WO2011104497A1 - Method for the preparation of a novel nanoparticle conjugate - Google Patents

Method for the preparation of a novel nanoparticle conjugate Download PDF

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Publication number
WO2011104497A1
WO2011104497A1 PCT/GB2011/000223 GB2011000223W WO2011104497A1 WO 2011104497 A1 WO2011104497 A1 WO 2011104497A1 GB 2011000223 W GB2011000223 W GB 2011000223W WO 2011104497 A1 WO2011104497 A1 WO 2011104497A1
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nanoparticle
antigen
conjugate
linker molecule
protein
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PCT/GB2011/000223
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French (fr)
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Richard William Titball
Andrew Mark Shaw
Anthony Edward Gregory
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University Of Exeter
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    • 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
    • A61K39/02—Bacterial antigens
    • A61K39/025—Enterobacteriales, e.g. Enterobacter
    • 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
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
    • 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
    • A61K47/6923—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 an inorganic particle, e.g. ceramic particles, silica particles, ferrite or synsorb
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • 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
    • A61K2039/62—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier
    • A61K2039/627—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier characterised by the linker

Definitions

  • the present invention relates to novel nanoparticle conjugates and their use in vaccine delivery, and to a method for the preparation of said nanoparticle conjugates.
  • Gold nanoparticles are sub-micrometre-sized particles of gold in fluid (normally water) that have unique molecular-recognition properties resulting in the particles being the subject of a large amount of research for a wide variety of applications.
  • fluid normally water
  • Gold nanoparticles have been investigated as carriers for drugs ⁇ Paclitaxel-Functionalized Gold Nanoparticles Jacob D. Gibson, Bishnu P. Khanal, and Eugene R. Zubarev. J. Am. Chem. Soc. 2007, 129, 1 1653-1 1661).
  • Vaccination (or immunisation) is the subject of vast amounts of research to enable the body to acquire immunity to a particular pathogen. Vaccines can prevent or reduce the effects of infection by many pathogens. For successful vaccination, it is necessary to provide a suitable antigen, dead microorganism or a microorganism with low virulence that will stimulate an immune response in a host cell, together with an adjuvant to potentiate that response. Vaccines have generally been administered as bulk proteins but problems can arise if the solution is not sufficiently pure, for example if lipopolysaccharides remain in the solution. It is also difficult to provide vaccines with multiple functionalities as the processes required to achieve such functionalities often leads to deterioration or contamination of the vaccine.
  • polysaccarides Lee, C.-J., Banks, S.D. & Li, J.P. 1991 Virulence, Immunity, and Vaccine Related to Streptococcus pneumoniae. Critical Reviews in Microbiology. 18, 89- 114).
  • a glycoconjugate vaccine By linking the polysaccharide to a protein a T-cell dependent response is generated but conventional methods for linking polysaccharide and protein are inefficient.
  • the bacterium Yersinia pestis belongs to the family Enterobacteriaceae and can infect both humans and animals. In humans, infection takes on three forms: pnuemonic, septicemic and bubonic plaques and the bacterium is considered to be a potential biological warfare agent. It is desirable to provide vaccines based on genetically engineered Fl and V antigens but it is necessary to obtain successful delivery of the antigen into the host cell to obtain long-term protection against the strain.
  • a first aspect of the present invention provides a novel nanoparticle conjugate comprising a nanoparticle linked to at least one antigen via a linker molecule.
  • linker molecule may be used to tether the antigen to the nanoparticle using conventional coupling techniques.
  • the linker molecule may comprise a thiol- carboxylic acid linker, such as a mercaptocarboxylic acid, for example 16- mercaptohexadecanoic acid (MHDA).
  • MHDA 16- mercaptohexadecanoic acid
  • the antigen preferably comprises a protein or sub-unit thereof having the desired antigen linked to the nanoparticle.
  • the antigen is the Fl protein of Yersinia pestis, having the amino acid sequence:
  • the nanoparticle comprises a gold nanoparticle but other nanoparticles, such as silver nanoparticles, may be used to provide the conjugate of the first aspect of the present invention. It is to be appreciated that any size of nanoparticle may be used to provide the conjugate of the first aspect of the present invention, although the particle should be dimensioned to enable access into a host cell, preferably being less than 200nm.
  • the nanoparticle conjugate according to the first aspect of the present invention may include additional functionalised groups, such as multiple proteins or sub-units thereof; polysaccharides, DNA or other ligands.
  • a delivery system for the delivery of an antigen to a host cell comprising at least one antigen linked to a nanoparticle by means of a linker molecule.
  • the delivery system of the second aspect of the present invention enables the promotion of an immune response in the host cell.
  • the nanoparticle conjugate according to the first aspect of the present invention maybe administered by itself for the promotion of an immune response or with an adjuvant to boost the response, such as alhydrogel (aluminium hydroxide).
  • a third aspect of the present invention provides a method for the preparation of a nanoparticle conjugate according to the first aspect of the present invention, the method comprising the steps of:
  • the method further comprising adding a surfactant to either or both steps (a) and (b) to stabilise the nanoconjugate.
  • the surfactant comprises a non-ionic surfactant/detergent that is biologically compatible, preferably Triton X-100 but other suitable detergents may include Triton XI 14, Brij35, Brij 58, Tween 20, Tween 80, Span 85 and Nonidet P-40, Octyl ⁇ Glucoside and MEGA 8, in particular Tween.
  • a non-ionic surfactant/detergent that is biologically compatible
  • suitable detergents may include Triton XI 14, Brij35, Brij 58, Tween 20, Tween 80, Span 85 and Nonidet P-40, Octyl ⁇ Glucoside and MEGA 8, in particular Tween.
  • surfactants include anionic surfactants, such as sodium dodecyl sulphate or deoxycholic acid or alternatively, zwitterionic surfactants, such as CHAPS.
  • a thiol-carboxylic acid linker molecule is attached to the nanoparticle to form a carboxylated gold nanoparticle and the antigen is added to form an antigen nanoconjugate linked by a peptide bond.
  • the linker molecule added to the nanoparticles in step (a) is preferably 16- mercaptodecanoic acid or other mercaptocarboxylic acid.
  • the antigen is preferably coupled to the nanoconjugate using the coupling reagents l-ethyl-3-(3- dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS).
  • the nanoconjugate particles are preferably separated from other constituents, such as unbound antigen, by means of centrifugation.
  • a non-ionic detergent or surfactant stabilises the conjugate to allow multiple centrifugation and resuspension of the nanoparticles thereby enabling enhanced purification of the conjugate and/or to allow multi-layer functionalisation of the immunogenic surface without effecting the biological integrity of the antigen.
  • Example 1 investigates the conjugation of the capsular protein Fl from the bacterium Yersinia pestis to gold nanoparticles according to one embodiment of the method of the present invention and Example 2 investigates the role of the Fl conjugate as a potential delivery vehicle for a vaccine against the bacteria; and with reference to the accompanying drawings in which: Figure 1 is an image of gold nanoparticles taken using transmission electron microscopy (Jeol JEM- 1400 transmission electron microscope);
  • Figures 2a to 2c illustrate optical extinction profiles of gold nanoparticles, carboxylated gold nanoparticles and Fl conjugated gold nanoparticles respectively, using a UV-visible spectrophotometer;
  • Figure 3 is a schematic diagram illustrating the coupling between a protein and a gold nanoparticle according to one embodiment of the present invention
  • Figure 4 shows SDS-PAGE gels illustrating the release of Fl protein from gold nanoparticles using acid hydrolysis
  • Figure 5 is a graph illustrating the immune response observed in mice following treatment with gold nanoparticles according to one embodiment of the present invention.
  • Figures 6a to 6d are schematic diagrams illustrating four types of potential vaccine conjugates according to one aspect of the present invention.
  • Figures 7a and 7b illustrate coupling techniques using lysine residues on the antigen to provide random orientation of the antigen on the nanoparticle
  • Figures 8a and 8b illustrate coupling techniques using cysteine residues on the antigen to provide an ordered orientation of the antigen on the nanoparticle
  • Figures 9a and 9b illustrate coupling of an antigen to the nanoparticle by means of the Diels Alder and Sharpless Click chemistry techniques respectively.
  • Example 1 Method of conjugating Y.pestis Fl antigen to gold nanoparticles. All glassware for use in the method was first washed with aqua regia (3 parts concentrated HC1 to one part concentrated HNO 3 ). In a conical flask, 90ml
  • the pellet was resuspended in 0.26 mg/ml aluminium hydroxide gel and left overnight.
  • the solution separated into two phases, of which the lower phase was kept for investigating the ability of the nanoparticle conjugate to immunize mice against Yersinia pestis (See Example 2 below).
  • conjugates were treated with 0.1 mM 1 1-mercapto 1-undecanol which displaced the MHDA from the gold surface.
  • the solution was then run on a SDS-PAGE gel and the density of the band was compared to a known standard, see Figure 4. From this, the concentration of protein conjugated to gold nanoparticles was estimated to be ⁇ 8 ⁇ g ml.
  • the method according to the third aspect of the present invention uses a non-ionic surfactant, in this embodiment Triton x-100, which has surprisingly been found to allow centrifugation and resus ' pension of the nanoparticles at each processing step. This ensures high efficiencies during the coupling processes and high yield of the
  • Antibody response to the conjugates prepared in Example 1 was assessed via intra-muscular injection into mice and assaying for the development of an IgG titre to rFl .
  • Groups of 5 BALB/c mice were dosed intramuscularly with approximately ⁇ g of Fl antigen on day 0.
  • Four groups were investigated as follows:
  • the Fl protein coupled to gold nanoparticles given with a well characterized vaccine adjuvant, alhydrogel (aluminium hydroxide).
  • the Fl protein coupled to gold nanoparticles in phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • Fl/PBS The non-conjugated Fl protein given with PBS.
  • FIG. 5 of the accompanying drawings illustrates the results obtained for each group. Mice dosed with nanoparticles alone showed no antibody response. Initially, non-conjugated Fl protein produced good titre, probably because the Fl was immediately available. However, by day 21 , this titre was significantly lower (P>0.09) than the gn+Fl/alhy group and by day 28, only the gn+Fl/alhy group was still increasing. The titres for the other groups appear to have plateaued. It is possible that an adjuvant is necessary for the conjugated nanoparticles to work well but further studies are to be carried out.
  • novel nanoconjugated vaccines according to the first aspect of the present invention are likely to provide increased immunity for a longer period of time than prior art vaccines.
  • nanoparticles are able to penetrate cell membranes and internalize without being endocysed (Gratton et al. 2008 The effect of particle design on cellular internalization pathways. Proceedings of the National Academy of Sciences of the United States of America 105, 1 1613-1 1618; Rothen- Rutishauser et al. 2006. Interaction of Fine Particles and Nanoparticles with Red Blood Cells Visualized with Advanced Microscopic Techniques. Environ. Sci. Technol. 40, 4353-4359; and Yuan et al. 2008.
  • the nanoconjugates of the present invention may include a single protein subunit tethered to the gold nanoparticle via a suitable linker to provide a non-living vaccine for delivery into a host.
  • multiple proteins may be displayed on the surface of the nanoparticle, see Figure 6a where A, B, C and D are different proteins.
  • the method of the present invention enables the production of such multi-functional nanoconjugates due to the presence of the non-ionic surfactant enabling multiple centrifiigation and resuspension steps to take place without cleavage of the antigen tethered to the nanoparticle.
  • the surfactant stabilises the conjugate enabling its purification and further manipulation.
  • a further embodiment could include linking a polysaccharide to the protein B to form a glycoconjugate (see Figure 6b) to elicit longstanding immunity and to elicit immune responses in children, whereby T-cell help is provided to the response to the polysaccharide.
  • Other combinations could also be produced according to the invention, such as combinations that include protein and lipopolysaccharides (LPS), see Figure 6c, and/or DNA (see Figure 6d).
  • FIGS. 7a and 7b illustrate coupling techniques using lysine residues on the antigen to provide random orientation of the antigen on the nanoparticle.
  • the lysine residues are abundant in the protein and therefore linkage via these residues provides for a random orientation.
  • Figure 7a illustrates a reductive animation via a labile schiff base stabilised by reduction with sodium cyanoborohydride.
  • Figure 7b illustrates a carboxylic acid activation of the nanoparticle using EDC and N-hydroxy succinimide (as per the example above).
  • Figures 8a and 8b of the accompanying drawings illustrate coupling techniques using cysteine residues on the antigen to provide an ordered orientation of the antigen on the nanoparticle. Cysteine residues are less abundant than lysine residues and therefore immobilisation of the antigen on the nanoparticle is more ordered.
  • Figure 8a illustrates a maleimide thiol michael addition reaction and Figure 8b shows a disulphide exchange reaction using pyridiyl disulfide functionality.
  • Figure 9a illustrates a Diels Alder cycloaddition reaction completed in water at room temperature (cyclopentadiene funtionalised to protein using linker function) and Figure 9b is an example of Sharpless Click chemistry involving 1,3-dipolar cycloaddition to form 1,2,3-triazoles in the presence of CuS0 4 (overnight at room temperature) wherein the protein is functionalised as azide through a linker chain (protein reacted through thiol-diphosphate substitution).
  • the nanoconjugates according to the present invention produce strong immune responses to antigens, as evidenced by the serum antibody titres of Example 2.
  • the ability of the nanoconjugates to modulate the Thl/Th2 bias of the immune system requires further investigation, together with the effect of particle size.
  • Ligands for example, Toll agonists
  • a nanoparticle conjugate according to the present invention may be selectively cleaved to optimise the immunogenic properties provided by the antigen tethered to the nanoparticle.
  • the antigen is tethered to the nanoparticle via a strong bond which enables a weaker natural bond linking two parts of the antigen to be cleaved.
  • the parts of the biomolecule that are cleaved from the nanoparticle conjugate can be tailored to optimise the immunogenic properties of the vaccine.

Abstract

A functionalised gold nanoparticle conjugate comprising a gold nanoparticle linked to at least one antigen, in particular the F1 protein of Yersinia pestis, via a thiol- carboxylic acid linker molecule, such as mercaptodecanoic acid. The reactants are coupled together in the presence of a non-ionic detergent to aid stabilisation of the nanoparticle conjugate to enable its purification and further manipulation.

Description

Method for the Preparation of a Novel Nanoparticle Conjugate.
DESCRIPTION
The present invention relates to novel nanoparticle conjugates and their use in vaccine delivery, and to a method for the preparation of said nanoparticle conjugates.
Gold nanoparticles are sub-micrometre-sized particles of gold in fluid (normally water) that have unique molecular-recognition properties resulting in the particles being the subject of a large amount of research for a wide variety of applications. Over recent years there has been a growing interest into the use of nanoparticles in medicine due largely to their unique chemical, physical and optical properties which have been exploited for bioimaging and for the development of novel diagnostic systems. Gold nanoparticles have been investigated as carriers for drugs {Paclitaxel-Functionalized Gold Nanoparticles Jacob D. Gibson, Bishnu P. Khanal, and Eugene R. Zubarev. J. Am. Chem. Soc. 2007, 129, 1 1653-1 1661).
Vaccination (or immunisation) is the subject of vast amounts of research to enable the body to acquire immunity to a particular pathogen. Vaccines can prevent or reduce the effects of infection by many pathogens. For successful vaccination, it is necessary to provide a suitable antigen, dead microorganism or a microorganism with low virulence that will stimulate an immune response in a host cell, together with an adjuvant to potentiate that response. Vaccines have generally been administered as bulk proteins but problems can arise if the solution is not sufficiently pure, for example if lipopolysaccharides remain in the solution. It is also difficult to provide vaccines with multiple functionalities as the processes required to achieve such functionalities often leads to deterioration or contamination of the vaccine.
There are currently a number of licensed vaccines that use microbial
polysaccarides (Lee, C.-J., Banks, S.D. & Li, J.P. 1991 Virulence, Immunity, and Vaccine Related to Streptococcus pneumoniae. Critical Reviews in Microbiology. 18, 89- 114). However, to elicit longstanding immunity in children it is necessary to link the polysaccharide to a protein (a glycoconjugate vaccine). By linking the polysaccharide to a protein a T-cell dependent response is generated but conventional methods for linking polysaccharide and protein are inefficient.
The bacterium Yersinia pestis belongs to the family Enterobacteriaceae and can infect both humans and animals. In humans, infection takes on three forms: pnuemonic, septicemic and bubonic plaques and the bacterium is considered to be a potential biological warfare agent. It is desirable to provide vaccines based on genetically engineered Fl and V antigens but it is necessary to obtain successful delivery of the antigen into the host cell to obtain long-term protection against the strain.
It is an aim of the present invention to provide novel nanoparticle conjugates for medical applications, in particular for providing more efficient vaccine delivery. It is a further aim of the present invention to provide improved methods for the conjugation of nanoparticles to proteins and/or polysaccharides.
A first aspect of the present invention provides a novel nanoparticle conjugate comprising a nanoparticle linked to at least one antigen via a linker molecule.
Any suitable linker molecule may be used to tether the antigen to the nanoparticle using conventional coupling techniques. The linker molecule may comprise a thiol- carboxylic acid linker, such as a mercaptocarboxylic acid, for example 16- mercaptohexadecanoic acid (MHDA).
The antigen preferably comprises a protein or sub-unit thereof having the desired antigen linked to the nanoparticle. In a preferred embodiment of the present invention, the antigen is the Fl protein of Yersinia pestis, having the amino acid sequence:
KKISSVIAIALFGTIATA AADLTASTTATATLVEPA ITLTYKEGAPITIMDNGNIDTELLVGTLTLG GYKTGTTSTSVNFTDAAGDP YLTFTSQDGN HQFTTKVIGKDSRDFDISPKVNGENLVGDDWLATGSQ DFFVRSIGSKGGKLAAGKYTDAVTVTVSNQ
(SEQ. ID No. 1).
Preferably, the nanoparticle comprises a gold nanoparticle but other nanoparticles, such as silver nanoparticles, may be used to provide the conjugate of the first aspect of the present invention. It is to be appreciated that any size of nanoparticle may be used to provide the conjugate of the first aspect of the present invention, although the particle should be dimensioned to enable access into a host cell, preferably being less than 200nm.
The nanoparticle conjugate according to the first aspect of the present invention may include additional functionalised groups, such as multiple proteins or sub-units thereof; polysaccharides, DNA or other ligands.
According to a second aspect of the present invention, there is provided a delivery system for the delivery of an antigen to a host cell, the system comprising at least one antigen linked to a nanoparticle by means of a linker molecule.
The delivery system of the second aspect of the present invention enables the promotion of an immune response in the host cell. The nanoparticle conjugate according to the first aspect of the present invention maybe administered by itself for the promotion of an immune response or with an adjuvant to boost the response, such as alhydrogel (aluminium hydroxide).
A third aspect of the present invention provides a method for the preparation of a nanoparticle conjugate according to the first aspect of the present invention, the method comprising the steps of:
(a) attaching a linker molecule to a nanoparticle to form a functionalised nanoparticle; and (b) adding an antigen to the functionalised nanoparticle to form an antigen nanoconjugate;
the method further comprising adding a surfactant to either or both steps (a) and (b) to stabilise the nanoconjugate.
Preferably, the surfactant comprises a non-ionic surfactant/detergent that is biologically compatible, preferably Triton X-100 but other suitable detergents may include Triton XI 14, Brij35, Brij 58, Tween 20, Tween 80, Span 85 and Nonidet P-40, Octyl β Glucoside and MEGA 8, in particular Tween.
Other possible surfactants include anionic surfactants, such as sodium dodecyl sulphate or deoxycholic acid or alternatively, zwitterionic surfactants, such as CHAPS.
In one embodiment of the method of the present invention, a thiol-carboxylic acid linker molecule is attached to the nanoparticle to form a carboxylated gold nanoparticle and the antigen is added to form an antigen nanoconjugate linked by a peptide bond.
The linker molecule added to the nanoparticles in step (a) is preferably 16- mercaptodecanoic acid or other mercaptocarboxylic acid. The antigen is preferably coupled to the nanoconjugate using the coupling reagents l-ethyl-3-(3- dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). However, it is to be appreciated that other coupling techniques could be used to attach the antigen to the nanoparticle, such as reductive amination using labile schiff base stabilised by reduction with sodium cyanoborohydride, maleimide thiol michael addition reaction, disulfide exchange reaction using pyridiyl disulfide functionality, Diels Alder cycloaddition (cyclopentadiene functionalised to antigen using linker function) and Sharpless Click chemistry (1,3-dipolar cycloaddition to form 1 ,2,3-triazoles in the presence of CuSC ). A non-ionic surfactant is included in the process to aid stabilisation of the nanoparticle conjugate formed by the coupling technique.
The nanoconjugate particles are preferably separated from other constituents, such as unbound antigen, by means of centrifugation. In this respect, it has surprisingly been found that the preparation of the nanoconjugate particles in the presence of a non-ionic detergent or surfactant stabilises the conjugate to allow multiple centrifugation and resuspension of the nanoparticles thereby enabling enhanced purification of the conjugate and/or to allow multi-layer functionalisation of the immunogenic surface without effecting the biological integrity of the antigen.
For a better understanding of the present invention and to show more clearly how it may be carried into effect reference will now be made to the following Examples in which Example 1 investigates the conjugation of the capsular protein Fl from the bacterium Yersinia pestis to gold nanoparticles according to one embodiment of the method of the present invention and Example 2 investigates the role of the Fl conjugate as a potential delivery vehicle for a vaccine against the bacteria; and with reference to the accompanying drawings in which: Figure 1 is an image of gold nanoparticles taken using transmission electron microscopy (Jeol JEM- 1400 transmission electron microscope);
Figures 2a to 2c illustrate optical extinction profiles of gold nanoparticles, carboxylated gold nanoparticles and Fl conjugated gold nanoparticles respectively, using a UV-visible spectrophotometer;
Figure 3 is a schematic diagram illustrating the coupling between a protein and a gold nanoparticle according to one embodiment of the present invention;
Figure 4 shows SDS-PAGE gels illustrating the release of Fl protein from gold nanoparticles using acid hydrolysis;
Figure 5 is a graph illustrating the immune response observed in mice following treatment with gold nanoparticles according to one embodiment of the present invention;
Figures 6a to 6d are schematic diagrams illustrating four types of potential vaccine conjugates according to one aspect of the present invention;
Figures 7a and 7b illustrate coupling techniques using lysine residues on the antigen to provide random orientation of the antigen on the nanoparticle;
Figures 8a and 8b illustrate coupling techniques using cysteine residues on the antigen to provide an ordered orientation of the antigen on the nanoparticle; and
Figures 9a and 9b illustrate coupling of an antigen to the nanoparticle by means of the Diels Alder and Sharpless Click chemistry techniques respectively.
Example 1 : Method of conjugating Y.pestis Fl antigen to gold nanoparticles. All glassware for use in the method was first washed with aqua regia (3 parts concentrated HC1 to one part concentrated HNO3 ). In a conical flask, 90ml
HAuCl4.3H20 (lmM) was heated to 90°C. Whilst stirring vigorously, 10ml 90mM
Na3C6H507 was quickly added. The solution immediately went colourless before a deep burgundy colour developed. Stirring continued for another 15 minutes before the solution was allowed to cool to room temperature and stored in a cool dark place.
Characterization of the particles was then carried out using transmission electron microscopy (TEM) to determine that the gold nanoparticles had an average diameter of 15.6 ± 2.3nm, as shown in Figure 1.
100 μΐ 1 mM 16-mercaptohexadecanoic acid (MHDA) was added to 880 μΐ gold nanoparticles placed in a centrifuge tube and allowed to stand for 10 minutes. 20 μΐ 5% (vol/vol) Triton® -x 100 was then added to the solution. The sample was centrifuged at 13400 rpm for 10 minutes, after which the supernatant was removed and the pellet resuspended in 842.5 μΐ phosphate buffered saline (PBS). In a separate tube, 100 μΐ 0.6mM l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) was added to ΙΟΟμΙ 0.15 mM N-hydroxysuccinimide (NHS) before adding ΙΟΟμΙ of this solution to the carboxylated particles. After leaving for 2 minutes, 57.5 μΐ of 1.74 mg/ml rFl was added to the particulate suspension, final protein concentration of 100 μξ/πύ. The amino acid sequence of Fl -antigen is as follows:
MKKISSVIAIALFGTIATANAADLTASTTATATLVEPARITLTY EGAPITIMDNGNIDTELLVGTLTLG GYKTGTTSTSWFTDAAGDPMYLTFTSQDG HQFTTKVIG DSRDFDISPKVISIGENLVGDDVVLATGSQ DFFVRSIGSKGGKLAAGKYTDAVTVTVSNQ
(SEQ. ID No. 1). The solution was left to incubate at room temperature for two hours and then centrifuged at 13400 rpm for 10 minutes. The supernatant was removed and the pellet was resuspended in 1 ml PBS. The solution was then re-spun under the same conditions to remove any unbound protein; this was repeated three more times. At each stage of the process, an absorption spectrum of the particles was determined using a UV-visible spectrophotometer. Figure 2a is the optical extinction profile of the gold nanoparticles wherein λ max is 514.8nm. Figure 2b is the profile for the carboxylated gold
nanoparticles. Evidence for functionalisation comes from a shift in λπιαχ to 523.9nm, and a broadening of the peak. Figure 2c shows the profile of Fl conjugated gold nanoparticles. λ max has shifted further to 551.5nm, with an even broader peak. After each washing step, the colloidal solution becomes more dilute resulting in a lower absorbance.
After the final centrifugation, the pellet was resuspended in 0.26 mg/ml aluminium hydroxide gel and left overnight. The solution separated into two phases, of which the lower phase was kept for investigating the ability of the nanoparticle conjugate to immunize mice against Yersinia pestis (See Example 2 below).
The main steps of the process are summarized in Figure 3 of the accompanying drawings wherein the circle represents a gold nanoparticle. A thiol -carboxylic acid linker (16-mercaptohexadecanoic acid) is attached to the gold nanoparticle via a sulphide bond. The vaccine sub-unit (eg. Fl protein) is then tethered to the nanoparticle via the linker by means of a peptide bond, using N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) as coupling reagents.
In order to determine how much protein was tethered to the nanoparticles, conjugates were treated with 0.1 mM 1 1-mercapto 1-undecanol which displaced the MHDA from the gold surface. The solution was then run on a SDS-PAGE gel and the density of the band was compared to a known standard, see Figure 4. From this, the concentration of protein conjugated to gold nanoparticles was estimated to be ~ 8 μg ml.
The method according to the third aspect of the present invention uses a non-ionic surfactant, in this embodiment Triton x-100, which has surprisingly been found to allow centrifugation and resus'pension of the nanoparticles at each processing step. This ensures high efficiencies during the coupling processes and high yield of the
functionalised product. Multiple resuspension events allow complex tethering chemistries to be used with effective purification, thereby allowing multi-layer functionalisation of the immunogenic surface. For example, linking a protein surface amine and activate carboxyl group using EDC NHS coupling as described above, linking sugar to surface amine group by Schiff base coupling, DNA coupling via ribose using Schiff base coupling and combining different motifs around the GNP controlled by the stochiometry of the association process. The stabilisation achieved by the non-ionic detergent also enables the monitoring of the addition of the ligands to the nanoparticle surface by monitoring the shift in absorbance profile using UV-visible
spectrophotometer. Example 2: Investigation into the antibody response to the Fl-nanoparticle conjugate.
Antibody response to the conjugates prepared in Example 1 was assessed via intra-muscular injection into mice and assaying for the development of an IgG titre to rFl . Groups of 5 BALB/c mice were dosed intramuscularly with approximately ^g of Fl antigen on day 0. Four groups were investigated as follows:
gn + Fl/alhy The Fl protein coupled to gold nanoparticles given with a well characterized vaccine adjuvant, alhydrogel (aluminium hydroxide).
gn + Fl/PBS The Fl protein coupled to gold nanoparticles in phosphate buffered saline (PBS).
Fl/alhy The non-conjugated Fl protein given with alhydrogel.
Fl/PBS The non-conjugated Fl protein given with PBS.
Figure 5 of the accompanying drawings illustrates the results obtained for each group. Mice dosed with nanoparticles alone showed no antibody response. Initially, non-conjugated Fl protein produced good titre, probably because the Fl was immediately available. However, by day 21 , this titre was significantly lower (P>0.09) than the gn+Fl/alhy group and by day 28, only the gn+Fl/alhy group was still increasing. The titres for the other groups appear to have plateaued. It is possible that an adjuvant is necessary for the conjugated nanoparticles to work well but further studies are to be carried out.
The novel nanoconjugated vaccines according to the first aspect of the present invention are likely to provide increased immunity for a longer period of time than prior art vaccines. There is evidence to suggest that nanoparticles are able to penetrate cell membranes and internalize without being endocysed (Gratton et al. 2008 The effect of particle design on cellular internalization pathways. Proceedings of the National Academy of Sciences of the United States of America 105, 1 1613-1 1618; Rothen- Rutishauser et al. 2006. Interaction of Fine Particles and Nanoparticles with Red Blood Cells Visualized with Advanced Microscopic Techniques. Environ. Sci. Technol. 40, 4353-4359; and Yuan et al. 2008. Cellular uptake of solid lipid nanoparticles and cytotoxicity of encapsulated paclitaxel in A549 cancer cells. International Journal of Pharmaceutics 348, 137-145). By using nanoparticles to deliver the subunit vaccine, the conjugates should penetrate the cell and subsequently be processed via MHC class 1 receptors, leading to the activation of CD8+ T cells. This not only confers greater immunity against Y.pestis infections but also provides individuals with a longer period of immunity since cellular immunity is important for developing immunological memory.
The nanoconjugates of the present invention may include a single protein subunit tethered to the gold nanoparticle via a suitable linker to provide a non-living vaccine for delivery into a host. Alternatively, multiple proteins may be displayed on the surface of the nanoparticle, see Figure 6a where A, B, C and D are different proteins. By conjugating more than one type of protein to the nanoparticle, there is the potential to have a vaccine that acts against several pathogens. The method of the present invention enables the production of such multi-functional nanoconjugates due to the presence of the non-ionic surfactant enabling multiple centrifiigation and resuspension steps to take place without cleavage of the antigen tethered to the nanoparticle. The surfactant stabilises the conjugate enabling its purification and further manipulation.
A further embodiment could include linking a polysaccharide to the protein B to form a glycoconjugate (see Figure 6b) to elicit longstanding immunity and to elicit immune responses in children, whereby T-cell help is provided to the response to the polysaccharide. Other combinations could also be produced according to the invention, such as combinations that include protein and lipopolysaccharides (LPS), see Figure 6c, and/or DNA (see Figure 6d).
The examples referred to above tether the antigen to the nanoparticle via a thiol- carboxylic acid linker. However, it is to be appreciated that other coupling techniques that are known in the art may be employed to attach an antigen to a nanoparticle. Figures 7a and 7b illustrate coupling techniques using lysine residues on the antigen to provide random orientation of the antigen on the nanoparticle. The lysine residues are abundant in the protein and therefore linkage via these residues provides for a random orientation. Figure 7a illustrates a reductive animation via a labile schiff base stabilised by reduction with sodium cyanoborohydride. Figure 7b illustrates a carboxylic acid activation of the nanoparticle using EDC and N-hydroxy succinimide (as per the example above). Figures 8a and 8b of the accompanying drawings illustrate coupling techniques using cysteine residues on the antigen to provide an ordered orientation of the antigen on the nanoparticle. Cysteine residues are less abundant than lysine residues and therefore immobilisation of the antigen on the nanoparticle is more ordered. Figure 8a illustrates a maleimide thiol michael addition reaction and Figure 8b shows a disulphide exchange reaction using pyridiyl disulfide functionality. Figure 9a illustrates a Diels Alder cycloaddition reaction completed in water at room temperature (cyclopentadiene funtionalised to protein using linker function) and Figure 9b is an example of Sharpless Click chemistry involving 1,3-dipolar cycloaddition to form 1,2,3-triazoles in the presence of CuS04 (overnight at room temperature) wherein the protein is functionalised as azide through a linker chain (protein reacted through thiol-diphosphate substitution).
The nanoconjugates according to the present invention produce strong immune responses to antigens, as evidenced by the serum antibody titres of Example 2. The ability of the nanoconjugates to modulate the Thl/Th2 bias of the immune system requires further investigation, together with the effect of particle size. Ligands (for example, Toll agonists) may be presented alongside the proteins on the nanoparticle surface to further modulate the immune response to the protein.
A nanoparticle conjugate according to the present invention may be selectively cleaved to optimise the immunogenic properties provided by the antigen tethered to the nanoparticle. The antigen is tethered to the nanoparticle via a strong bond which enables a weaker natural bond linking two parts of the antigen to be cleaved. The parts of the biomolecule that are cleaved from the nanoparticle conjugate can be tailored to optimise the immunogenic properties of the vaccine.

Claims

1. A functionalised nanoparticle conjugate comprising a nanoparticle linked to at least one antigen via a linker molecule.
2. A conjugate as claimed in claim 1 wherein the antigen is a protein or sub-unit thereof linked to the nanoparticle.
3. A conjugate as claimed in claim 1 or claim 2 wherein the nanoparticle is selected from the group consisting of a gold nanoparticle and a silver nanoparticle.
4. A conjugate as claimed in claim 2 or claim 3 wherein the antigen is the Fl protein of Yersinia pestis, the protein having the amino acid sequence of SEQ. ID No. 1.
5. A conjugate as claimed in any one of claims 1 to 4 wherein the linker molecule is a thiol-carboxylic acid linker molecule.
6. A conjugate as claimed in claim 5 wherein the thiol-carboxylic linker molecule comprises a mercaptocarboxylic acid.
7. A conjugate as claimed in claim 6 wherein the thiol-carboxylic acid linker molecule is 16-mercaptohexadecanoic acid (MHDA).
8. A conjugate as claimed in any one of claims 1 to 7 further comprising additional functionalised groups selected from the groups consisting of multiple proteins or sub- units thereof, polysaccharides, DNA and other ligands.
9. A delivery system for the delivery of an antigen to a host cell, the system comprising an antigen conjugated to a nanoparticle by means of a linker molecule.
10. The delivery system of claim 9 wherein the antigen is the Fl protein of Yersinia pestis, the protein having the amino acid sequence of SEQ. ID No. 1.
11. The delivery system of claim 9 or claim 10 further comprising an adjuvant to boost the immune response.
12. The delivery system of claim 1 1 wherein the adjuvant is alhydrogel (aluminium hydroxide).
13. A method for the preparation of a nanoparticle conjugate, the method comprising the steps of:
(a) attaching a linker molecule to a nanoparticle to form a functionalised nanoparticle; and
(b) adding an antigen to the functionalised nanoparticle to form an antigen nanoconjugate;
the method further comprising adding a surfactant to either or both steps (a) and (b) to stabilise the nanoconjugate.
14. The method according to claim 13 wherein the surfactant is a non-ionic detergent.
15. The method according to claim 14 wherein the non-ionic detergent is Triton xlOO.
16. The method according to claim 14 wherein the non-ionic detergent is selected from the group consisting of Triton XI 14, Brij35, Brij 58, Tween 20, Tween 80 and Nonidet P-40, Octyl β Glucoside and MEGA 8.
17. The method according to any one of claims 13 to 16 wherein the linker molecule added to the nanoparticles in step (a) is a mercaptocarboxylic acid.
18. The method according to claim 17 wherein the linker molecule is 16- mercaptodecanoic acid.
19. The method according to any one of claims 13 to 18 wherein the antigen is coupled to the nanoconjugate using the coupling reagents l-ethyl-3-(3- dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS).
20. The method according to any one of claims 13 to 19 wherein the desired nanoconjugate particles are separated from other constituents by means of centrifugation.
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