with 10% FBS.
  • Figure 4 shows confocal fluorescence microscopy images of (A) control DSPC- PEG40S microbubbles and (B) functionalised DSPC-PEG40S-DGS-NTA(Ni) microbubbles which have both been exposed to his-GFP.
  • His-GFP green
  • nickelated lipid black
  • his-GFP binds specifically to microbubbles containing nickelated lipis in image (B).
  • Figure 5 shows confocal fluorescence microscopy images of A549s cells incubated with CellMask Deep Red and subsequently with DSPC-PEG40S-DGS-NTA(Ni)-His- GFP microbubbles.
  • A shows the control, where cells are not exposed to ultrasound, whereas
  • B shows the sample exposed to ultrasound as described in the Examples.
  • Confocal fluorescence images were obtained from the Zeiss 780 microscope at a magnification of 63x.
  • Figure 7 shows confocal microscopy images of A549s incubated with CellMask Deep Red (magenta) and subsequently with -9x10 7 DSPC-PEG40S-18:1 DGS- NTA(Ni)-His-AF488 bubbles (green).
  • A shows the control, where cells are not exposed to ultrasound (US)
  • B which shows the sample exposed to 1 MHz CW at -0.2 MPa peak to peak.
  • Confocal fluorescence images were obtained from the Zeiss 780 microscope at a magnification of 63x.
  • a batch sonication protocol as previously reported by De Cock et al. (Ultrasound and microbubble mediated drug delivery: acoustic pressure as determinant for uptake via membrane pores or endocytosis, 2015, Journal of Controlled Release, 197, 20-28) has been employed to prepare microbubbles (MBs).
  • the protocol is as follows: 1,2-distearoyl-sn-glycero-3-phosphocholine (18:0 PC, Avanti Polar Lipids), commonly referred to as DSPC, polyoxyethylene (40) stearate (PEG40S, Sigma Aldrich, UK), and 18:1 nickel-nitrilotriacetic acid (Ni-NTA)- functionalised lipids (DGS-NTA(Ni), Avanti Polar Lipids) were dissolved in chloroform (Sigma Aldrich, UK) and mixed in a glass vial at a molar ratio of 9 : 0.5 : 0.2, containing 20 mg in total of lipid constituents.
  • the chloroform solution was covered with perforated parafilm (Bemis Company, Inc., Neenah, Wl, USA) and the mixture was subsequently heated overnight on a hotplate set to 50 °C, facilitating evaporation of chloroform and the formation of a homogenous lipid film.
  • perforated parafilm Bemis Company, Inc., Neenah, Wl, USA
  • the obtained dry lipid film was suspended in 5 ml of Dulbecco’s phosphate-buffered saline (DPBS, pH 7.4, Life Technologies, Paisley, UK) and stirred at 100 °C on a magnetic stirrer hotplate for a minimum of 30 minutes.
  • Phosphate-buffered saline consists of 137 mM NaCI, 2.7 mM KCI, 10 mM Na 2 HP0 4 , and 1.8 mM KH 2 P0 4 in H 2 0.
  • Lipids were then homogeneously dispersed for 150 s using a sonicator (Microson XL 2000, probe diameter 3 mm, 20 W, 22.5 kHz, QSonica, NewTown, Ct, USA) with the tip completely immersed in the lipid solution (power setting 3).
  • MBs were subsequently formed by placing the sonicator tip at the air-water interface under constant sulphur hexafluoride flow (The BOC Group pic, UK) and sonicated for 30 s to form a cloudy suspension of microbubbles (power setting 14).
  • the vial containing the DSPC-PEG40S-DGS-NTA(Ni) MB suspension was capped and placed in ice for approximately 5 minutes.
  • Microbubbles were washed twice to eliminate the excess of free (Ni-NTA)- functionalised lipid. Microbubbles were washed using the centrifugation method without size isolation, as reported by Feshitan et al. (Microbubble size isolation by differential centrifugation, 2009, Journal of Colloid and Interface Science, 329(2), 316-324). In summary, MBs were loaded into a 5 ml syringe and centrifuged for 5 minutes at 300g, corresponding to 1000 rpm. Following centrifugation, the subnatentwas discarded and MBs were either resuspended in 1 ml of fresh DPBS or in 1 ml of a solution containing a His-tagged protein, e.g. His-GFP (25 pg/ml, Stratech, UK), in DPBS.
  • His-GFP 25 pg/ml, Stratech, UK
  • Microbubbles were incubated with His-GFP and a third washing step was performed. After 5 minutes of incubation, centrifugation was performed, again for 5 minutes at 300g, to eliminate the excess unbound His-GFP and improve signal-to- noise ratio for fluorescence microscopy experiments.
  • Microbubble characterisation concentration, specificity and stability
  • Figure 1B shows an exemplary field of view, clearly visualising the size distribution of the microbubbles. After performing two washing steps and incubation with His-GFP, the size distribution changed accordingly, as seen in Figure 2. A.
  • the SAT3 as designed by Dr Michael Gray, comprises of a water tank system, consisting of either a 0.5 MHz or 1.7 MHz transducer, a purpose-built sample holder, acoustic absorber, and a passive cavitation detector (PCD).
  • the acoustic absorber is placed opposite of the transducer and above the sample holder to attenuate any reflections.
  • the sample holder is designed to hold a 6.5 mm transwell (Corning ® , Sigma Aldrich, UK), upright, in which various types of cells can be cultured in cell culture medium and, subsequently, exposed to ultrasound in the presence and absence of microbubbles.
  • a plug is used to shield the cells and small volumes of culture medium, -100 pi, from the surrounding water.
  • A549s cells were seeded approximately 24 hours prior to imaging in 6.5 mm diameter transwells in 24-well plates (Corning ® , Sigma Aldrich, UK) in 100 mI culture medium. Sub-confluent cells were chosen to enable proper membrane segmentation. Cells were washed twice with PBS and incubated with 2 pg/ml CellMask Deep Red for 10 minutes. Subsequently, cells were washed twice before incubation with 100 mI of DSPC-PEG40S-DGS-NTA(Ni)-His-GFP microbubbles and either exposed to ultrasound or simply only placed in the 37 °C bath
  • the protocol is as follows: 1,2- distearoyl-sn-glycero-3-phosphocholine (18:0 PC, Avanti Polar Lipids), commonly referred to as DSPC, polyoxyethylene (40) stearate (PEG40S, Sigma Aldrich, UK), and either 18:1 or 16:0 nickel-nitrilotriacetic acid (Ni-NTA)-functionalised lipids (DGS-NTA(Ni), Avanti Polar Lipids) were dissolved in chloroform (Sigma Aldrich, UK) and mixed in a glass vial at a molar ratio of 9 : 0.5 : 0.2, containing 20 mg in total of lipid constituents.
  • the chloroform solution was covered with perforated parafilm (Bemis Company, Inc., Neenah, Wl, USA) and the mixture was subsequently heated overnight on a hotplate set to 50 °C, facilitating evaporation of chloroform and the formation of a homogenous lipid film.
  • perforated parafilm Bemis Company, Inc., Neenah, Wl, USA
  • the obtained dry lipid film was suspended in 5 ml of Dulbecco’s phosphate-buffered saline (DPBS, pH 7.4, Life Technologies, Paisley, UK) and stirred at 100 °C on a magnetic stirrer hotplate for a minimum of 45 minutes.
  • Phosphate-buffered saline consists of 137 mM NaCI, 2.7 mM KCI, 10 mM Na2HP04, and 1.8 mM KH2P04 in H20.
  • Lipids were then homogeneously dispersed for 150 s using a sonicator (Microson XL 2000, probe diameter 3 mm, 20 W, 22.5 kHz, QSonica, NewTown, Ct, USA) with the tip completely immersed in the lipid solution (power setting 3).
  • MBs were subsequently formed by placing the sonicator tip at the air-water interface under constant sulphur hexafluoride flow (The BOC Group pic, UK) and sonicated for 30 s to form a cloudy suspension of microbubbles (power setting 14).
  • the vial containing the DSPC-PEG40S-DGS-NTA(Ni) MB suspension was capped and placed in ice for approximately 5 minutes.
  • Microbubbles were washed once to eliminate the excess of free (Ni-NTA)- functionalised lipid. Microbubbles were washed using the centrifugation method without size isolation, as reported by Feshitan et al. (supra). In summary, MBs were loaded into a 5 ml syringe and centrifuged for 5 minutes at 300g, corresponding to 1000 rpm. Following centrifugation, the subnatent was discarded and MBs were resuspended in 5 ml of a freshly made up His-tagged AF488 DPBS solution containing 4 ug of His-AF488 ([AF488]-HHHHHH-acid, Cambridge Research Biochemicals).
  • the sample holder was designed to hold cell culture dishes (m-Dishes, Ibidi) with plastic substrates much thinner (-100 pm) than the acoustic wavelength in water enclosed with a custom polydimethylsiloxane (PDMS) lid (-1.2 mm thick). Characterization of the acoustical properties of the PDMS lid has been described in detail by Carugo et al. (Biologically and acoustically compatible chamber for studying ultrasound-mediated delivery of therapeutic compounds (2015) Ultrasound in medicine & biology, 41(7), 1927-1937). The ultrasound transducer was driven by a 1 MHz sinusoidal signal from a function generator passed through a 1.9 MHz low- pass filter and amplified 55 dB by a radiofrequency amplifier.
  • A549s were grown in a 35 mm-diameter low Ibidi m-Dish at 20% confluence 17 hrs prior to exposure. A549s were incubated with Cell Mask Deep Red at 1 pg/ml for 8 minutes at 37 °C. The Ibidi m-Dish was subsequently enclosed by a thin PDMS lid designed for ultrasound exposure as described by Carugo et al. (2015). A 12 ml solution of washed MBs were prepared at room temperature and then injected through the acoustically-compatible PDMS lid, ensuring that there were no bubbles trapped in the p-Dish. The inlet and outlet of the PDMS lid were subsequently plugged using 1.4 mm diameter plastic rods.
  • the m-Dish was then inverted to increase microbubble-cell proximity, transferred into the water tank setup filled with filtered, distilled, deionised, and degassed water at room temperature, and exposed to 30 s of ultrasound at 1MHz, 0.25 MPa peak to peak, continuous wave, during which acoustic emissions monitoring was performed.

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Abstract

The invention provides a method for presenting a macromolecule of interest at the outer surface of the plasma membrane of a eukaryotic cell, said method comprising contacting a eukaryotic cell with a microbubble comprising a gas core and a shell, wherein said shell comprises a lipid-modified chelator and wherein a metal cation is immobilised by said chelator and said macromolecule is bound to the metal cation via one or more coordinate bonds, and whilst in contact with one another, exposing the cell and the microbubble to ultrasound conditions effective to result in presentation of said macromolecule at the outer surface of the plasma membrane of the eukaryotic cell. The method may be used in immunotherapy. Also provided are microbubbles and kits of use in the methods of the invention.

Description

Functionalised microbubble mediated cell tagging
The present invention provides a method for presenting macromolecules of interest at the outer surface of the plasma membrane of eukaryotic cells. More specifically, the method uses microbubbles carrying the macromolecule of interest bound to a functionalised lipid component of the microbubble shell together with focused low intensity ultrasound to transfer the macromolecule from the microbubble to a site in or on the outer surface of the plasma membrane and/or to induce the uptake of the lipid-macromolecule complex by the plasma membrane of eukaryotic cells to which the microbubbles have been exposed. The method may be used to incorporate macromolecules of interest into the plasma membrane of cells in vivo, ex vivo or in vitro and thus therapeutic and non-therapeutic uses are envisaged, in particular the combat of hyperproliferative disorders. The invention further provides said functionalised microbubbles, with or without the macromolecule of interest bound thereto, and kits for the preparation of the same.
Gas microbubbles, coated with a surfactant or polymer shell, have become established as an effective type of contrast agent available for diagnostic ultrasound imaging. The gas core of the microbubbles scatters ultrasound both more efficiently and over a wider range of frequencies than biological cells, producing strong contrast between the vasculature and the surrounding tissue.
More recently, it has been shown that microbubbles may be used in the targeted delivery of a drug or therapeutic agent to a specific organ or tissue, or cell thereof. Therapeutic agents may be incorporated into the encapsulating shell of the microbubbles. The microbubbles can then be introduced into the body at their intended site of action or introduced intravenously and their passage through the bloodstream can be traced using low intensity imaging ultrasound. Once the microbubbles have found their way to their intended location the therapeutic agent can be released by applying focused low intensity therapeutic ultrasound (typically longer pulses and higher frequencies than imagining ultrasound) to rupture the microbubbles selectively. It has been shown that the rupture of the microbubbles may also enhance the cellular uptake of the therapeutic agent or drug, e.g. macromolecules such as proteins and nucleic acids, by temporarily increasing the membrane permeability of nearby cells (Rahim et al; Ultrasound Med. Biol., 2006; 32(8): 1269-79). This effect is known as sonoporation.
In addition to the delivery of therapeutic agents to the interior of cells where they may exert their effects, there are instances in which the delivery of macromolecules to the external surface of a cell could provide novel therapeutic, diagnostic and research applications. For instance, the cells of an animal subject may be tagged selectively with a macromolecule which is recognised by the subject's immune system and which induces the immune system to eliminate the cells displaying such a label. In another example, target cells of an animal subject may be tagged selectively with a macromolecule which is part of a specific and selective binding pair and which is not found naturally in the subject or the local region in which such target cells are found and administration of a drug conjugated to a specific binding partner of the macromolecule will direct that drug to those cells that display the label. In research, delivery of macromolecules to the cell surface will allow the study of cell surface biology. Many other applications of such a technique would be readily apparent to the skilled person.
It has now been recognised that microbubbles of a particular composition may be engineered to carry macromolecules in a manner which permits the presentation of said macromolecules at the outer surface of the plasma membrane of eukaryotic cells when said microbubbles are exposed to particular ultrasound conditions.
Thus, in a first aspect the invention provides a method for presenting a macromolecule of interest at the outer surface of the plasma membrane of a eukaryotic cell, said method comprising
(i) contacting a eukaryotic cell with a microbubble comprising a gas core and a shell, wherein said shell comprises a lipid-modified chelator and wherein a metal cation is immobilised by said chelator and said macromolecule is bound to the metal cation via one or more coordinate bonds, and
(ii) whilst in contact with one another, exposing the cell and the microbubble to ultrasound conditions effective to result in presentation of said macromolecule at the outer surface of the plasma membrane of the eukaryotic cell. Expressed alternatively the invention also provides a microbubble comprising a gas core and a shell, wherein said shell comprises a lipid-modified chelator and wherein a metal cation is immobilised by said chelator and a macromolecule of interest is bound to the metal cation via one or more coordinate bonds for use in a method for presenting said macromolecule at the outer surface of the plasma membrane of a eukaryotic cell in or on a subject, said method comprising
(i) contacting a eukaryotic cell whilst in or on a subject with the microbubble, and
(ii) whilst in contact with one another, exposing the cell and the microbubble to ultrasound conditions effective to result in presentation of said macromolecule at the outer surface of the plasma membrane of the eukaryotic cell.
Expressed alternatively the invention also provides the use of a microbubble comprising a gas core and a shell, wherein said shell comprises a lipid-modified chelator and wherein a metal cation is immobilised by said chelator and a macromolecule of interest is bound to the metal cation via one or more coordinate bonds in the manufacture of a medicament for presenting said macromolecule at the outer surface of the plasma membrane of a eukaryotic cell in or on a subject, said method comprising
(i) contacting a eukaryotic cell with the microbubble whilst in or on a subject, and
(ii) whilst in contact with one another, exposing the cell and the microbubble to ultrasound conditions effective to result in presentation of said macromolecule at the outer surface of the plasma membrane of the eukaryotic cell.
The macromolecule may be therapeutically or diagnostically effective in that its presentation at the outer surface of the plasma membrane of a eukaryotic cell can result in a therapeutic effect, e.g. an immunotherapeutic effect, either directly or indirectly, or can provide information useful in the diagnosis or monitoring of disease. Thus, the above methods may be defined as therapeutic and/or diagnostic methods.
Presentation of a macromolecule at the outer surface of the plasma membrane may be expressed alternatively as the display or showing of said macromolecule at this location. From other perspectives, the act of using the microbubbles of the invention to present a macromolecule at the outer surface of the plasma membrane may be described as incorporating a macromolecule into the plasma membrane, or introducing, delivering or transferring a macromolecule to the plasma membrane, more specifically from the microbubble. From still further perspectives, the act of using the microbubbles of the invention to present a macromolecule at the outer surface of the plasma membrane may be described as tagging, labelling or marking the plasma membrane with the macromolecule.
More specifically, “presentation of the macromolecule at the outer surface of the plasma membrane of a eukaryotic cell” may be a result of the transfer of the macromolecule from the microbubble to a site in or on the outer surface of the plasma membrane of a eukaryotic cell and/or a result of the introduction of the macromolecule-lipid complex of the microbubble into the outer surface of the plasma membrane of a eukaryotic cell upon exposure to suitable ultrasound conditions. This latter process may be due to fusion of the microbubble shell with the plasma membrane or by selective transfer of the macromolecule-lipid complex from the microbubble shell to the plasma membrane or by any other means. The term “contact” therefore includes sufficiently close proximity between the eukaryotic cell and the microbubble to permit the transfer of the macromolecule from the microbubble to a site in or on the outer surface of the plasma membrane of a eukaryotic cell and/or the introduction of the macromolecule-lipid complex of the microbubble into the outer surface of the plasma membrane of the eukaryotic cell upon exposure to suitable ultrasound conditions.
In the methods disclosed herein there is preferably no meaningful delivery of the macromolecule to the interior of the cell, e.g. the cytoplasm and/or the nucleus. In other words, immediately following presentation of the macromolecule at the outer surface of the plasma membrane of a eukaryotic cell essentially none of the macromolecule carried by the cell will be found in the interior of the cell. Put differently the macromolecule will preferably not be in the interior of the cell in detectable amounts immediately following presentation of the macromolecule at the outer surface of the plasma membrane of a eukaryotic cell. Immediately following presentation of the macromolecule at the outer surface of the plasma membrane of a eukaryotic cell maybe considered less than 5 minutes following completion of the ultrasound exposure. Microbubbles of use in the invention comprise a gas core and a shell, wherein said shell comprises a lipid-modified chelator and wherein a metal cation is immobilised by said chelator.
In accordance with the invention a chelator is an organic compound which carries one or more polar groups capable of forming at least one, preferably at least two, stable coordinate bonds with a metal cation. Such compounds may be referred to as chelants, chelating agents, or sequestering agents. In certain embodiments the chelator may be nitrilotriacetic acid (NTA), iminodiacetic acid (IDA), diethylenetriaminepentaacetic acid (DTPA), 1,4,7,10-tetraazacyclododecane;
1.4.7.10-tetraacetic acid (DOTA); 1,4, 8,11-tetraazacyclotetradecane (cyclam);
1.4.7.10-tetraazacyclododecane (cyclen); 1,4-ethano-1,4,8,11- tetraazacyclotetradecane (et-cyclam); 1 ,4,7,11-tetraazacyclotetradecane (isocyclam); 1,4,7,10- tetraazacyclotridecane ([13]aneN4); 1,4,7,10- tetraazacyclododecane-1,7-diacetic acid (D02A); 1,4,7,10- tetraazacyclododecane- 1,4,7-triacetic acid (D03A); 1,4,7,10-tetraazacyclododecane-1,7- di(methanephosphonic acid) (D02P); 1,4,7,10-tetraazacyclododecane-1,4,7- tri(methanephosphonic acid) (D03P); 1,4,7,10-tetraazacyclododecane-1,4,7,10- tetra(methanephosphonic acid) (DOTP); ethylenediaminetetraacetic acid (EDTA); 4-(1 ,4,8,11-tetraazacyclotetradec-1-yl) methylbenzoic acid (CPTA); cyclohexanediaminetetraacetic acid (CDTA); ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA); hydroxyethyl ethylenediamine triacetic acid (HEDTA); triethylene tetraamine hexaacetic acid (TTHA); 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), carboxymethylated-aspartic acid (CM-Asp), tris(2-aminoethyl)amine (TREN), ortho- phosphoserine, and N,N,N’-tris(carboxymethyl)ethylenediamine. NTA , DTPA and IDA may be advantageous in accordance with the invention, especially NTA
In certain embodiments, the chelator is not a lipophilic chelator. The chelators described above are considered not to be lipophilic chelators. However, in other embodiments lipophilic chelators may be used so long as the chelator selected does not interfere with the capacity of the microbubble to present macromolecules of interest at the outer surface of the plasma membrane of eukaryotic cells under suitable ultrasound conditions. Lipophilic chelators include, but are not limited to, 2-hydroxyquinoline-4-carboxylic acid; 6-chloro-2-hydroxyquinoline; 8-chloro-2-hydroxyquinoline; carbostyril 124; carbostyril 165; 4,6-dimethyl-2-hydroxyquinoline; 4,8-dimethyl-2-hydroxyquinoline; or other 2-quinolinol compounds; 8- hydroxyquinoline (oxine); 8-hydroxyquinoline b- D-galactopyranoside; 8- hydroxyquinoline b-D-glucopyranoside; 8-hydroxyquinoline glucuronide; 8- hydroxyquinoline-5-sulfonic acid; 8-hydroxyquinoline^-D- glucuronide sodium salt; 8-quinolinol hemisulfate salt; 8-quinolinol N-oxide; 2- amino-8-quinolinol; 5,7-dibromo-8-hydroxyquinoline; 5,7-dichloro-8- hydroxyquinoline; 5,7-diiodo-8-hydroxyquinoline; 5,7-dimethyl-8-quinolinol; 5- amino-8-hydroxyquinoline dihydrochloride; 5-chloro-8-quinolinol; 5-nitro-8- hydroxyquinoline; 7-bromo-5-chloro-8-quinolinol; N-butyl-2,2'-imino-di(8-quinolinol); 8-hydroxyquinoline benzoate; 2-benzyl-8-hydroxyquinoline; 5- chloro-8- hydroxyquinoline hydrochloride; 2-methyl-8-quinolinol; 5-chloro-7-iodo-8-quinolinol; 8-hydroxy-5-nitroquinoline; 8-hydroxy-7-iodo-5-quinolinesulfonic acid; 5,7-dichloro- 8-hydroxy-2-methylquinoline; 1-azanaphthalene; 1-benzazine; and other quinolone containing chemical compounds; A23187; HMPAO (hexamethyl propylene amine oxime); HYNIC (6-hydrazinopyridine-3-carboxylic acid); BMEDA (N-N-bis (2- mercaptoethyl)-N’,N’-diethylethylenediamine), DISIDA (diisopropyl iminodiacetic acid); phthaldialdehyde, 2,4-dinitrophenol; di- benzo-18-crown-6; o-xylylenebis(N, N-diisobutyldithiocarbamate); N,N,N',N'-tetracyclohexyl-2,2'-thiodiacetamide; 2- (1,4,8,11 -tetrathiacyclotetradec-6-yloxy)hexanoic acid; 2-(3,6, 10,13- tetrathiacyclotetradec-1-oxy)hexanoic acid; N,N-bis (2-mercaptoethyl)-N',N'- diethylethylenediamine; ionomycin; pyridoxal isonicotinoyl hydrazone (PIH); salicylaldehyde isonicotinoyl hydrazone (SIH), 1,4,7-trismercaptoethyl-1,4,7- triazacyclononane; N,N',N"-tris(2-mercaptoethyl)-1,4,7-triaza-cyclononane; monensis; DP-b99; DP-109; BAPTA, pyridoxal isonicotinoyl hydrazone (PIH), alamethicin, di-2-pyridylketone thiosemicarbazone (HDpT), carbonyl cyanide m- chlorophenyl hydrazone (CCCP), lasalocid A (X-537A), 5-bromo derivative of lasalocid; cyclic depsipeptides; cyclic peptides: DECYL-2; N,N,N',N'-tetrabutyl-3,6- dioxaoctanedi[thioamide]); N,N,N',N'-tetracyclohexyl-3-oxa-pentanediamide; N,N- dicyclohexyl-N',N'-dioctadecyl-diglycolic-diamide; N,N'-diheptyl-N,N'-dimethyl-1- butanediamide; N,N"-octamethylene-bis[N'-heptyl-N'-methyl-malonamide]; N,N- dioctadecyl-N',N'-dipropyl-3,6-dioxaoctanediamide; N-[2-(1H-pyrrolyl-methyl)]-N'-(4- penten-3-on-2)-ethane-1 ,2-diamine (MRP20). The lipid of the lipid-modified chelator may be any hydrophobic or amphiphilic hydrocarbon-containing compound which, when linked to a chelator with an immobilised metal ion carrying, bound to, or associated with a macromolecule in accordance with the invention, is capable of stable incorporation into the shell of the microbubble and the outer layer of the lipid bilayer of the plasma membrane of a eukaryotic cell. The lipid may be saturated or unsaturated. Typically, these are hydrophobic or amphiphilic hydrocarbon-containing compounds of at least 10 carbon residues. This may include representatives from the cationic lipids, zwitterionic lipids, neutral lipids, or anionic lipids. Thus, the lipid may be a fatty acid, a monoacylglycerol, a diacylgylcerol, a triacylglycerol, a sterol, a glycolipid, or a phospholipid (e.g. a glycerophospholipids or a sphingolipid). The lipid may be an amphiphilic lipid, i.e. any lipid composed of a hydrophilic portion and a hydrophobic portion (typically a hydrophilic head and a hydrophobic tail). The hydrophilic portion of useful amphiphilic lipids may comprise polar or charged groups such as carbohydrates, phosphate, carboxylic, sulfato, amino, sulfhydryl, nitro, hydroxy and other like groups. The hydrophobic portion may comprise apolar groups that include without limitation long chain saturated and unsaturated aliphatic hydrocarbon groups and groups substituted by one or more aromatic, cyclo-aliphatic or heterocyclic group(s). Examples of amphipathic lipid compounds include, but are not limited to, phospholipids, aminolipids, glycolipids and sphingolipids.
When lipids with acyl groups, e.g. monoacylglycerol, diacylgylcerol, or triacylglycerol lipids, are used, it may be advantageous if one or more, e.g. all, of the acyl groups are unsaturated.
In certain embodiments, the lipids may be anionic or neutral (including zwitterionic and polar) lipids, e.g. anionic or neutral phospholipids.
Neutral lipids exist in an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, for example, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides and diacyl glycerols (e.g. 1,2-dioleoyl-sn- glycerol (DG)). Suitable zwitterionic lipids include, without limitation, diacylphosphatidylcholine (e.g. dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC)). An anionic lipid is a lipid that is negatively charged at physiological pH. These lipids include, without limitation, phosphatidylglycerol (e.g. dioleoylphosphatidylglycerol (DOPG)), diacylphosphatidylethanolamine, cardiolipin, diacylphosphatidylserine (e.g. dioleoylphosphatidylserine (DOPS), diacylphosphatidic acid, N-dode-canoyl phosphatidylethanolamines, N-succinyl phosphatidylethanolamines, N- glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifying groups joined to neutral lipids.
Anionic and neutral lipids may be referred to herein as non-cationic lipids. Such lipids may contain phosphorus. Examples of non-cationic lipids of use in the invention include lecithin, lysolecithin, phosphatidylethanolamine, lysophosphatidylethanolamine, dioleoylphosphatidylethanolamine (DOPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DM PE), distearoylphosphatidy 1-ethanolamine (DSPE), palmitoyloleoylphosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), 1,2- dioleoyl-sn-glycerol (DG), 1,2-dioleoyl-sn-glycero-3-succinate (DGS); 1 ,2-distearoyl- sn-glycerol (18:0 DG); dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleyolphosphatidylglycerol (POPG), 16-0-monomethyl PE, 16-0- dimethyl PE, 18-1- trans PE, palmitoyloleoyl-phosphatidylethanolamine (POPE), 1-stearoyl- 2-oleoylphosphatidyethanolamine (SOPE), phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetylphosphate, and cholesterol.
The lipid of the lipid-modified chelator of the present invention can also be a cationic lipid. Cationic lipids contain positively charged functional groups under physiological conditions. Cationic lipids include, but are not limited to, N,N-dioleyl- N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), 2,3-dioleoyloxy trimethylammonium propane (DOTAP), 2,3-di- (oleyloxy)propyl trimethyl ammonium (DOTMA), N-[1-(2,3,-ditetradecyloxy)propyl]- N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N-[1- (2,3,dioleyloxy)propyl]-N,N-dimethyl-N-hydroxy ethylammonium bromide (DORIE), 3b-[N-(N',N'^ίhΐqΐI^ΐ3GTΐίhoqΐΐΊ3hq) carbamoyl]cholesterol (DC-Choi), dimethyldioctadecylammonium (DDAB), dioctadecylamidoglycyl spermine (DOGS) and N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA).
In other embodiments the amphiphilic lipid may be selected from phosphatidylcholines, e.g. 1,2-dioleoyl-phosphatidylcholine, 1,2-dipalmitoyl- phosphatidylcholine, 1 ,2-dimyristoyl-phosphatidylcholine, 1 ,2-distearoyl- phosphatidylcholine, 1-oleoyl-2-palmitoyl-phosphatidylcholine, 1-oleoyl-2-stearoyl- phosphatidylcholine, 1-palmitoyl-2-oleoyl-phosphatidylcholine and 1-stearoyl-2- oleoyl-phosphatidylcholine; phosphatidylethanolamines, e.g. 1,2-dioleoyl- phosphatidylethanolamine, 1,2- dipalmitoylphosphatidylethanolamine, 1,2- dimyristoylphosphatidylethanolamine, 1,2- distearoylphosphatidylethanolamine, 1- oleoyl-2-palmitoyl-phosphatidylethanolamine, 1-oleoyl-2-stearoyl- phosphatidylethanolamine, 1-palmitoyl-2-oleoyl-phosphatidylethanolamine, 1- stearoyl-2-oleoyl-phosphatidylethanolamine and N-succinyl-dioleoyl- phosphatidylethanolamine; phosphatidylserines, e.g. 1,2-dioleoyl- phosphatidylserine, 1 ,2-dipalmitoyl-phosphatidylserine, 1 ,2-dimyristoyl- phosphatidylserine, 1,2-distearoyl-phosphatidylserine, 1-oleoyl-2-palmitoyl- phosphatidylserine, 1-oleoyl-2-stearoyl-phosphatidylserine, 1-palmitoyl-2-oleoyl- phosphatidylserine and 1-stearoyl-2-oleoyl-phosphatidylserine; phosphatidylglycerols, e.g.1 ,2-dioleoyl-phosphatidylglycerol, 1 ,2-dipalmitoyl- phosphatidylglycerol, 1,2- dimyristoyl-phosphatidylglycerol, 1,2-distearoyl- phosphatidylglycerol, 1-oleoyl-2-palmitoyl-phosphatidylglycerol, 1-oleoyl-2-stearoyl- phosphatidylglycerol, 1-palmitoyl-2-oleoyl-phosphatidylglycerol and 1-stearoyl-2- oleoyl-phosphatidylglycerol; pegylated lipids; pegylated phospoholipids, e.g. phophatidylethanolamine-N-[methoxy(polyethyleneglycol)-1000], phophatidylethanolamine-N-[methoxy(polyethyleneglycol)-2000], phophatidylethanolamine-N-[methoxy(polyethylene glycol)-3000], phophatidylethanolamine-N-[methoxy(polyethyleneglycol)-5000]; pegylated ceramides, e.g. N-octanoyl- sphingosine-1-
{succinyl[methoxy(polyethyleneglycol)1000]}, N-octanoyl-sphingosine-1- {succinyl[methoxy(polyethylene glycol)2000]}, N-octanoyl-sphingosine-1- {succinyl[methoxy(polyethyleneglycol)3000]}, N-octanoyl-sphingosine-1- {succinyl[methoxy(polyethyleneglycol)5000]}; lyso-phosphatidylcholines; lyso- phosphatidylethanolamines; lyso-phosphatidylglycerols; lyso-phosphatidylserines; ceramides; sphingolipids; glycolipids, e.g. ganglioside GM1; glucolipids; sulphatides; phosphatidic acids, e.g. di-palmitoyl-glycerophosphatidic acid; palmitic fatty acids; stearic fatty acids; arachidonic fatty acids; lauric fatty acids; myristic fatty acids; lauroleic fatty acids; physeteric fatty acids; myristoleic fatty acids; palmitoleic fatty acids; petroselinic fatty acids; oleic fatty acids; isolauric fatty acids; isomyristic fatty acids; isostearic fatty acids; sterol and sterol derivatives, e.g. cholesterol, cholesterol hemisuccinate, cholesterol sulphate, and cholesteryl-(4- trimethylammonio)- butanoate, ergosterol, lanosterol; polyoxyethylene fatty acids esters and polyoxyethylene fatty acids alcohols; polyoxyethylene fatty acids alcohol ethers; polyoxyethylated sorbitan fatty acid esters; glycerol polyethylene glycol oxy- stearate; glycerol polyethylene glycol ricinoleate; ethoxylated soybean sterols; ethoxylated castor oil; polyoxyethylene polyoxypropylene fatty acid polymers; polyoxyethylene fatty acid stearates; di-oleoyl-sn-glycerol; 1,2-dioleoyl-sn-glycero- 3-succinate; dipalmitoyl-succinylglycerol; 1,3-dipalmitoyl-2- succinylglycerol; 1-alkyl- 2-acyl-phosphatidylcholines, e.g. 1-hexadecyl-2-palmitoyl-phosphatidylcholine; 1- alkyl-2-acyl-phosphatidylethanolamines, e.g. as 1-hexadecyl-2- palmitoyl- phosphatidylethanolamine; 1-alkyl-2-acyl-phosphatidylserines, e.g. 1- hexadecyl-2- palmitoyl-phosphatidylserine; 1-alkyl-2-acyl-phosphatidylglycerols, e.g. 1- hexadecyl-2-palmitoyl-phosphatidylglycerol; 1-alkyl-2-alkyl-phosphatidylcholines, e.g. 1 -hexadecyl-2-hexadecyl-phosphatidylcholine; 1 -alkyl-2-alkyl- phosphatidylethanolamines, e.g. 1-hexadecyl-2-hexadecyl- phosphatidylethanolamine; 1-alkyl-2-alkyl-phosphatidylserines, e.g. 1-hexadecyl-2- hexadecyl-phosphatidylserine; 1-alkyl-2-alkyl-phosphatidylglycerols, e.g. 1- hexadecyl-2-hexadecyl-phosphatidylglycerol; N-Succinyl-dioctadecylamine; palmitoylhomocysteine; lauryltrimethylammonium bromide; cetyltrimethyl- ammonium bromide; myristyltrimethylammonium bromide; 1,2-dioleoyl-sn-glycerol; 1,2-dioleoyl-sn-glycero-3-succinate (DGS); DOTMA; DOTAP; 1,2-dioleoyl-c-(4'- trimethylammonium)-butanoyl-sn-glycerol (DOTB); 1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[amino(polyethylene glycol)-2000]; 1 ,2-distearoyl-sn- glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (DSPE- PEG-2000); DSPE-PEG2000-TATE (1,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000]-TATE); and 1- tetradecanoyl-2-octadecanoyl-sn-glycero-3-phosphocholine (MSPC). In certain embodiments the lipid may be a diglyceride, e.g. 1,2-dioleoyl-sn-glycerol or 1,2-dioleoyl-sn-glycero-3-succinate or 1,2-distearoyl-sn-glycerol (18:0 DG).
A lipid-modified chelator is a chelator which is linked to a lipid via a direct covalent bond or a covalent molecular linker. A direct covalent bond between the lipid and the chelating agent is a covalent bond formed by an atom of the lipid and an atom of the chelating agent. The atoms contributing to the bond may together or independently be carbon, oxygen, sulphur, nitrogen and/or phosphorous. The bond may be single, double or triple. In certain embodiments, the bond is part of an organic functional group. The skilled person would be entirely familiar with the options available for suitable organic functional groups which could act as linkers between the lipid and the chelating agent. Non-limiting examples thereof may include ester, carbonate ester, orthoester, ketone, ketal, hemiketal, ketene, ether, acetal, hemiacteal, peroxy, methylenedioxy, carbamate, amide, amine, amine oxide, hydroxamic acid, imine, imide, imidate, azide, azo, oxime, carbodiimide, carbazone, hydrozone, sulfide, disulfide, sulfinyl, sulfonyl, carbonothioyl, thioamide, thioester, thioether, thioketone, thioketal, sulphonate ester, dithiocarbamate, semicarbazone, phosphine or phosphodiester functional groups. Ester and amide bonds may be used in particular.
The covalent molecular linker may be any molecule, typically an organic molecule, or part thereof, which has a structure formed from covalently bonded atoms which is capable of bonding covalently with the lipid and the chelating agent. Within the lipid-chelator conjugate there will be a continuous series of covalently bonded atoms from the lipid to chelating agent via the molecular linker. In preferred embodiments, at least one of the covalent bonds in said series is as defined above. The molecular linker may however further comprise non-covalent, e.g. ionic bonds, in parts of the molecule which are not contributing to the covalent linkage between the chelating agent and the lipid.
The covalent molecular linker may be linear, circular or branched. In certain embodiments, the molecular linker will have a molecular weight of equal to or less than 1500 Daltons, e.g. equal to or less than 1250, 1000, 900, 800, 700, 600, 500, 400, 300, 200 or 100 Daltons. In certain embodiments, at least one direct covalent bond between the lipid and the covalent molecular linker is as defined above. In certain embodiments, at least one direct covalent bond between the chelating agent and the covalent molecular linker is as defined above. Each bond may be the same or different. The covalent linker molecule may comprise at least one covalent bond as defined above, preferably in the part of that molecule which contributes to the continuous series of covalently bonded atoms from the lipid to the chelating agent via the molecular linker.
The covalent molecular linker may be or comprise an amino acid or a peptide, e.g. of equal to or fewer than 15 amino acid residues, e.g. of equal to or fewer than 12, 10, 8, 6, 5, 4, 3 or 2 amino acid residues. Specific examples of peptide linkers which may be used include but are not limited to peptides of Gly and/or Ser residues (e.g. (Gly)2-8, (Ser)2-8, (GGGGS)1-3); (EAAAK)1-3; A(EAAAK)1-3A; Leu-Glu; (Xaa-Pro)1-6 (e.g. (Glu-Pro)1-6, (Lys-Pro)1-6, (Ala-Pro)1-6).
The covalent molecular linker may be or comprise a monosaccharide or an oligosaccharide or a polymer formed therefrom, e.g. a saccharide of equal to or fewer than 12 amino acid residues, e.g. equal to or fewer than 10, 8, 6, 5, 4, 3 or 2 amino acid residues. Thus, the covalent molecular linker may be a monosaccharide, disaccharide or trisaccharide or sugar derivatives thereof, e.g. aldonic and uronic acids, deoxy or amino sugars, sulfated sugars, and sugar alcohols. The monosaccharide or one or more of the monosaccharide residues of the disaccharide or trisaccharide may be a those, a tetrose, a pentose, a hexose, a heptose, an octose, a nonose or a decose in pyranose or furanose form and/or L- or D- form where appropriate and/or sugar derivatives thereof. Pentose or hexose saccharides/residues are preferred, e.g. mannose (e.g. D-mannose), galactose (e.g. D- galactose), glucose (e.g. D-glucose), fructose, fucose (e.g. L-fucose), N- acetyl-glucosamine, N-acetylgalactosamine, rhamnose, galactosamine, glucosamine (e.g. D-glucosamine), galacturonic acid, glucuronic acid, mannuronate, guluronate, N-acetylneuraminic acid, methyl D-mannopyranoside (mannoside), a-methyl-glucoside, galactoside, ribose, xylose, arabinose, saccharate, mannitol, sorbitol, inositol, glycerol and derivatives of these monomers. The disaccharide may be exemplified by acarviosin, allolactose, cellobiose, chitobiose, galactose-alpha-1, 3-galactose, dentiobiose, isomalt, isomaltose, isomaltulose, kojibiose, lactitol, lactobionic acid, lactose, lactulose, laminaribiose, maltitol, maltose, mannobiose, melibiose, melibiulose, neohesperidose, nigerose, robinose, rutinose, sambubiose, sophorose, sucralfate, sucralose, sucrose, sucrose acetate isobutyrate, sucrose octaacetate, trehalose, truranose, xylobiose or derivatives of these disaccharides.
The covalent molecular linker may be or comprise a nucleotide or an oligonucleotide, i.e. a nucleic acid, e.g. a ribonucleotide or a deoxyribonucleotide.
The linker may also be or comprise a straight chain, branched or cyclic, substituted or unsubstituted, alkyl, alkenyl or alkynl group (typically C2-8) or derivative thereof, e.g. aminohexanoic acid or one of a range of commercially available PEG (polyethylene glycol) linkers. Further examples of suitable covalent linker molecules include but are not limited to acetyl, glycol, succinyl, aconityl (c/s or trans), glutaryl, methylsuccinyl, trimellityl cysteamine, penicillamine, N-(2- mercaptopropionyl)glycine, 2-mercaptopropionic acid, homocysteine, 3- mercaptopropionic acid and deamino-penicillamine groups.
In certain embodiments, the covalent linker molecule may be a plurality of the molecules and/or groups described above.
In some embodiments, the covalent linker may have one or more polar groups capable of forming one or more coordinate bonds with a metal cation and said groups may form a stable coordinate bond with the metal cation of the microbubble of use in the invention.
The metal cation may be any metal cation which when immobilised by the lipid modified chelator is capable of binding a macromolecule carrying a metal ion binding motif, e.g. a metal ion binding amino acid sequence as defined herein. In certain embodiments, the metal ion may be a monovalent, diavalent, trivalent or tetravalent metal cation. In certain embodiments, the metal cation is a transition metal cation. For in vivo use, the cation should be non-toxic in the amounts used.
In certain embodiments, the metal cation is not radioactive. The metal cation may be Zn2+, Cu2+, Cd2+, Hg2+, Co2+, Ni2+, Fe2+, Fe3+, Pd3+, Ga3+, Al3+. Zn2+, Cu2+, Ni2+, and Fe2+ are preferred. Ni2+ and Zn2+ is most preferred. In this context a reference to a metal cation “immobilised” by a chelator is a reference to the arrangement of a metal cation and the chelator group of the lipid- modified chelator of use in the invention such that there exists at least one, preferably at least two, stable coordinate bonds between one or more polar groups of the chelator and the metal ion.
Notable lipid-modified chelators of use in the invention are 18:1 and 16:0 DGS- NTA(Ni).
Microbubbles comprise a shell which surrounds an internal void comprising a gas. Generally, these are approximately spherical in shape, although the shape of the microbubble is not essential in carrying out the invention and is therefore not to be considered limiting. The size of the microbubble may vary depending on its intended application. For in vivo applications the microbubble may be of a size such as to permit its passage through the vascular system following administration, e.g. by intravenous injection, but in embodiments in which the microbubbles are administered directly to a target site such constraints are not so limiting. Likewise, in vitro and/or ex vivo applications the size of the microbubble need not be constrained in this way. In general, microbubbles typically have a diameter of less than about 200 pm and greater than about 0.1 pm, preferably in the range from about 0.5 to about 100 pm. Particularly suitable for use in the invention are microbubbles having a diameter of less than about 10 pm and greater than about 0.5 pm, more preferably 1 to 8 pm, particularly preferably up to 5 pm, e.g. about 2 pm. The shell of the microbubble will vary in thickness and will typically range from about 10 to about 200 nm. The precise thickness is not essential provided that the shell performs the desired function of retaining the gas core and carrying the lipid modified chelator and the microbubble is functional in the methods of the invention described herein. Size selection and size reduction techniques may be employed to select/reduce the size of microbubbles (e.g. sonication, size exclusion chromatography, filtration). Individual microbubble diameter may be measured by microscopy. The size of a plurality of microbubbles, e.g. the weight-average particle size, may be determined according to a dynamic light scattering method (e.g., quasi-elastic light scattering method). For example, microbubble particle sizes can be measured using dynamic light scattering instruments (e.g. Zetasizer Nano ZS model manufactured by Malvern Instruments Ltd. and ELS-8000 manufactured by Otsuka Electronics Co., Ltd.). The instruments measure Brownian motion of the particles and particle size is determined based on established dynamic light scattering methodological theory.
Materials which may be used to form the shell of the microbubbles of the invention should be biocompatible and suitable materials are well known in the art. Typically, the shell of the microbubble will comprise a surfactant or a polymer. Surfactants which may be used include any material which is capable of forming and maintaining a microbubble by forming a layer at the interface between the gas within the core and an external medium, e.g. an aqueous solution which contains the microbubble. A surfactant or combination of surfactants may be used. Those which are suitable include lipids, in particular phospholipids. Such lipids are present in the microbubbles in addition to the lipid-modified chealtor.
Suitable microbubble shell forming lipids include phospholipids and/or glycolipids. Examples of suitable lipids include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidic acid, phosphatidylinositol, dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dimyristoyiphosphatidylcholine (DMPC), dioleylphosphatidylcholine (DOPE), dibehenoylphosphatidylcholine (dibehenoyl-sn-glycero-3-phosphocholine; DBPC), dimyristoylphosphatidylethanolamine, dipalmitolphosphatidylethanolamine, distearoylphosphatidylethanolamine, DSPE-PEG-2000, cardiolipin, sphingomyelin, glycosphingolipids, glucolipids, glycolipids, sulphatides, lipids with ether and ester- linked fatty acids, polymerizable lipids, and combinations thereof. The lipids used may be of either natural or synthetic origin. There are also usable phospholipids derived from plants and animals, e.g. egg yolk or soybeans (e.g. egg yolk lecithin or soya bean lecithin) and their hydrogenation products or hydroxide derivatives, so- called semi-synthetic phospholipids. Fatty acids constituting a phospholipid are not specifically limited, and saturated and unsaturated fatty acids are usable. Suitable lipids may include any of those described above in the context of the lipid modified chelator component.
In preferred embodiments the microbubble of use in the invention comprises a phosphatidylcholine or a phosphatidylethanolamine, e.g. distearoylphosphatidylcholine (DSPC), dimyristoyiphosphatidylcholine (DMPC), dibehenoylphosphatidylcholine (DBPC) or 1,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (DSPE-PEG-2000)
Polymer materials which are suitable for use in forming the shell of the microbubble include proteins, in particular albumin, particularly human serum albumin. Other biocompatible polymers which may be used include poly(vinyl alcohol) (PVA), poly(D,L-lactide-co-glycolide) (PLGA), cyanoacrylate, poloxamers (Pluronics) or combinations thereof.
The shell materials, e.g. lipids, may also be functionalised microbubble shell forming compounds. Representative, non-limiting examples of the above mentioned materials include sialic acid derivatives, glucuronic acid derivatives; glutaminic acid derivatives; polyglycerin derivatives; polyethylene glycol derivatives (including methoxypolyethylene glycol condensates, etc.), e.g. polyethylene glycol (40) stearate, N-[carbonyl-methoxy polyethylene glycol-2000]-1,2-dipalmitoyl-sn-glycero- 3-phosphoethanolamine, N-[carbonyl-methoxy polyethylene glycol-5000]-1,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine, N-[carbonyl-methoxy polyethylene glycol-750]-1 ,2-distearoyl-sn glycero-3-phosphoethanolamine, N-[carbonyl-methoxy polyethylene glycol-2000]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (MPEG 2000-distearoyl phosphatidylethanolamine), N-[carbonyl-methoxy polyethylene glycol-5000]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, DSPE-PEG-2000 and DSPE-PEG2000-TATE.
In preferred embodiments the microbubble of use in the invention, more specifically its shell, comprises a polyethylene glycol modified lipid, polyethylene glycol (40) stearate or DSPE-PEG-2000.
The amounts of shell components and the ratio between the amounts of shell components may vary depending on the number and identity of those selected.
The skilled person would be able to select appropriate shell components in appropriate quantities to prepare a microbubble of use in the invention, i.e. a microbubble as defined herein and which is able to carry macromolecules in a manner which permits the presentation of said macromolecules at the outer surface of the plasma membrane of eukaryotic cells when said microbubbles are exposed to the particular ultrasound conditions of the invention. In one embodiment, the microbubble of use in the invention, more specifically its shell, comprises DSPC, polyethylene glycol (40) stearate (PEG40S), and DGS-NTA (18:1 and/or 16:0). In a more specific embodiment, at least a portion of said DGS- NTA of said microbubble has an immobilised Ni2+ or Zn2+ ion. In these embodiments the ratio of DSPC, PEG40S and DGS-NTA is about 9 : about 0.5 : about 0.2, respectively, e.g. 5-14 : 0.25-0.75 : 0.1-0.3 or 7-11 : 0.4-0.6 ; 0.15-0.25.
In more specific embodiments of these microbubbles of use in the invention, PEG40S will be present in the shell at about 2 to about 10 % w/w, e.g. about 3, 4,
5, 6, 7, 8 or 9 to about 10%, or about 2 to about 34, 5, 6, 7, 8 or 9 % w/w.
In more specific embodiments of these microbubbles of use in the invention, DGS- NTA will be present in the shell at less than 5% w/w, e.g. at about 0.1 to about 5 % w/w, e.g. about 0.5, 1 , 2, 3 or 4 to about 5%, or about 0.1 to about 0.5, 1 , 2, 3 or about 4 % w/w.
In these specific embodiments, the remainder of the shell mass may be DSPC.
“% w/w” (or “percentage weight by weight”) is a commonly used expression of the amount of a compound in a solid. 1% w/w equates to 1 gram of compound per 100g of solid, 2% w/w equates to 2g of compound per 100g of solid, and so on. Accordingly, % w/w may be expressed as g/1 OOg, grams per 100 grams and g 100g_1. 1% w/w also equates to 10 gram of compound per kilogram of solid. In the present context, the term refers to the amount of shell component component in question in grams per 100g of microbubble shell.
The microbubble shells may comprise single or multiple layers of the same or different materials. Multiple layers may, for example, be formed in cases where the basic shell material (e.g. a lipid) bears one or more polymers or polysaccharides. Examples of such polymers include polyethylene glycol and polyvinylpyrrolidone.
The microbubble shell may also be coated with polymers, e.g. poly-L-lysine and PLGA, and/or polysaccharides, e.g. alginate, dextran, diethylamino-ethyl- dextran hydrochloride (DEAE) or chitosan. Methods for attaching these coating materials may involve electrostatic or covalent interactions. Different coating materials (polymers, polysaccharides, proteins, etc.) may be used in order to improve the properties of the microbubble, for example by increasing the rigidity, stability in circulation and/or tissue permeation capability of the microbubble-based reagents, by manipulating the net surface charge of the microbubble and by increasing its payload capacity.
The gas within the core of the microbubble should be biocompatible. The term "gas" encompasses not only substances which are gaseous at ambient temperature and pressure, but also those which are in liquid form under these conditions. Where the "gas" is liquid at ambient temperature this will generally undergo a phase change to a gas at a temperature of 30°C or above, more preferably 35°C or above. For any gas which is a liquid at ambient temperature, it is generally preferred that this will undergo a phase change to a gas at a temperature between about 30 and 37°C, preferably at around normal body temperature. Any reference herein to "gas" should thus be considered to encompass not only gases and liquids, but also liquid vapours and any combination thereof, e.g. a mixture of a liquid vapour in a gas.
Gases which are suitable for incorporation within the microbubbles for use according to the invention include air, nitrogen, oxygen, carbon dioxide, hydrogen; inert gases, e.g. helium, argon, xenon or krypton; sulphur fluorides (e.g. sulphur hexafluoride, disulphur decafluoride); low molecular weight hydrocarbons e.g. alkanes (e.g. methane, ethane, propane, butane), cycloalkanes (e.g. cyclopropane, cyclobutane, cyclopentane), alkenes (e.g. ethylene, propene); and alkynes (e.g. acetylene or propyne); ethers; esters; halogenated low molecular weight hydrocarbons; and mixtures thereof. Examples of suitable halogenated hydrocarbons are those which contain one or more fluorine atoms and include, for example, bromochlorodifluoromethane, chlorodifluoromethane, dichlorodifluoromethane, bromotrifluoromethane, chlorotrifluoromethane, chloropentafluoroethane, dichlorotetrafluoroethane, chlorotrifluoroethylene, fluoroethylene, ethyl fluoride, 1,1-difluoroethane and perfluorocarbons. Perfluorocarbons include perfluoroalkanes (e.g. perfluoromethane, perfluoroethane, perfluoropropanes, perfluorobutanes, perfluoropentanes, perfluorohexanes and peril uoroheptanes); perfluoroalkenes (e.g. perfluoropropene, perfluorobutenes); and perfluorocycloalkanes (e.g. perfluorocyclobutane). Microbubbles containing perfluorinated gases, in particular, perfluorocarbons (e.g. perfluoropropanes, perfluorobutanes, perfluoropentanes and peril uorohexanes) are suitable for use in the invention due to their stability in the bloodstream.
In other preferred embodiments, the microbubble of use in the invention comprises a high molecular weight gas, e.g. a perfluorocarbon or a sulphur fluoride.
The microbubble of the use in the invention further comprises a macromolecule bound to (associated with) the metal cation immobilised by the chelator via one or more coordinate bonds. In certain embodiments, the number and/or strength of the one or more coordinate bonds are sufficient to ensure a sufficiently persistent relationship exists between the macromolecule and the lipid-modified chelator such that macromolecule-lipid complex is capable of stable incorporation into both the shell of the microbubble and the outer layer of the lipid bilayer of the plasma membrane of a eukaryotic cell following appropriate ultrasound exposure. In other embodiments, the number and/or strength of the one or more coordinate bonds are sufficient to ensure a sufficiently persistent relationship exists between the macromolecule and the lipid-modified chelator such that macromolecule-lipid complex is capable of stable incorporation into the shell of the microbubble but that the macromolecule may still be transferred from the microbubble to a site in or on the outer surface of the plasma membrane of a cell following appropriate ultrasound exposure. The relationship between the macromolecule and the lipid-modified chelator need not be permanent however, and some embodiments may be designed to permit a gradual release of the macromolecule from the lipid-modified chelator.
The macromolecule may be a protein (including peptides), a carbohydrate, a nucleic acid or a combination or complex thereof. The macromolecule may be therapeutically or diagnostically effective in that its presentation at the outer surface of the plasma membrane of a eukaryotic cell can result in a therapeutic effect, e.g. an immunotherapeutic effect, either directly or indirectly, or can provide information useful in the diagnosis or monitoring of disease. If the macromolecule does not have a molecular motif capable of forming one or more coordinate bonds with the immobilised metal cation of sufficient stability, the macromolecule must be engineered to comprise or carry such a metal cation binding motif. Examples of such a motif include, but are not limited to a histidine residue, a cysteine residue, a tryptophan residue, an amino acid analogue comprising an imidazole side chain or a combination and/or a plurality thereof, i.e. a contiguous sequence of subunits selected from the aforementioned amino acid residues and analogues. In certain embodiments, the metal cation binding motif is a contiguous sequence of subunits selected from histidine residues and amino acid analogues comprising an imidazole side chain. The contiguous sequence may consist of 2-10 of such subunits, e.g. 3-10, 4-10, 5-10, 6-10, 7-10, 8-10 or 9-10 of such subunits. The contiguous sequence may consist of 2, 3, 4, 5, 6, 7, 8, 9, or 10 of such subunits. In certain embodiments the sequence will be entirely histidine residues and in this context the motif may be described as a His tag.
In certain embodiments, the macromolecule of use in the invention will comprise a metal cation binding motif comprising a contiguous sequence of 3-10 amino acids selected from histidine, cysteine, tryptophan or an amino acid analogue comprising an imidazole side chain or a combination thereof, preferably a contiguous sequence of 5-10 histidine residues.
Thus, in certain embodiments the macromolecule may be a fusion protein comprising, e.g. consisting of, at least one protein of interest and at least one heterologous sequence of amino acids which is capable of forming one or more coordinate bonds with the immobilised metal cation of sufficient stability, e.g. a metal cation binding motif as described above. The amino acids of the protein of interest may be contiguous with the amino acids of the metal cation binding motif, but in other embodiments at least one amino acid spacer sequence may be included. Such spacer sequences may be up to 10, e.g. up to 9, 8, 7, 6, 5, 4, 3, 2 or 1 , amino acids in length. The spacer amino acids may be any of the proteinogenic amino acids or structurally similar analogues. The metal cation binding motif of the fusion protein is “heterologous” in the sense that it is a sequence of amino acids which is not present in the consensus wild type sequence of the at least one protein of interest of the fusion protein. The macromolecule, including any heterologous metal cation binding motif, will typically be water-soluble. Water-soluble macromolecules can be considered to be macromolecules for which less than 1000 parts pure water are required to solubilise 1 part of the macromolecule, e.g. less than 500, 250, 100, 50, 40, 30, 20, 10, 9, 8,
7, 6, 5, 4, 3, 2 or 1 parts pure water are required to solubilise 1 part of the macromolecule.
The identity of the macromolecule is limited only insofar as the skilled person is interested in presenting that macromolecule, or a part thereof, at the outer surface of the plasma membrane of a eukaryotic cell.
In certain embodiments, the macromolecule may be, or comprise, a tumour antigen, e.g. those listed in the Cancer Antigenic Peptide Database hosted by the Cancer Immunity Journal (https://caped.icp.ucl.ac.be/Peptide/list).
In certain embodiments, the macromolecule may be, or comprise, a hydrophilic portion of a macromolecule with hydrophilic and hydrophobic regions, or one or more hydrophilic portions of a macromolecule complex with hydrophilic and hydrophobic regions. In certain embodiments, the macromolecule may be selected from hydrophilic portions, e.g. extracellular domains or a portion thereof, of a cluster of differentiation (CD) protein (e.g. CD58 (lymphocyte function-associated antigen 3), CD106 (VCAM-1), CD54 (ICAM-1), CD2, CD4, CD19), an interleukin receptor protein (e.g. IL-4 receptor, IL-2 receptor or IL-12 receptor), a MUC protein (e.g. MUC16/CA-125, MUC5AC and MUC1), a major histocompatibility complex (MHC), preferably an peptide loaded MHC capable of inducing, maintaining or propagating an immune response thereto, a T cell receptor or a B cell receptor.
In certain embodiments the macromolecule may be, or comprise, a portion, or all, of a caspase, carcinoembryonic antigen (CEA), alphafetoprotein (AFP), tyrosinase, epithelial tumour antigen (ETA), melanoma-associated antigen (MAGE), an antibody (including fragments thereof, e.g. Fab, Fab', F(ab')2, Fv, scFv, dAb, minibody), preferably wherein said antibody can induce, maintain or propagate an immune response when bound to its target antigen, or Protein A/G (preferably bound to an antibody, e.g. those mentioned above). In certain embodiments, the antibody specifically binds any of the above-mentioned macromolecules, e.g. a tumour antigen, or an extracellular portion of CD58, CD106, CD54, CD2, CD4, or CD19.
References to the above named proteins extends to mutations thereof, e.g. mutations that result in the formation of a tumour antigen.
The microbubbles of the invention are specifically designed to be used in ultrasound-mediated tagging of eukaryotic cell plasma membranes.
The ultrasound conditions which may be used, i.e. applied to the target cell and the microbubble when each are in contact, will be appropriate to result in the presentation of the macromolecule of interest at the outer surface of the plasma membrane of the target eukaryotic cell in the relevant context. Conditions for use in vitro or ex vivo may vary from those required in vivo. The skilled person would be able to adjust the various conditions to achieve successful results. Adjusting the frequency and/or the peak rarefaction pressure and/or the pulse length may give the most effective control. The ultrasound may be provided in continuous or pulsed mode. Pulsed mode may be helpful in in vivo contexts where there may be concerns over prolonged exposures.
In certain embodiments, the frequency of the ultrasound will be about 0.25 to about
2.5 MHz, e.g. any of about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, or 2.0 to about
2.5 MHz or about 0.25 to any of about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, or 2.0 MHz. Any and all ranges which may be formed from any of these range endpoints are specifically contemplated. A range of about 0.25 to about 0.75 MHz, e.g. about 0.4 to about 0.6 MHz, or about 0.5 MHz, may be advantageous. In other embodiments a range of about 0.5 to about 1.5 MHz, e.g. about 0.7 to about 1.2 MHz, or about 1.0 MHz, may be advantageous.
In certain embodiments, the peak rarefaction pressure will be about 0.10 to about
1.5 MPa, e.g. any of about 0.15, 0.2, 0.25, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, or 1.25 to about 1.5 MPa or about 0.10 to any of about 0.15, 0.2, 0.25, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, or 1.25 MPa. Any and all ranges which may be formed from any of these range endpoints are specifically contemplated. A range of about 0.1 to about 0.3 MPa, e.g. about 0.15 to about 0.25 or about 0.18 MPa, may be advantageous. In other embodiments, a range of about 0.22 to about 0.28 MPa, or about 0.25 MPa, may be advantageous.
In certain embodiments, the number of cycles per pulse may be about 1 to about 10 million, e.g. about 1 to about 2, 3, 4, 5, 6, 7, 8, 9 million or about 1, 2, 3, 4, 5, 6, 7,
8, 9 to about 10 million or about 5 million. In other embodiments, the cycles per pulse may however be different and the skilled person would be able to determine appropriate values for the specific needs in question.
In certain embodiments, the duty cycle will be about 0.5 to about 100%, e.g. any of about 1, 10, 20, 30, 40, 50, 60, 70, 80 or 90 to about 100% or about 0.5 to about 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100%. Any and all ranges which may be formed from any of these range endpoints are specifically contemplated. A range of about 70 to about 100%, or about 100% may be advantageous.
In certain embodiments, the duration of exposure will be about 10 to about 300 seconds, e.g. any of about 20, 40, 60, 80, 100, 120, 150, 180, 210, 240, 270 to about 300 seconds or about 10 to about 20, 40, 60, 80, 100, 120, 150, 180, 210, 240, or 270 seconds. Any and all ranges which may be formed from any of these range endpoints are specifically contemplated. A range of about 10 to about 120 seconds, e.g. about 30 to about 90 seconds, or about 60 seconds may be advantageous.
Any source capable of producing ultrasound may be used in the methods herein described. The source should be capable of directing the energy to the target site and may include, for example, a probe or device capable of directing energy to the target tissue from the surface of the body. In certain embodiments, the equipment used may comprise an focused ultrasound transducer, a transformer, an amplifier, a waveform generator, a digitiser, a pre-amplifier, a high-pass filter, and a passive cavitation detector (PCD).
The method may be performed in vitro, e.g. on cultured cells or tissue. Such cell cultures may be planktonic cultures or a solid support bound culture. The contacting step may take place on a suspension of cells (which may include cells which have previously been cultured on a solid support and are in suspension transiently) or on cells associated with a solid support (which may include cells which have previously been cultured in suspension and are associated with a solid support transiently). The cells/tissue will typically be maintained in a suitable buffered aqueous medium and then be exposed to microbubbles provided in an aqueous medium. The microbubble may be applied to the cells/tissue or vice versa. Upon contact or shortly thereafter the cells/tissue and microbubbles will be exposed to ultrasound as described above. Following ultrasound treatment, the cells/tissue may be washed to remove excess microbubbles and/or tested to assess, e.g. quantify, the presentation of the macromolecule of interest at the outer surface of the plasma membrane of the target eukaryotic cell.
Such methods are expected to represent a highly advantageous investigational tool for use to investigate the role or cell membrane components and their impact upon interactions with other cells, pathogens, drug molecules, and so on.
The method may also be performed on cells or tissue which are ex vivo, i.e. cells or tissue which have been removed from a subject in order to have the method of the invention performed thereon and then to be returned to the subject or be administered to a different subject.
Such methods are expected to have utility in therapeutic and diagnostic contexts. For instance, immune cells, in particular cell-eliminating immune cells, e.g. T cells (in particular Tc cells), natural killer cells, monocytes and macrophages, may be tagged with macromolecules which function to modulate their (cell-eliminating, e.g. cytotoxic and/or cytophagic) activities and/or target such activities towards a target cell type, e.g. a hyperproliferative cell. In other embodiments, B cells and dendritic cells may be tagged with macromolecules which function to modulate their immunogenic activities, in particular towards a target cell type, e.g. a hyperproliferative cell.
In other embodiments immune cells involved in immunotolerance, e.g. T cells (in particular Th and Treg cells), dendritic cells, mast cells and B cells may be tagged with macromolecules which function to modulate their immunotolerance activities towards a target cell type, e.g. those cells associated with autoimmune disease, inflammatory disease or allergy.
In other embodiments, aberrant cells from a subject may be tagged with immunostimulatory macromolecules which are designed to induce a targeted immune response to those aberrant cells and others like them within the subject, thereby promoting their elimination from the subject undergoing treatment.
In still further embodiments, it is envisaged that particular cells of interest may be tagged ex vivo with a detectable macromolecule, e.g. radioactive or fluorescent macromolecule, and their distribution within the subject may be monitored upon their return to the subject.
These methods may also be performed on cells/tissues which are in vivo. In such embodiments, aberrant cells in or on a subject may be tagged with immunostimulatory macromolecules which are designed to induce a targeted immune response to those aberrant cells and others like them within the subject and promote their elimination.
In other embodiments, target cells may be tagged with macromolecules which are part of a specific and selective binding pair and which are not found naturally in the subject or the local region in which such target cells are found. A suitable drug may then be targeted to those cells if administered as a conjugate with the relevant specific and selective binding partner.
The invention further provides a microbubble of the invention for use in such methods and the use of a microbubble of the invention in the manufacture of a medicament for use in such methods.
In these in vivo methods a pharmaceutically acceptable suspension of the microbubbles of the invention is generally introduced directly into the region comprising the target cells (e.g. intratumourally), or intradermally, subcutaneously, intraperitoneally or intravenously, or into lymph vessels at or near the area to be treated. Preferably, the suspension of microbubbles is introduced intravenously or directly. The target area is then exposed to ultrasound to image the area and to determine the location of the microbubbles. Once the microbubbles are at the intended target, they are exposed to the ultrasound conditions described above. The optional step of monitoring the microbubbles using ultrasound is generally performed with ultrasound of a lower intensity (typically corresponding to a lower frequency) than that used to introduce the macromolecule-lipid complex of the microbubble into the outer surface of the plasma membrane of a eukaryotic cell and/or to transfer of the macromolecule from the microbubble to a site in or on the outer surface of the plasma membrane of a eukaryotic cell.
To make the in vivo methods more selective for the target aberrant cells, the microbubbles, more specifically the shell forming materials, may be derivatised with receptor affinity molecules specific to receptors on the target aberrant cells. Such affinity molecules include receptor specific polypeptides, nucleic acids and carbohydrates, e.g. antibodies, antibody fragments, peptide growth factors, DNA and RNA. Said receptors may be polypeptides, nucleic acids or carbohydrates. A plethora of receptor-ligand pairs are known to the skilled person and may be identified without undue burden and any of these pair could provide the basis of the choice of receptor affinity molecule for use in the invention.
In an alternative or additional approach the microbubbles of the invention may be rendered magnetic, e.g. by further comprising magnetic nanoparticles (e.g. as described in US 9427396), and magnetic fields may be applied at the region containing the target aberrant cells prior to or during exposure to the ultrasound conditions recited described above.
The quantity of microbubbles that may be administered to the subject will depend on the specific microbubble formulation used and the macromolecule that is carried by the microbubble. Nevertheless, it is, in general, preferred that a dose of the microbubble formulation should not exceed 1 cc/kg, preferably 0.3 cc/kg, and more preferably 0.15 cc/kg.
In more specific embodiments the invention provides a method of immunotherapy of a human or non-human animal subject, said method comprising presenting a macromolecule at the outer surface of the plasma membrane of a target cell on or in the subject, wherein said macromolecule promotes elimination of cells displaying said macromolecule on their surface by the immune system of the subject, wherein said method comprises contacting said target cell whilst on or in the subject with a microbubble of the invention carrying the macromolecule as defined herein and, whilst in contact with one another, exposing the cell and the microbubble to ultrasound conditions effective to result in presentation of said macromolecule at the outer surface of the plasma membrane of the cell.
Expressed alternatively the invention provides a microbubble of the invention carrying a macromolecule as defined herein, wherein said macromolecule promotes elimination of cells displaying said macromolecule on their surface by the immune system of a human or non-human animal subject for use in a method of immunotherapy of a human or non-human animal subject, said method comprising presenting said macromolecule at the outer surface of the plasma membrane of a target cell on or in the subject by contacting said target cell whilst on or in the subject with the microbubble and, whilst in contact with one another, exposing the cell and the microbubble to ultrasound conditions effective to result in presentation of said macromolecule at the outer surface of the plasma membrane of the cell.
Expressed in another way, the invention provides for the use of a microbubble of the invention carrying a macromolecule as defined herein, wherein said macromolecule promotes elimination of cells displaying said macromolecule on their surface by the immune system of a human or non-human animal subject in the manufacture of a medicament for use in a method of immunotherapy of a human or non-human animal subject, said method comprising presenting said macromolecule at the outer surface of the plasma membrane of a target cell on or in the subject by contacting said target cell whilst on or in the subject with the microbubble and, whilst in contact with one another, exposing the cell and the microbubble to ultrasound conditions effective to result in presentation of said macromolecule at the outer surface of the plasma membrane of the cell.
Elimination of cells by the immune system refers to any mechanism by which the subject’s immune system recognises cells presenting the macromolecule in question and then acts to reduce the number of viable forms of such cells in the subject. This may be via a cytotoxic and/or a cytophagic mechanism, e.g. involving T cells (in particular Tc cells), natural killer cells, phagocytes and/or macrophages. The term encompasses complete eradication of viable forms of such cells from the subject but also the eradication of a portion of viable forms of such cells. The elimination may be transient or permanent. The term “cell-eliminating” should be construed accordingly.
Examples of macromolecules which may be presented on the target cells in this embodiment may be, or comprise, full length or functional portions, e.g. extracellular domains of portions thereof, of CD58, CD106, CD54, an MHC (preferably an peptide loaded MHC capable of inducing, maintaining or propagating an immune response thereto), a MUC protein (e.g. MUC16/CA-125, MUC5AC and MUC1), a caspase, carcinoembryonic antigen (CEA), alphafetoprotein (AFP), tyrosinase, epithelial tumour antigen (ETA), melanoma-associated antigen (MAGE).
The step of exposing the target cell on or in a subject to ultrasound conditions which are effective to result in presentation of said macromolecule at the outer surface of the plasma membrane of said cell may be as described above.
In other more specific embodiments the invention provides a method of immunotherapy of a human or non-human animal subject, said method comprising providing an ex vivo sample of immune cells, preferably immune cells of the subject; contacting said immune cells with a microbubble of the invention carrying a macromolecule as defined herein, wherein said macromolecule functions to target the cell-eliminating activity of the immune cell towards a target cell type in or on the subject or to modulate the cell-eliminating activity of the immune cell; and, whilst in contact with one another, exposing the cells and the microbubble to ultrasound conditions effective to result in presentation of said macromolecule at the outer surface of the plasma membrane of one or more of the immune cells; and administering at least a portion of the immune cells presenting the macromolecule to the subject.
Expressed alternatively the invention provides a microbubble of the invention carrying a macromolecule as defined herein, wherein said macromolecule functions to target the cell-eliminating activity of an immune cell towards a target cell type in or on a human or non-human animal subject or to modulate the cell-eliminating activity of an immune cell for use in a method of immunotherapy of a human or non- human animal subject, said method comprising providing an ex vivo sample of said immune cells, preferably immune cells of the subject; contacting said immune cells with the microbubble and, whilst in contact with one another, exposing the cells and the microbubble to ultrasound conditions effective to result in presentation of said macromolecule at the outer surface of the plasma membrane of one or more of the immune cells; and administering at least a portion of the immune cells presenting the macromolecule to the subject.
Expressed in another way, the invention provides for the use of a microbubble of the invention carrying a macromolecule as defined herein, wherein said macromolecule functions to target the cell-eliminating activity of an immune cell towards a target cell type in or on a human or non-human animal subject or to modulate the cell-eliminating activity of an immune cell in the manufacture of a medicament for use in a method of immunotherapy of a human or non-human animal subject, said method comprising providing an ex vivo sample of said immune cells, preferably immune cells of the subject; contacting said immune cells with the microbubble and, whilst in contact with one another, exposing the cells and the microbubble to ultrasound conditions effective to result in presentation of said macromolecule at the outer surface of the plasma membrane of one or more of the immune cells; and administering at least a portion of the immune cells presenting the macromolecule to the subject.
The invention also provides an ex vivo method useful in the immunotherapy of a human or non-human animal subject said method comprising providing an ex vivo sample of immune cells, preferably immune cells of the subject; contacting said immune cells with a microbubble of the invention carrying a macromolecule as defined herein, wherein said macromolecule functions to target the cell-eliminating activity of the immune cell towards a target cell type in or on the subject or to modulate the cell-eliminating activity of the immune cell; and, whilst in contact with one another, and exposing the cells and the microbubble to ultrasound conditions effective to result in presentation of said macromolecule at the outer surface of the plasma membrane of one or more of the immune cells.
The immune cells of use in this aspect of the invention are typically cytotoxic and/or cytophagic immune cells, e.g. T cells (in particular Tc cells), natural killer cells, monocytes and macrophages, but in other embodiments, B cells and dendritic cells may be used. Modulation of the cell-eliminating activity of an immune cell refers to any effect which stimulates, maintains, prolongs or enhances an immune cell’s role in promoting the elimination of a target cell from the body of a subject, e.g. by cytotoxic or cytophagic mechanisms. It may also refer to any effect which inhibits, reduces or prevents an immune cell’s role in suppressing the elimination of a target cell from the body of a subject, e.g. by cytotoxic or cytophagic means. These roles of an immune cell in cell elimination mechanisms may be direct and/or indirect.
The sample of immune cells of use in this aspect of the invention may be provided by any of the convenient cell separation/isolation techniques known in the art or which may be developed in the future. Such techniques may involve one or more steps of blood fractionation, centrifugation, precipitation, filtration, cell sorting. In other embodiments, the immune cells may be provided from cell culture.
Examples of macromolecules which may be presented on the immune cells in this embodiment may be, or comprise, full length or functional portions, e.g. extracellular domains of portions thereof, of CD2, CD4, CD19, IL-4 receptor, IL-2 receptor, IL-12 receptor, an antibody (including fragments thereof, e.g. Fab, Fab', F(ab')2, Fv, scFv, dAb, minibody) directed to CD106, CD58 or CD54, Protein A/G (preferably bound to an antibody, e.g. those mentioned above), a T cell receptor or a B cell receptor.
The step of exposing ex vivo samples of immune cells to ultrasound conditions which are effective to result in presentation of said macromolecules at the outer surface of the plasma membrane of one or more of the immune cells may be as described above.
Once presenting the macromolecule of interest the immune cells may be administered to the subject by any convenient means. Many approaches are available and are performed routinely, but may, for instance, include parental administration of a liquid or gel composition comprising the cells or implantation of a solid support, e.g. a natural or artificial polymer scaffold, seeded with the cells. Administration may be to a particular site at which the immune cells are designed to exert their effects or could be via systemic delivery to the blood and/or lymph circulatory systems. Transfusion based approaches may be used. In such embodiments, the immune cells may be mixed with blood or plasma and then transfused to the patient. Delivery may take the form of a bone marrow transplant or injection into the bone marrow.
It will be seen that the above two methods may be combined. It may be particularly advantageous if a binding pair of macromolecules are used to tag the target cells with one member of the pair and the immune cells are tagged with the other member of the pair.
Thus, in still more specific embodiments, the invention provides a method of immunotherapy of a human or non-human animal subject, said method comprising
(a) providing an ex vivo sample of immune cells, preferably immune cells of the subject; contacting said immune cells with a microbubble of the invention carrying a first macromolecule as defined herein, wherein said first macromolecule functions to target the cell-eliminating activity of the immune cell towards target cells presenting a second macromolecule as defined herein and, whilst in contact with one another, exposing the immune cells and the first microbubble to ultrasound conditions effective to result in presentation of said first macromolecule at the outer surface of the plasma membrane of one or more of the immune cells;
(b) administering at least a portion of the immune cells presenting the first macromolecule to the subject;
(c) contacting a target cell on or in the subject with a microbubble of the invention carrying the second macromolecule and, whilst in contact with one another, exposing the target cell and the second microbubble to ultrasound conditions effective to result in presentation of said second macromolecule at the outer surface of the plasma membrane of the target cell, wherein step (c) may be performed before, after or simultaneously with step
(a) and/or step (b).
The invention further provides a microbubble of the invention for use in one or other part of such methods (i.e. a microbubble carrying the first or the second macromolecule) and the use of a microbubble of the invention in the manufacture of a medicament for use in one or other part of said methods (i.e. a microbubble carrying the first or the second macromolecule). The invention still further provides a microbubble of the invention carrying the first macromolecule and a microbubble of the invention carrying the second macromolecule for use in such methods and the use of a microbubble of the invention carrying the first macromolecule and a microbubble of the invention carrying the second macromolecule in the manufacture of a medicament for use in said methods.
A plethora of specific binding pairs (also referred to a receptor-ligand pairs) is known to the skilled person and may be identified without undue burden and any of these pairs could provide the basis of the choice of macromolecule for use in this aspect of the invention. In certain embodiments the binding pair may be a specific antibody (or antibody fragment, e.g. Fab, Fab', F(ab')2, Fv, scFv, dAb, minibody) and antigen pair. In other embodiments the pair may be selected from following pairs, preferably in the arrangement target cell : immune cell; CD58:CD2,
CD106:anti-CD106, CD54:anti-CD54.
It will be apparent to the skilled person that in the above immunotherapy methods of the invention the target cell/cell type is the cell/cell type the immunotherapy method is designed to eliminate. Such cells will be aberrant, abnormal or dysfunctional in some way and thus give rise to or drive a clinical disease or condition which would benefit from treatment. In certain embodiments, the target cell type is a hyperproliferative or neoplastic cell, e.g. a cancer cell, in particular a tumour cell. In certain embodiments, it is a cell from the hyperproliferative or neoplastic diseases or conditions mentioned herein. These immunotherapy methods of the invention may therefore be described as methods for the elimination of a target cell from a subject or methods for the immunotherapeutic elimination of a target cell from a subject.
The above immunotherapy methods of the invention may further comprise the administration of further immunostimulatory therapeutic agents, e.g. agents which stimulate or enhance the subject’s immune system directly or those which do so indirectly by inhibiting, reducing or preventing immunosuppression pathways in the subject’s immune system. These may include immune cell checkpoint inhibitors and cytokines . In certain embodiments the immune checkpoint inhibitor may be selected from ipilimumab (anti-CTLA4), tremelimumab (anti-CTLA4), MDX-1106 (also known as BMS-936558 and nivolumab; anti-PD1 antibody), MK3475 (also known as pembrolizumab; anti-PD1 antibody), CT-011 (anti-PD1 antibody), AMP-224 (anti- PD1 fusion protein - PDL2-lg fusion protein), MDX-1105 (anti-PDL1 antibody), RG7446 (anti-PDL1 antibody), BMS-936559 (anti-PDL1 antibody), MGA271 (anti- B7-H3 antibody), atezolizumab (also known as MPDL3280A; anti-PDL1 antibody), avelumab (also known as MSB0010718C; anti-PDL1 antibody) and durvalumab (anti-PDL1 antibody).
In certain embodiments the immunostimulatory cytokine may be selected from cytokines e.g. TNF, IL-1, IL-2, IL-4, IL-6, IL-8, IL-12 and IFNa,
The disease or condition treatable by the above immunotherapy methods of the invention may be any hyperproliferative or neoplastic disease or condition, which terms include diseases or conditions caused by any malignant, pre-malignant or non-malignant (benign) neoplastic entities. The term therefore encompasses, inter alia, the treatment of cancers, tumours, malignancies, sarcomas, carcinomas, germinomas, lymphomas, leukaemias, blastomas, papillomas and adenomas. In these various embodiments the hyperproliferative or neoplastic disease or condition may be selected from colorectal cancer (also known as colon cancer, rectal cancer or bowel cancer), prostate cancer, kidney (renal) cancer (e.g. Wilm’s tumour), pancreatic cancer, testicular cancer, skin cancer (e.g. melanoma and non melanoma (e.g. basal-cell cancer, squamous-cell cancer)), breast cancer, ovarian cancer, vulvar cancer, cervical cancer, endometrial cancer, bladder cancer, stomach (gastric) cancer, intestinal cancer (e.g. duodenal cancer, ileal cancer, jejunal cancer, small intestine cancer), liver (hepatic) cancer, lung (pulmonary) cancer, oesophageal cancer, oral cancer, throat cancer, brain cancer (e.g. glioblastoma, medulloblastoma), adrenal cancer (e.g. adrenocortical cancer), thyroid cancer (e.g. anaplastic thyroid carcinoma), uterine cancer (e.g. uterine carcinosarcoma), haematological cancer (also known as the haematological malignancies) (e.g. haematopoietic and lymphoid cancer malignancies, e.g. leukemia, lymphoma and myeloma), including metastatic forms thereof, and non- malignant neoplasm or tumour in these anatomical sites (e.g. colorectal polyps, pilomatrixoma, hemangioma, osteoma, chondroma, lipoma, fibroma, lymphangioma, leiomyoma, rhabdomyoma, astrocytoma, meningioma, ganglioneuroma, papilloma, adenoma).
In other embodiments the immunotherapy methods of the invention may be used to treat autoimmune disease.
In still further embodiments the invention provides a method of immunotherapy of a human or non-human animal subject, said method comprising providing an ex vivo sample of immune cells, preferably immune cells of the subject; contacting said immune cells with a microbubble of the invention carrying a macromolecule as defined herein, wherein said macromolecule functions to modulate the immunotolerance activity of the immune cell; and, whilst in contact with one another, exposing the cell and the microbubble to ultrasound conditions effective to result in presentation of said macromolecule at the outer surface of the plasma membrane of one or more of the immune cells; and administering at least a portion of the immune cells presenting the macromolecule to the subject.
The invention further provides a microbubble of the invention for use in such methods and the use of a microbubble of the invention in the manufacture of a medicament for use in said methods.
The immune cells of use in this embodiment of the invention are immune cells involved in immunotolerance, e.g. T cells (in particular Th and Treg cells), dendritic cells, mast cells and B cells. Modulation of the immunotolerance activity of an immune cell refers to any effect which stimulates, maintains, prolongs or enhances an immune cell’s role in promoting immunotolerance towards a target cell from the body of a subject. It may also refer to any effect which inhibits, reduces or prevents an immune cell’s role in promoting immunotolerance towards a target cell from the body of a subject. These roles of an immune cell in immunotolerance mechanisms may be direct and/or indirect.
In this embodiment the macromolecule is, or comprises, a functional portion, e.g. extracellular domains of portions thereof, of the IL-2 receptor, IL-4 receptor, IL-12 receptor, CD19 or CD4. In this embodiment the disease or condition treatable by the above immunotherapy method of the invention may rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease (including Crohn’s disease and ulcerative colitis), multiple sclerosis, type 1 diabetes mellitus, Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, psoriasis, Graves' disease, Hashimoto's thyroiditis, myasthenia gravis, autoimmune vasculitis, autoimmune hepatitis, alopecia areata, celiac disease, eczema, atopic dermatitis, urticara, asthma, allergic conjunctivitis, allergic rhinitis, food allergies and other allergies.
In other more specific embodiments the invention provides a method of targeted drug delivery to a human or non-human animal subject, said method comprising presenting a macromolecule at the outer surface of the plasma membrane of a target cell of the subject, wherein said macromolecule is part of a specific binding pair and is not found in the subject or a region or tissue of the subject in which the target cells are found, wherein said method comprises contacting said target cell with a microbubble of the invention carrying the macromolecule as defined herein and, whilst in contact with one another, exposing the cell and the microbubble to ultrasound conditions effective to result in presentation of said macromolecule at the outer surface of the plasma membrane of the cell.
A plethora of specific binding pairs (also referred to a receptor-ligand pairs) is known to the skilled person and may be identified without undue burden and any of these pairs could provide the basis of the choice of macromolecule for use in this aspect of the invention. In certain embodiments the binding pair may be a specific antibody (or antibody fragment, e.g. Fab, Fab', F(ab')2, Fv, scFv, dAb, minibody) and antigen pair.
The method may further comprise administering to the subject a conjugate of a drug and the binding partner of said macromolecule. In accordance with the invention, such drug conjugates will typically be administered, e.g. orally, by inhalation or by injection (e.g. intravenous, intratumoural, intradermal, subcutaneous, intraperitoneal or intramuscular) in the form of a pharmaceutically acceptable composition together with at least one pharmaceutically acceptable carrier or excipient. Examples of suitable carriers or excipients are disclosed below. In some embodiments the the drug conjugate will be directly administered to the region or the tissue of the subject in which the target cells are found.
In this embodiment the drug may be a therapeutic agent selected from: anti-allergic agents (e.g. amelexanox); anti-anginals (e.g. diltiazem, nifedipine, verapamil, erythrityl tetranitrate, isosorbide dinitrate, nitroglycerin (glyceryl trinitrate) and pentaerythritol tetranitrate); anticoagulants (e.g. phenprocoumon, heparin); antibodies, (e.g. a polyclonal antibody or a monoclonal antibody, including ranibizumab, pembrolizumab); antibiotics (e.g. dapsone, chloramphenicol, neomycin, cefaclor, cefadroxil, cephalexin, cephradine erythromycin, clindamycin, lincomycin, amoxicillin, ampicillin, bacampicillin, carbenicillin, dicloxacillin, cyclacillin, picloxacillin, hetacillin, methicillin, nafcillin, oxacillin, penicillin G, penicillin V, ticarcillin, rifampin and tetracycline); anti-cancer agents (e.g. platinum compounds (spiroplatin, cisplatin and carboplatin) methotrexate, adriamycin, taxol, mitomycin, ansamitocin, bleomycin, cytosine arabinoside, arabinosyl adenine, mercaptopolylysine, vincristine, busulfan, chlorambucil, melphalan and phenylalanine mustard (PAM)), mercaptopurine, mitotane, procarbazine hydrochloride, dactinomycin (actinomycin D), daunorubicin hydrochloride, doxorubicin hydrochloride, mitomycin, plicamycin (mithramycin), paclitaxel aminoglutethimide, estramustine phosphate sodium, flutamide, leuprolide acetate, megestrol acetate, tamoxifen citrate, testolactone, trilostane, amsacrine (m-AMSA), asparaginase (L-asparaginase) Erwina asparaginase, etoposide (VP-16), interferon a-2a, interferon a-2b, teniposide (VM-26), vinblastine sulfate (VLB), vincristine sulfate, bleomycin, bleomycin sulfate, methotrexate, adriamycin, carzelesin, arabinosyl, aziridinylbenzoquinone, muramyl-tripeptide and 5-fluoruracil), anti coagulation agents (e.g. phenprocoumon and heparin); anti-fungal agents, (e.g. ketoconazole, nystatin, griseofulvin, flucytosine, miconazole, amphotericin B); anti- parasitics; anti-protozoans (e.g. chloroquine, hydroxychloroquine, metronidazole, quinine and meglumine antimonate); anti-rheumatics (e.g. penicillamine); antituberculars (e.g. para-aminosalicylic acid, isoniazid, capreomycin sulfate cycloserine, ethambutol hydrochloride ethionamide, pyrazinamide, rifampin, and streptomycin sulfate); anti-virals (e.g. acyclovir, amantadine, azidothymidine, e.g. as AZT or Zidovudine, ribavirin, amantadine, vidarabine and vidarabine monohydrate); anti-inflammatories (e.g. difunisal, ibuprofen, indomethacin, meclofenamate, mefenamic acid, naproxen, oxyphenbutazone, phenylbutazone, piroxicam, sulindac, tolmetin, aspirin and salicylates); vitamins (e.g. cyanocobalamin neinoic acid, retinoids and derivatives, e.g. retinol palmitate, a-tocopherol, naphthoquinone, cholecalciferol, folic acid, and tetrahydrofolate); blood products (e.g. parenteral iron, hemin, hematoporphyrins and their derivatives); cardiac glycosides (e.g. deslanoside, digitoxin, digoxin, digitalin and digitalis); circulatory drugs (e.g. propranolol); hormones or steroids (e.g. growth hormone, melanocyte stimulating hormone, estradiol, beclomethasone dipropionate, betamethasone, betamethasone acetate and betamethasone sodium phosphate, vetamethasone disodium phosphate, vetamethasone sodium phosphate, cortisone acetate, dexamethasone, dexamethasone acetate, dexamethasone sodium phosphate, flunsolide, hydrocortisone, hydrocortisone acetate, hydrocortisone cypionate, hydrocortisone sodium phosphate, hydrocortisone sodium succinate, methylprednisolone, methylprednisolone acetate, methylprednisolone sodium succinate, paramethasone acetate, prednisolone, prednisolone acetate, prednisolone sodium phosphate, prednisolone tebutate, prednisone, triamcinolone, triamcinolone acetonide, triamcinolone diacetate, triamcinolone hexacetonide, fludrocortisone acetate, progesterone, testosterone, and adrenocorticotropic hormone); narcotics (e.g. paregoric and opiates, e.g. codeine, heroin, methadone, morphine and opium); neuromuscular blockers (e.g. atracurium besylate, gallamine triethiodide, hexafluorenium bromide, metocurine iodide, pancuronium bromide, succinylcholine chloride, tubocurarine chloride and vecuronium bromide); peptides (e.g. angiostatin, manganese super oxide dismutase, tissue plasminogen activator, glutathione, insulin, dopamine, human chorionic gonadotropin, corticotropin release factor, cholecystokinins, bradykinins, elastins, vasopressins, pepsins, glucagon, integrins, adrenocorticotropic hormone, oxytocin, calcitonins, IgG, IgA, IgM, thrombin, streptokinase, urokinase, Protein Kinase C, interferon, colony stimulating factors, granulocyte colony stimulating factors, granulocyte-macrophage colony stimulating factors, tumour necrosis factors, nerve growth factors, platelet derived growth factors, lymphotoxin, epidermal growth factors, fibroblast growth factors, vascular endothelial cell growth factors, erythropoietin, transforming growth factors, oncostatin M, interleukins (interleukin 1, interleukin 2, interleukin 3, interleukin 4, interleukin 5, interleukin 6, interleukin 7, interleukin 8, interleukin 9, interleukin 10, interleukin 11, and interleukin 12), metalloprotein kinase ligands, and collagenases); angiotensin converting enzyme (ACE) inhibitors (captopril, enalapril, and lisinopril), and sedatives (e.g. amobarbital, amobarbital sodium, aprobarbital, butabarbital sodium, chloral hydrate, ethchlorvynol, ethinamate, flurazepam hydrochloride, glutethimide, methotrimeprazine hydrochloride, methyprylon, midazolam hydrochloride, paraldehyde, pentobarbital, pentobarbital sodium, phenobarbital sodium, secobarbital sodium, talbutal, temazepam and triazolam).
The method of targeted drug delivery of the invention may be considered a method for the treatment of a disease or condition in a human or non-human animal which is responsive to a drug, e.g. those disclosed above, in which said drug undergoes targeted drug delivery to target cells, said method comprising performing the above described method of targeted drug delivery and administering to the subject a conjugate of said drug and the binding partner of said macromolecule.
Alternatively, this aspect of the invention may be expressed as method of targeted drug delivery in the treatment of a disease or condition in a human or non-human animal which is responsive to a drug, e.g. those disclosed above, said method comprising performing the above described method of targeted drug delivery and administering to the subject a conjugate of said drug and the binding partner of said macromolecule
The invention further provides a microbubble of the invention for use in such methods and the use of a microbubble of the invention in the manufacture of a medicament for use in said methods.
In another aspect, the invention provides a microbubble of the invention, or a suspension thereof, for use in the medical (e.g. therapeutic, diagnostic or surgical) treatment of a human or non-human animal subject. The invention also relates to methods of medical treatment involving the use of a microbubble of the invention, or a suspension thereof, and also the use of a microbubble of the invention, or a suspension thereof, in the manufacture of a medicament for use in the medical treatment of a human or non-human animal subject.
The subject may be any human or non-human animal subject, but more particularly may be a human or a non-human vertebrate, e.g. a non-human mammal, bird, amphibian, fish or reptile. In a preferred embodiment, the subject is a mammalian subject. The animal may be a livestock or a domestic animal or an animal of commercial value, including laboratory animals or an animal in a zoo or game park. Representative animals therefore include dogs, cats, rabbits, mice, guinea pigs, hamsters, horses, pigs, sheep, goats and cows. Veterinary uses of the invention are thus covered. The subject may be viewed as a patient. Preferably, the subject is a human.
"Treatment" when used in relation to the treatment of a disease or medical condition in a subject in accordance with the invention is used broadly herein to include any therapeutic effect, i.e. any beneficial effect in relation to the disease or the condition. Thus, not only included is eradication or elimination of the disease or the condition, or cure of the subject, but also an improvement in the disease or the condition of the subject. Thus included for example, is an improvement in any symptom or sign of the disease or the condition, or in any clinically accepted indicator of the disease or the condition (for example a decrease in tumour size (volume, area and/or cell number), a decrease in tumour invasion, a reduction in general discomfort or pain in the surrounding tissue, or a reduction in inflammation). Treatment thus includes both curative and palliative therapy, e.g. of a pre-existing or diagnosed disease or condition, i.e. a reactionary treatment.
In other more specific embodiments the invention provides a method for the diagnosis or monitoring of a disease or condition in a human or non-human animal subject said method comprising providing an ex vivo sample of a cell of interest, preferably immune cells, preferably cells of the subject; contacting said cells with a microbubble of the invention carrying a detectable macromolecule as defined herein (e.g. a fluorescent or radioactive macromolecule) and, whilst in contact with one another, exposing the cells and the microbubble to ultrasound conditions effective to result in presentation of said macromolecule at the outer surface of the plasma membrane of one or more of the cells of interest; administering at least a portion of the cells presenting the macromolecule to the subject and detecting said macromolecule in the body of said subject and/or assessing the distribution of said macromolecule in the body of said subject. In these embodiments, the detection of said macromolecule in the body of said subject and/or assessing the distribution of said macromolecule in the body of said subject allows the skilled person to monitor or diagnose a disease or condition in the subject. Methods for the formation of microbubbles are known in the art. Such methods include the formation of a suspension of the gas in an aqueous medium, e.g. PBS, in the presence of the selected shell materials. In certain embodiments the macromolecule-lipid complex described herein is present as part of the selected shell materials. In other embodiments, the macromolecule is added to preformed microbubbles of the invention, i.e. microbubbles comprising a gas core and a shell, wherein said shell comprises the lipid-modified chelator and wherein a metal cation is immobilised by said chelator.
Techniques used to form the microbubbles include sonication, shaking, high speed mixing (mechanical agitation), coaxial electrohydrodynamic atomisation and microfluidic processing using a T-junction (see e.g. Stride & Edirisinghe, Med. Biol. Eng. Comput., 47: 883-892, 2009). Sonication is widely used and generally preferred. This technique may be carried out using an ultrasound transmitting probe. Sonication or shaking may be performed until formation of a suspension of bubbles.
These techniques are typically carried out under an atmosphere of the gas that is to be trapped as the gas core of the microbubbles, e.g. sulfur hexafluoride. In other approaches, the gas for the gas core of the microbubbles may be bubbled through the aqueous solution used to prepare the suspension in addition to, or as an alternative to, carrying out sonication and/or shaking under an atmosphere of the desired gas.
Typically, the microbubble suspension so formed is allowed to settle before a lower part of the suspension is isolated.
Other methods which may be used to form the microbubbles include vaporisation of a nanodroplet core in a nanoemulsion (see e.g. Rapoport et al, Bubble Sci. Eng. Technol. 1 : 31 -39, 2009). The core of such nanodroplets will typically be formed by an organic perfluorocompound which is encased by walls of a biodegradable amphiphilic block copolymer such as poly(ethylene oxide)-co-poly(L-lactide) or poly(ethylene oxide)-co-caprolactone. Alternatively, nanoemulsions may be prepared by extrusion through sizing membranes, for example using albumin as the shell material. The droplet-to-bubble transition may be induced by physical and/or mechanical means which include heat, ultrasound and injection through a fine- gauge needle. Such microbubbles may be formed at the point of administration to the subject (e.g. during the step of administration using a fine-gauge needle) or in vivo at the desired target cells or tissues (e.g. by subjecting the nanoemulsion to ultrasound). Accordingly, in the above methods and uses, the step of administering a microbubble of the invention to a human or non-human animal subject includes administration of a nanoemulsion of the microbubble shell materials and the gas under conditions suitable for the formation of the microbubbles of the invention.
A suspension of microbubbles, which may have been prepared according to the sonication/shaking method outlined above, or according to a method known in the art, may be filtered to obtain a suspension having a desired distribution of optimised microbubbles.
In a further aspect of the invention there is provided a suspension of the microbubbles of the invention in an aqueous phase. Such suspensions will contain a distribution of microbubbles having different particle sizes. It is preferred that the distribution is centred on the desired weight average particle size. The standard deviation of microbubble diameters in a distribution representing a suspension of the microbubbles is preferably ±3 pm, more preferably ±1 pm.
The term “aqueous” is used herein to describe a liquid composition which is comprised mainly, e.g. substantially, predominantly or essentially of water, e.g. at least 80%, 90%, 95%, 99% or 100% of the solvent in the composition is water. In other embodiments less than 20%, 10%, 5%, or 1% of the solvent in the composition is a non-polar solvent, or at least the amount of non-polar solvent is insufficient to interfere with vesicle formation and integrity. Expressed differently the “aqueous suspension” can be considered to comprise, in addition to any microbubbles of the invention or the non-assembled components thereof, less than 20%, 10%, 5%, or 1% of a non-polar organic phase, or at least an amount of non polar organic phase that is insufficient to interfere with microbubble integrity. Preferably, the aqueous phase is devoid of non-polar solvent/phase. Consistent with this the presence of other molecules dissolved or suspended in the aqueous phase is not excluded, again as long as they are present in amounts that are insufficient to interfere with microbubble integrity. In certain embodiments, the aqueous suspension of the microbubbles of the invention is a suspension in water or a saline solution, e.g. phosphate-buffered saline.
The macromolecule may be loaded onto preformed microbubbles of the invention by any convenient means. Typically this may by combining an aqueous suspension of microboubbles of the invention comprising a gas core and a shell, wherein said shell comprises a lipid-modified chelator and wherein a metal cation is immobilised by said chelator, with an aqueous solution of the macromolecule and incubating the mixture under conditions suitable for the metal ion binding motif of the macromolecule to form at least one stable coordinate bond with the immobilised metal ion. Suitable washing and/or isolation steps may follow, e.g. as described above.
For use in any of the in vivo methods herein described, the microbubbles of the invention, or the aqueous suspension thereof, will generally be provided in a pharmaceutical composition, e.g. together with at least one pharmaceutically acceptable carrier or excipient. Such compositions form a further aspect of the invention.
The pharmaceutical compositions comprising microbubbles for use according to the invention may be formulated using techniques well known in the art. The route of administration will depend on the intended use. Typically, these will be administered systemically or directly to an internal body location and may thus be provided in a form adapted for parenteral administration, e.g. by intratumoural, intradermal, subcutaneous, intraperitoneal or intravenous injection. Suitable pharmaceutical forms include suspensions and solutions which contain the microbubbles of the invention, or the aqueous suspension thereof, together with one or more inert carriers or excipients. Suitable carriers include saline, sterile water, phosphate buffered saline and mixtures thereof.
The compositions may additionally include other agents, e.g. emulsifiers, suspending agents, viscosity modifiers, dispersing agents, solubilisers, stabilisers, buffering agents, preserving agents, etc. The compositions may be sterilised by conventional sterilisation techniques.
The non-gaseous components of the microbubbles of the invention may be supplied in the form of a dry mixture, e.g. a lyophilised powder or dehydrated film, for reconstitution together with the gaseous component at the point of use, e.g. for reconstitution in water, saline or PBS.
In further aspects, the invention provides a dry mixture of a lipid-modified chelator, wherein a metal cation is immobilised by said chelator and one or more additional microbubble shell forming components. These components of the mixture may be any of the examples described herein. In more specific embodiments, the dry mixtures of the invention may further comprise a macromolecule as defined herein.
The dry mixtures of the invention may be in the form of a powder or a film. In certain embodiments, the mixture is a lyophilised mixture.
The dry mixtures of the invention are substantially, e.g. essentially, water-free (moisture-free). This may be expressed as a water content of less than 10% w/w, e.g. less than 9%, 8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5% or 1% w/w as measured by weight loss on drying or chemically by the Karl Fischer method (United States Pharmacopeia; European Pharmacopoeia).
In other aspects, the invention provides kits comprising the dry mixtures of the invention and a gas suitable for forming a microbubble, e.g. any of the examples described herein.
In one particular embodiment the kit comprises, in separate containers, (i) a dry mixture of a lipid-modified chelator, wherein a metal cation is immobilised by said chelator and one or more additional microbubble shell forming components; (ii) a macromolecule as defined herein and (iii) a gas suitable for forming a microbubble.
In another particular embodiment, the kit comprises, in separate containers, (i) a dry mixture of a lipid-modified chelator, wherein a metal cation is immobilised by said chelator and one or more additional microbubble shell forming components; and (ii) a gas suitable for forming a microbubble.
In another particular embodiment, the kit comprises, in separate containers, (i) a dry mixture of a lipid-modified chelator, wherein a metal cation is immobilised by said chelator, one or more additional microbubble shell forming components and a macromolecule as defined herein and (ii) a gas suitable for forming a microbubble.
In a still further aspect the invention provides kits comprising the components of the microbubbles of the invention, or combinations thereof, in separate containers. These components of the kit may be any of the examples described herein.
In one particular embodiment the kit comprises, in separate containers, (i) a lipid- modified chelator, wherein a metal cation is immobilised by said chelator, and (ii) a gas suitable for forming a microbubble, and optionally (iii) a macromolecule as defined herein and optionally (iv) one or more additional microbubble shell forming components.
In one particular embodiment the kit comprises, in separate containers, (i) a lipid- modified chelator, wherein a metal cation is immobilised by said chelator and (ii) a macromolecule as defined herein, and optionally (iii) a gas suitable for forming a microbubble and optionally (iv) one or more additional microbubble shell forming components.
In one particular embodiment the kit comprises, in separate containers, (i) a lipid- modified chelator, wherein a metal cation is immobilised by said chelator and one or more additional microbubble shell forming components and (ii) a macromolecule as defined herein, and optionally (iii) a gas suitable for forming a microbubble.
In one particular embodiment the kit comprises, in separate containers, (i) a lipid- modified chelator, wherein a metal cation is immobilised by said chelator and one or more additional microbubble shell forming components and (ii) a gas suitable for forming a microbubble, and optionally (iii) a macromolecule as defined herein. BRIEF DESCRIPTION OF THE FIGURES
Figure 1 shows (A) DSPC-PEG40S-DGS-NTA(Ni) MB size distribution histogram (N = 19413) straight after production, giving a mean diameter of 2.34 ± 1.37 pm and a concentration of 1.24x109 bubbles per ml (white bars include all bubbles obtained from the images that are excluded because of them being too small (< 0.5 pm) or too big (> 10 pm)); and (B) a representative brightfield microscopy image, which contributed to the statistics in panel (A).
Figure 2 shows (A) DSPC-PEG40S-DGS-NTA(Ni) MB size distribution histogram (N = 12145) after two wash cycles, giving a mean diameter of 1.62 ± 1.36 pm and a concentration of 2.56x108 bubbles per ml (white bars include all bubbles obtained from the images excluded because of them being too small (< 0.5 pm) or too big (> 10 pm)); and (B) a representative brightfield microscopy image, which contributed to the statistics in panel (A).
Figure 3 shows representative confocal fluorescence microscopy images of DSPC- PEG40S-DGS-NTA(Ni) MB in (A) DPBS and (B) DPBS supplemented with 10% FBS.
Figure 4 shows confocal fluorescence microscopy images of (A) control DSPC- PEG40S microbubbles and (B) functionalised DSPC-PEG40S-DGS-NTA(Ni) microbubbles which have both been exposed to his-GFP. As can clearly be observed, there is no non-specific binding of his-GFP (green) to microbubbles lacking nickelated lipid (black) in image (A), whereas his-GFP binds specifically to microbubbles containing nickelated lipis in image (B).
Figure 5 shows confocal fluorescence microscopy images of A549s cells incubated with CellMask Deep Red and subsequently with DSPC-PEG40S-DGS-NTA(Ni)-His- GFP microbubbles. (A) shows the control, where cells are not exposed to ultrasound, whereas (B) shows the sample exposed to ultrasound as described in the Examples. Confocal fluorescence images were obtained from the Zeiss 780 microscope at a magnification of 63x. Figure 6 shows confocal microscopy images of DSPC-PEG40S-16:0 DGS-NTA(Ni)- His-AF488 bubbles (green) at -4.6x108/ml with a mean diameter at t = 0 of -2.6 pm. Confocal fluorescence images were obtained from the Zeiss 780 microscope at a magnification of 40x.
Figure 7 shows confocal microscopy images of A549s incubated with CellMask Deep Red (magenta) and subsequently with -9x107 DSPC-PEG40S-18:1 DGS- NTA(Ni)-His-AF488 bubbles (green). (A) shows the control, where cells are not exposed to ultrasound (US), whereas (B), which shows the sample exposed to 1 MHz CW at -0.2 MPa peak to peak. Confocal fluorescence images were obtained from the Zeiss 780 microscope at a magnification of 63x.
EXAMPLES
Example 1
Microbubble preparation
A batch sonication protocol as previously reported by De Cock et al. (Ultrasound and microbubble mediated drug delivery: acoustic pressure as determinant for uptake via membrane pores or endocytosis, 2015, Journal of Controlled Release, 197, 20-28) has been employed to prepare microbubbles (MBs). Briefly, the protocol is as follows: 1,2-distearoyl-sn-glycero-3-phosphocholine (18:0 PC, Avanti Polar Lipids), commonly referred to as DSPC, polyoxyethylene (40) stearate (PEG40S, Sigma Aldrich, UK), and 18:1 nickel-nitrilotriacetic acid (Ni-NTA)- functionalised lipids (DGS-NTA(Ni), Avanti Polar Lipids) were dissolved in chloroform (Sigma Aldrich, UK) and mixed in a glass vial at a molar ratio of 9 : 0.5 : 0.2, containing 20 mg in total of lipid constituents. The chloroform solution was covered with perforated parafilm (Bemis Company, Inc., Neenah, Wl, USA) and the mixture was subsequently heated overnight on a hotplate set to 50 °C, facilitating evaporation of chloroform and the formation of a homogenous lipid film.
The obtained dry lipid film was suspended in 5 ml of Dulbecco’s phosphate-buffered saline (DPBS, pH 7.4, Life Technologies, Paisley, UK) and stirred at 100 °C on a magnetic stirrer hotplate for a minimum of 30 minutes. Phosphate-buffered saline consists of 137 mM NaCI, 2.7 mM KCI, 10 mM Na2HP04, and 1.8 mM KH2P04 in H20. Lipids were then homogeneously dispersed for 150 s using a sonicator (Microson XL 2000, probe diameter 3 mm, 20 W, 22.5 kHz, QSonica, NewTown, Ct, USA) with the tip completely immersed in the lipid solution (power setting 3). MBs were subsequently formed by placing the sonicator tip at the air-water interface under constant sulphur hexafluoride flow (The BOC Group pic, UK) and sonicated for 30 s to form a cloudy suspension of microbubbles (power setting 14). Immediately after production, the vial containing the DSPC-PEG40S-DGS-NTA(Ni) MB suspension was capped and placed in ice for approximately 5 minutes.
Microbubbles were washed twice to eliminate the excess of free (Ni-NTA)- functionalised lipid. Microbubbles were washed using the centrifugation method without size isolation, as reported by Feshitan et al. (Microbubble size isolation by differential centrifugation, 2009, Journal of Colloid and Interface Science, 329(2), 316-324). In summary, MBs were loaded into a 5 ml syringe and centrifuged for 5 minutes at 300g, corresponding to 1000 rpm. Following centrifugation, the subnatentwas discarded and MBs were either resuspended in 1 ml of fresh DPBS or in 1 ml of a solution containing a His-tagged protein, e.g. His-GFP (25 pg/ml, Stratech, UK), in DPBS.
Microbubbles were incubated with His-GFP and a third washing step was performed. After 5 minutes of incubation, centrifugation was performed, again for 5 minutes at 300g, to eliminate the excess unbound His-GFP and improve signal-to- noise ratio for fluorescence microscopy experiments.
Microbubble characterisation: concentration, specificity and stability
For quantification of the MB size and concentration, 10 pL of the produced microbubble suspension was transferred onto a Neubauer improved cell counting chamber (Hausser Scientific Company) under a 24 mm c 24 mm glass coverslip (VWR International). Roughly 40 images of MBs were acquired at 40x magnification using a Leica DM500 microscope (Leica Microsystems GmbH, Germany) coupled with a CCD camera (Leica Microsystems GmbH, Germany). MB sizing and counting was performed using a purpose-written code in MATLAB® (The MathWorks Inc.). Figure 1A shows an example of a microbubble histogram prior to washing (N = 19413), showing microbubbles with a mean diameter of 2.34 ± 1.37 pm and a concentration of 1.24x109 bubbles per ml, and Figure 1B shows an exemplary field of view, clearly visualising the size distribution of the microbubbles. After performing two washing steps and incubation with His-GFP, the size distribution changed accordingly, as seen in Figure 2. A.
Corresponding confocal fluorescence images to assess the success of microbubble shell labelling with His-GFP (25 pg/ml) were obtained using a confocal microscope (LSM 780, Carl Zeiss AG, Germany), with magnifications varying from 20x to 63x. For this purpose, 2 pi of the microbubble sample was placed on a plasma-cleaned 75 mm c 25 mm c 0.17 mm glass coverslip (Logitech Ltd., Scotland). The results are shown in Figure 3, both simply in PBS (Figure 3A) as well as in PBS supplemented with 10% Fetal Bovine Serum (FBS) (Figure 3.B) to mimic the conditions of the blood.
Specificity of His-GFP for microbubbles containing 18:1 DGS-NTA(Ni) was investigated using confocal fluorescence microscopy, where bubbles containing nickelated lipid were compared to DSPC-PEG40S bubbles, which were made following the same protocol as described above with a molar ratio of 9 : 0.5, respectively. The results are shown in Figure 4. As can clearly be observed, His- GFP (green) binds to bubbles containing nickelated lipid (Figure 4B), whereas no binding can be observed for the control bubbles without nickelated lipid in Figure 4A.
Tagging eukaryotic cell plasma membrane with DSPC-PEG40S-DGS-NTA(Ni)-His- GFP microbubbles
The SAT3, as designed by Dr Michael Gray, comprises of a water tank system, consisting of either a 0.5 MHz or 1.7 MHz transducer, a purpose-built sample holder, acoustic absorber, and a passive cavitation detector (PCD). The acoustic absorber is placed opposite of the transducer and above the sample holder to attenuate any reflections. The sample holder is designed to hold a 6.5 mm transwell (Corning®, Sigma Aldrich, UK), upright, in which various types of cells can be cultured in cell culture medium and, subsequently, exposed to ultrasound in the presence and absence of microbubbles. A plug is used to shield the cells and small volumes of culture medium, -100 pi, from the surrounding water.
Experiments were performed exposing A549 cells, stained using CellMask Deep Red to DSPC-PEG40S-DGS-NTA(Ni)-His-GFP microbubbles and, subsequently, exposing the cells and microbubbles to ultrasound (continuous 60 second exposure, 0.5 MHz, peak rarefaction pressure of -0.18 MPa). Controls were treated in a similar fashion, but without actual ultrasound exposure. .
2x105 A549s cells were seeded approximately 24 hours prior to imaging in 6.5 mm diameter transwells in 24-well plates (Corning®, Sigma Aldrich, UK) in 100 mI culture medium. Sub-confluent cells were chosen to enable proper membrane segmentation. Cells were washed twice with PBS and incubated with 2 pg/ml CellMask Deep Red for 10 minutes. Subsequently, cells were washed twice before incubation with 100 mI of DSPC-PEG40S-DGS-NTA(Ni)-His-GFP microbubbles and either exposed to ultrasound or simply only placed in the 37 °C bath
The results are shown in Figure 5. As can clearly be observed, following ultrasound exposure, transfer of GFP and/or fusion has taken place from the microbubbles to the cellular membrane (Figure 5B), as indicated by colocalisation of both CellMask and His-GFP, whereas this is absent if no exposure to US has taken place (Figure 5A). These results indicate a greatly promising avenue of using microbubbles in immunomodulation.
Example 2
Microbubble preparation
A batch sonication protocol as previously reported by De Cock et al. (supra) has been employed to prepare microbubbles (MBs). The protocol is as follows: 1,2- distearoyl-sn-glycero-3-phosphocholine (18:0 PC, Avanti Polar Lipids), commonly referred to as DSPC, polyoxyethylene (40) stearate (PEG40S, Sigma Aldrich, UK), and either 18:1 or 16:0 nickel-nitrilotriacetic acid (Ni-NTA)-functionalised lipids (DGS-NTA(Ni), Avanti Polar Lipids) were dissolved in chloroform (Sigma Aldrich, UK) and mixed in a glass vial at a molar ratio of 9 : 0.5 : 0.2, containing 20 mg in total of lipid constituents. The chloroform solution was covered with perforated parafilm (Bemis Company, Inc., Neenah, Wl, USA) and the mixture was subsequently heated overnight on a hotplate set to 50 °C, facilitating evaporation of chloroform and the formation of a homogenous lipid film.
The obtained dry lipid film was suspended in 5 ml of Dulbecco’s phosphate-buffered saline (DPBS, pH 7.4, Life Technologies, Paisley, UK) and stirred at 100 °C on a magnetic stirrer hotplate for a minimum of 45 minutes. Phosphate-buffered saline consists of 137 mM NaCI, 2.7 mM KCI, 10 mM Na2HP04, and 1.8 mM KH2P04 in H20. Lipids were then homogeneously dispersed for 150 s using a sonicator (Microson XL 2000, probe diameter 3 mm, 20 W, 22.5 kHz, QSonica, NewTown, Ct, USA) with the tip completely immersed in the lipid solution (power setting 3). MBs were subsequently formed by placing the sonicator tip at the air-water interface under constant sulphur hexafluoride flow (The BOC Group pic, UK) and sonicated for 30 s to form a cloudy suspension of microbubbles (power setting 14). Immediately after production, the vial containing the DSPC-PEG40S-DGS-NTA(Ni) MB suspension was capped and placed in ice for approximately 5 minutes.
Microbubbles were washed once to eliminate the excess of free (Ni-NTA)- functionalised lipid. Microbubbles were washed using the centrifugation method without size isolation, as reported by Feshitan et al. (supra). In summary, MBs were loaded into a 5 ml syringe and centrifuged for 5 minutes at 300g, corresponding to 1000 rpm. Following centrifugation, the subnatent was discarded and MBs were resuspended in 5 ml of a freshly made up His-tagged AF488 DPBS solution containing 4 ug of His-AF488 ([AF488]-HHHHHH-acid, Cambridge Research Biochemicals). After 5 minutes of incubation, centrifugation was performed twice more, again for 5 minutes at 300g, to eliminate the excess unbound His-AF488 and improve signal-to-noise ratio for fluorescence microscopy experiments, after which MBs were resuspended in 5 ml of fresh DPBS.
The resultant microbubbles as prepared by this protocol are shown in Figure 6. As can clearly be observed, His- AF488 (green) binds to bubbles containing nickelated lipid. Tagging eukaryotic cell plasma membrane with DSPC-PEG40S-18:1 DGS- NTA(Ni)-His-AF488 microbubbles
Experiments corresponding to the results in Figure 7 were conducted in the water tank system (~1 L) consisting of a 1 MHz unfocused ultrasound transducer (Imasonic 8233 A101, 40 mm diameter, 120 mm radius of curvature), a purpose- built sample holder, acoustic absorber, and passive cavitation detector (7.5 MHz centre frequency, V320 Panametrics, Olympus) as designed by Dr. Michael Gray. The acoustic absorber was placed opposite the transducer to attenuate reflections. The sample holder was designed to hold cell culture dishes (m-Dishes, Ibidi) with plastic substrates much thinner (-100 pm) than the acoustic wavelength in water enclosed with a custom polydimethylsiloxane (PDMS) lid (-1.2 mm thick). Characterization of the acoustical properties of the PDMS lid has been described in detail by Carugo et al. (Biologically and acoustically compatible chamber for studying ultrasound-mediated delivery of therapeutic compounds (2015) Ultrasound in medicine & biology, 41(7), 1927-1937). The ultrasound transducer was driven by a 1 MHz sinusoidal signal from a function generator passed through a 1.9 MHz low- pass filter and amplified 55 dB by a radiofrequency amplifier.
A549s were grown in a 35 mm-diameter low Ibidi m-Dish at 20% confluence 17 hrs prior to exposure. A549s were incubated with Cell Mask Deep Red at 1 pg/ml for 8 minutes at 37 °C. The Ibidi m-Dish was subsequently enclosed by a thin PDMS lid designed for ultrasound exposure as described by Carugo et al. (2015). A 12 ml solution of washed MBs were prepared at room temperature and then injected through the acoustically-compatible PDMS lid, ensuring that there were no bubbles trapped in the p-Dish. The inlet and outlet of the PDMS lid were subsequently plugged using 1.4 mm diameter plastic rods. The m-Dish was then inverted to increase microbubble-cell proximity, transferred into the water tank setup filled with filtered, distilled, deionised, and degassed water at room temperature, and exposed to 30 s of ultrasound at 1MHz, 0.25 MPa peak to peak, continuous wave, during which acoustic emissions monitoring was performed.
Upon removal from the water tank, the acoustically-compatible lid was removed from the m-Dish and the cells were washed two times with PBS. Transfer/fusion of DSPC-PEG40S-DGS-NTA(Ni)-His-AF488 MBs with the A549’s target cellular membranes was investigated using confocal fluorescence microscopy (LSM 780, Carl Zeiss AG, Germany).
The results are shown in Figure 7. Following ultrasound exposure, transfer of AF488 and/or fusion has taken place from the microbubbles to the cell membrane
(Figure 7B), as evidenced by the colocalisation of CellMask and His-AF488. In contrast, in the absence of ultrasound, no such colocalisation takes place (Figure 7A).

Claims

Claims
1. A method for presenting a macromolecule of interest at the outer surface of the plasma membrane of a eukaryotic cell, said method comprising
(i) contacting a eukaryotic cell with a microbubble comprising a gas core and a shell, wherein said shell comprises a lipid-modified chelator and wherein a metal cation is immobilised by said chelator and said macromolecule is bound to the metal cation via one or more coordinate bonds, and
(ii) whilst in contact with one another, exposing the cell and the microbubble to ultrasound conditions effective to result in presentation of said macromolecule at the outer surface of the plasma membrane of the eukaryotic cell.
2. The method of claim 1 wherein said eukaryotic cell is in vitro or ex vivo.
3. A microbubble comprising a gas core and a shell, wherein said shell comprises a lipid-modified chelator and wherein a metal cation is immobilised by said chelator and a macromolecule of interest is bound to the metal cation via one or more coordinate bonds for use in a method for presenting said macromolecule at the outer surface of the plasma membrane of a eukaryotic cell in or on a subject, said method comprising
(i) contacting a eukaryotic cell whilst in or on a subject with the microbubble, and
(ii) whilst in contact with one another, exposing the cell and the microbubble to ultrasound conditions effective to result in presentation of said macromolecule at the outer surface of the plasma membrane of the eukaryotic cell.
4. The method or the microbubble for use of any one of claims 1 to 3, wherein said macromolecule is therapeutically or diagnostically effective.
5. The method or the microbubble for use of any one of claims 1 to 5, wherein the ultrasound conditions include (i) a frequency of about 0.25 to about 1.5 MHz, e.g. about 0.4 to about 0.6 MHz, or about 0.5 MHz, or about 0.7 to about 1.2 MHz, or about 1 MHz;
(ii) a peak rarefaction pressure of about 0.1 to about 0.3 MPa, e.g. about 0.15 to about 0.25 MPa, or about 0.18 MPa or about 0.22 to about 0.28 MPa, or about 0.25 MPa;
(iii) a duration of exposure of about 10 to about 120 seconds, e.g. about 30 to about 90 seconds, or about 20 to 60 seconds; and/or
(iv) a duty cycle of about 70 to about 100%.
6. The method or the microbubble for use of any one of claims 1 to 5, wherein the macromolecule is, or comprises
(i) a tumour antigen;
(ii) an extracellular domain, or a portion thereof, of a cluster of differentiation (CD) protein, an interleukin receptor protein, a MUC protein, a major histocompatibility complex (MHC), a T cell receptor or a B cell receptor; or
(iii) a portion, or all, of a caspase, carcinoembryonic antigen (CEA), alphafetoprotein (AFP), tyrosinase, epithelial tumour antigen (ETA), melanoma-associated antigen (MAGE), an antibody, or Protein A/G.
7. The method or the microbubble for use of any one of claims 1 to 6, wherein said macromolecule promotes elimination of cells displaying said macromolecule on their surface by the immune system of a human or non-human animal subject, and said method is a method of immunotherapy of a human or non-human animal subject, said method comprising presenting said macromolecule at the outer surface of the plasma membrane of a target cell on or in the subject by contacting said target cell whilst on or in the subject with the microbubble and, whilst in contact with one another, exposing the cell and the microbubble to ultrasound conditions effective to result in presentation of said macromolecule at the outer surface of the plasma membrane of the cell.
8. The method or the microbubble for use of claim 7, wherein the macromolecule is, or comprises, full length or a functional portion, e.g. an extracellular domain of portion thereof, of CD58, CD106, CD54, an MHC, a MUC protein, a caspase, carcinoembryonic antigen, alphafetoprotein, tyrosinase, an epithelial tumour antigen, or a melanoma-associated antigen (MAGE).
9. The method or the microbubble for use of any one of claims 1 to 6, wherein said macromolecule functions to target the cell-eliminating activity of an immune cell towards a target cell type in or on a human or non-human animal subject or to modulate the cell-eliminating activity of an immune cell, and said method is a method of immunotherapy of a human or non-human animal subject, said method comprising
(i) providing an ex vivo sample of said immune cells, preferably immune cells of the subject; contacting said immune cells with the microbubble and,
(ii) whilst in contact with one another, exposing the cells and the microbubble to ultrasound conditions effective to result in presentation of said macromolecule at the outer surface of the plasma membrane of one or more of the immune cells; and
(iii) administering at least a portion of the immune cells presenting the macromolecule to the subject.
10. The method or the microbubble for use of claim 9, wherein the immune cells are cytotoxic and/or cytophagic immune cells, B cells or dendritic cells, preferably wherein the cytotoxic and/or cytophagic immune cells are Tc cells, natural killer cells, monocytes or macrophages.
11. The method or the microbubble for use of claim 9 or claim 10, wherein the macromolecule is, or comprises, full length or a functional portion, e.g. an extracellular domain of portion thereof, of CD2, CD4, CD19, IL-4 receptor, IL-2 receptor, IL-12 receptor, an antibody directed to CD106, CD58 or CD54, Protein A/G, a T cell receptor or a B cell receptor.
12. The method or the microbubble for use of any one of claims 7 to 11 , wherein said immunotherapy is to treat a hyperproliferative or neoplastic disease or condition, preferably selected from colorectal cancer, prostate cancer, kidney cancer, pancreatic cancer, testicular cancer, skin cancer, breast cancer, ovarian cancer, vulvar cancer, cervical cancer, endometrial cancer, bladder cancer, stomach cancer, intestinal cancer, liver cancer, lung cancer, oesophageal cancer, oral cancer, throat cancer, brain cancer, adrenal cancer, thyroid cancer, uterine cancer, haematological cancer, including metastatic forms thereof.
13. The method or the microbubble for use of any one of claims 1 to 6, wherein said macromolecule functions to modulate the immunotolerance activity of an immune cell, and said method is a method of immunotherapy of a human or non human animal subject, said method comprising
(i) providing an ex vivo sample of said immune cells, preferably immune cells of the subject;
(ii) contacting said immune cells with the microbubble and,
(iii) whilst in contact with one another, exposing the cells and the microbubble to ultrasound conditions effective to result in presentation of said macromolecule at the outer surface of the plasma membrane of one or more of the immune cells;
(iv) and administering at least a portion of the immune cells presenting the macromolecule to the subject.
14. The method or the microbubble for use of claim 13, wherein the immune cells are immune cells involved in immunotolerance, preferably Th cells, Treg cells, dendritic cells, mast cells or B cells.
15. The method or the microbubble for use of claim 13 or claim 14, wherein the macromolecule is, or comprises, a functional portion, e.g. an extracellular domain of portion thereof, of the IL-2 receptor, IL-4 receptor, IL-12 receptor, CD19 or CD4
16. The method or the microbubble for use of any one of claims 13 to 15, wherein said immunotherapy is to treat rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, multiple sclerosis, type 1 diabetes mellitus, Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, psoriasis, Graves' disease, Hashimoto's thyroiditis, myasthenia gravis, autoimmune vasculitis, autoimmune hepatitis, alopecia areata, celiac disease, eczema, atopic dermatitis, urticara, asthma, allergic conjunctivitis, allergic rhinitis, food allergies or other allergies.
17. The method or the microbubble for use of any one of claims 1 to 6, wherein said method is a method of immunotherapy of a human or non-human animal subject, said method comprising
(a) providing an ex vivo sample of immune cells, preferably immune cells of the subject; contacting said immune cells with a first microbubble as defined in claims 1 to 5 carrying a first macromolecule, wherein said first macromolecule functions to target the cell-eliminating activity of an immune cell towards target cells presenting a second macromolecule and, whilst in contact with one another, exposing the cell and the first microbubble to ultrasound conditions effective to result in presentation of said first macromolecule at the outer surface of the plasma membrane of one or more of the immune cells;
(b) administering at least a portion of the immune cells presenting the first macromolecule to the subject;
(c) contacting a target cell on or in the subject with a second microbubble as defined in claims 1 to 5 carrying the second macromolecule and, whilst in contact with one another, exposing the target cell and the second microbubble to ultrasound conditions effective to result in presentation of said second macromolecule at the outer surface of the plasma membrane of the target cell, wherein step (c) may be performed before, after or simultaneously with step
(a) and/or step (b).
18. The method or the microbubble for use of any one of claims 7 to 17, wherein wherein the ultrasound conditions include
(i) a frequency of about 0.25 to about 1.5 MHz, e.g. about 0.4 to about 0.6 MHz, or about 0.5 MHz, or about 0.7 to about 1.2 MHz, or about 1 MHz;
(ii) a peak rarefaction pressure of about 0.1 to about 0.3 MPa, e.g. about 0.15 to about 0.25 MPa, or about 0.18 MPa or about 0.22 to about 0.28 MPa, or about 0.25 MPa; (iii) a duration of exposure of about 10 to about 120 seconds, e.g. about 30 to about 90 seconds, or about 20 to 60 seconds; and/or
(iv) a duty cycle of about 70 to about 100%.
19. A microbubble as defined in claims 7 to 16.
20. The method, or the microbubble for use, or the microbubble of any one of claims 1 to 19, wherein wherein the chelator is nitrilotriacetic acid (NTA), iminodiacetic acid (IDA), diethylenetriaminepentaacetic acid (DTPA) 1,4,7,10- tetraazacyclododecane; 1,4,7,10-tetraacetic acid (DOTA); 1,4,8,11- tetraazacyclotetradecane (cyclam); 1,4,7,10-tetraazacyclododecane (cyclen); 1,4- ethano-1,4,8,11- tetraazacyclotetradecane (et-cyclam); 1,4,7,11- tetraazacyclotetradecane (isocyclam); 1,4,7,10- tetraazacyclotridecane ([13]aneN4); 1,4,7,10-tetraazacyclododecane-1,7-diacetic acid (D02A); 1,4,7,10- tetraazacyclododecane-1,4,7-triacetic acid (D03A); 1,4,7,10- tetraazacyclododecane-1,7-di(methanephosphonic acid) (D02P); 1,4,7,10- tetraazacyclododecane-1,4,7-tri(methanephosphonic acid) (D03P); 1,4,7,10- tetraazacyclododecane-1,4,7,10-tetra(methanephosphonic acid) (DOTP); ethylenediaminetetraacetic acid (EDTA); 4-(1 ,4,8,11-tetraazacyclotetradec-1-yl) methylbenzoic acid (CPTA); cyclohexanediaminetetraacetic acid (CDTA); ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA); hydroxyethyl ethylenediamine triacetic acid (HEDTA); triethylene tetraamine hexaacetic acid (TTHA); 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), carboxymethylated-aspartic acid (CM-Asp), tris(2-aminoethyl)amine (TREN), ortho- phosphoserine, or N,N,N’-tris(carboxymethyl)ethylenediamine.
21. The method, or the microbubble for use, or the microbubble of claim 20, wherein the chelator is nitrilotriacetic acid (NTA), iminodiacetic acid (IDA), or diethylenetriaminepentaacetic acid (DTPA).
22. The method, or the microbubble for use, or the microbubble of any one of claims 1 to 21, wherein the lipid of the lipid-modified chelator is a cationic lipid, zwitterionic lipid, neutral lipid, or and anionic lipid.
23. The method, or the microbubble for use, or the microbubble of any one of claims 1 to 22, wherein the lipid of the lipid-modified chelator is a fatty acid, a monoacylglycerol, a diacylgylcerol, a triacylglycerol, a sterol, a glycolipid, or a phospholipid, preferably a glycerophospholipid or a sphingolipid.
24. The method, or the microbubble for use, or the microbubble of any one of claims 1 to 23, wherein the lipid of the lipid-modified chelator is a monoacylglycerol, diacylgylcerol, or triacylglycerol lipid in which one or more of the acyl groups are unsaturated.
25. The method, or the microbubble for use, or the microbubble of any one of claims 1 to 21, wherein the lipid of the lipid-modified chelator is selected from lecithin, lysolecithin, phosphatidylethanolamine, lysophosphatidylethanolamine, dioleoylphosphatidylethanolamine (DOPE), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidy 1- ethanolamine (DSPE), palmitoyloleoylphosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG),1,2- dioleoyl-sn-glycerol (DG), 1,2-dioleoyl-sn-glycero-3-succinate (DGS); 1,2-distearoyl- sn-glycerol (18:0 DG); dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleyolphosphatidylglycerol (POPG), 16-0-monomethyl PE, 16-0- dimethyl PE, 18-1- trans PE, palmitoyloleoyl-phosphatidylethanolamine (POPE), 1-stearoyl- 2-oleoylphosphatidyethanolamine (SOPE), phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetylphosphate, and cholesterol.
26. The method, or the microbubble for use, or the microbubble of claim 25, wherein the lipid of the lipid-modified chelator is 1,2-dioleoyl-sn-glycerol or 1,2- dioleoyl-sn-glycero-3-succinate or 1,2-distearoyl-sn-glycerol (18:0 DG).
27. The method, or the microbubble for use, or the microbubble of any one of claims 1 to 26, wherein the chelator is linked to the lipid via a direct covalent bond or a covalent molecular linker.
28. The method, or the microbubble for use, or the microbubble of claim 27, wherein the covalent molecular linker is an acetyl, glycol, succinyl, aconityl (cis or trans), glutaryl, methylsuccinyl, trimellityl cysteamine, penicillamine, N-(2- mercaptopropionyl)glycine, 2-mercaptopropionic acid, homocysteine, 3- mercaptopropionic acid or deamino-penicillamine group.
29. The method, or the microbubble for use, or the microbubble of any one of claims 1 to 28, wherein the lipid modified chelator is DGS-NTA.
30. The method, or the microbubble for use, or the microbubble of any one of claims 1 to 29, wherein the metal cation is Zn2+, Cu2+, Cd2+, Hg2+, Co2+, Ni2+, Fe2+, Fe3+, Pd3+, Ga3+, or Al3+, preferably Ni2+ and Zn2+.
31. The method, or the microbubble for use, or the microbubble of any one of claims 1 to 30, wherein the shell comprises a microbubble shell forming phospholipid or glycolipid, preferably selected from phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidic acid, phosphatidylinositol, dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dimyristoyiphosphatidylcholine (DMPC), dioleylphosphatidylcholine (DOPE), dibehenoylphosphatidylcholine (dibehenoyl-sn- glycero-3-phosphocholine; DBPC), dimyristoylphosphatidylethanolamine, dipalmitolphosphatidylethanolamine, distearoylphosphatidylethanolamine, cardiolipin, sphingomyelin, lecithin.
32. The method, or the microbubble for use, or the microbubble of any one of claims 1 to 31 , wherein the shell comprises a microbubble shell forming polyethylene glycol derivative of a lipid, preferably polyethylene glycol (40) stearate, N-[carbonyl-methoxy polyethylene glycol-2000]-1 ,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine, N-[carbonyl-methoxy polyethylene glycol-5000]-1 ,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine, N-[carbonyl-methoxy polyethylene glycol-750]-1 ,2-distearoyl-sn glycero-3-phosphoethanolamine, N-[carbonyl-methoxy polyethylene glycol-2000]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (MPEG 2000-distearoyl phosphatidylethanolamine), N-[carbonyl-methoxy polyethylene glycol-5000]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, DSPE-PEG-2000 or DSPE-PEG2000-TATE.
33. The method, or the microbubble for use, or the microbubble of any one of claims 1 to 32, wherein the microbubble shell comprises DSPC, polyethylene glycol (40) stearate (PEG40S), and DGS-NTA, preferably wherein at least a portion of said DGS-NTA of said microbubble has an immobilised Ni2+ or Zn2+ ion.
34. The method, or the microbubble for use, or the microbubble of any one of claims 1 to 33, wherein said gas is sulphur hexafluoride, a perfluorobutane, or a perfluoropentane.
35. The method, or the microbubble for use, or the microbubble of any one of claims 1 to 34, wherein the macromolecule is a protein, a carbohydrate, a nucleic acid or a combination or complex thereof.
36. The method, or the microbubble for use, or the microbubble of any one of claims 1 to 35, wherein the macromolecule comprises a metal cation binding motif comprising a contiguous sequence of 3-10 amino acids selected from histidine, cysteine, tryptophan or an amino acid analogue comprising an imidazole side chain or a combination thereof.
37. A pharmaceutical composition comprising a microbubble as defined in any one of claims 19 to 36.
38. A dry mixture of a lipid-modified chelator, wherein a metal cation is immobilised by said chelator, one or more additional microbubble shell forming components, and a macromolecule as defined in claims 7 to 16, preferably wherein the mixture is in the form of a film or a powder.
36. A kit comprising a dry mixture as claimed in claim 38 and a gas suitable for forming a microbubble.
37. A kit comprising:
(a) in separate containers (i) a lipid-modified chelator, wherein a metal cation is immobilised by said chelator, and one or more additional microbubble shell forming components, (ii) a macromolecule as defined in any one of claims 7 to 16 and optionally (iii) a gas suitable for forming a microbubble; or
(b) in separate containers, (i) a lipid-modified chelator, wherein a metal cation is immobilised by said chelator, and (ii) a macromolecule as defined in any one of claims 7-19, and optionally (iii) a gas suitable for forming a microbubble and optionally (iv) one or more additional microbubble shell forming components; or
(c) in separate containers, (i) a lipid-modified chelator, wherein a metal cation is immobilised by said chelator and a macromolecule as defined in any one of claims 7-19, (ii) a gas suitable for forming a microbubble and optionally (iii) one or more additional microbubble shell forming components; or
(d) in separate containers, (i) a lipid-modified chelator, wherein a metal cation is immobilised by said chelator, (ii) a macromolecule as defined in any one of claims 7-19, (iii) one or more additional microbubble shell forming components; and optionally (iv) a gas suitable for forming a microbubble or
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