WO2023219502A1 - Immunotherapeutic compositions and adjuvants - Google Patents

Immunotherapeutic compositions and adjuvants Download PDF

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Publication number
WO2023219502A1
WO2023219502A1 PCT/NL2023/050257 NL2023050257W WO2023219502A1 WO 2023219502 A1 WO2023219502 A1 WO 2023219502A1 NL 2023050257 W NL2023050257 W NL 2023050257W WO 2023219502 A1 WO2023219502 A1 WO 2023219502A1
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Prior art keywords
elp
immunomodulator
peptide
micelles
micelle
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PCT/NL2023/050257
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French (fr)
Inventor
Alexander Kros
René OLSTHOORN
Jolinde VAN STRIEN
Romain LEBOUX
Ronald Van Ree
Hans WARMENHOVEN
Wim Jiskoot
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Universiteit Leiden
Academisch Medisch Centrum
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Publication of WO2023219502A1 publication Critical patent/WO2023219502A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • 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
    • A61K39/0005—Vertebrate antigens
    • 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
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • C07K14/77—Ovalbumin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • 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
    • A61K2039/55516—Proteins; Peptides
    • 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
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention provides compositions for modulating an immune response in a subject.
  • the compositions include micelles formed from elastin-like peptides (ELPs) that may be bound to immunomodulators.
  • ELPs elastin-like peptides
  • the invention also provides uses of such micelles as adjuvants.
  • pharmaceutical formulations including the compositions for use as medicaments, particularly for use in treating cancers, allergy and infectious disease.
  • Vaccination is an effective method of preventing infectious diseases and decreasing associated mortality.
  • Traditional vaccination strategies are based on live attenuated or inactivated pathogens. These conventional vaccines are linked to various adverse effects.
  • In the search for safer vaccines research is becoming increasingly focused on subunit vaccines, containing antigenic components of pathogens and omitting any unnecessary and harmful elements.
  • Subunit vaccines are considered safer than traditional vaccines, but at the cost of lower efficacy.
  • subunit vaccines do not generate the same level of immunity, they require an adjuvant.
  • the most commonly used adjuvant is alum, which refers to micron-sized particles based on aluminium salts.
  • injections with alum can cause local adverse effects and are also associated with some systemic side effects.
  • alum is unsuitable for inducing immunity against intracellular targets such as tuberculosis, legionella and malaria, because it primarily triggers a strong humoral response.
  • SCIT subcutaneous allergen immunotherapy
  • Allergen immunotherapy (AIT) and SCIT aim to induce lgG1 and lgG4 (in humans) or lgG1 and lgG2a (in mice), which compete with IgE to bind allergens, to prevent IgE-mediated effector mechanisms.
  • These so-called blocking antibodies are produced by regulatory B-cells.
  • the induction of regulatory T-cells is also considered a beneficial effect of AIT.
  • Both types of regulatory cells produce interleukin 10 (IL-10).
  • IL-10 interleukin 10
  • a switch from a Th2- to a Th1-biased immune response is generally favourable for AIT. This switch is measured by the induction of interferon y (IFN-y).
  • SCIT generally contains aluminium-based adjuvants (alum) such as aluminium hydroxide or aluminium phosphate.
  • alum aluminium-based adjuvants
  • alum has been regarded as a safe adjuvant in vaccines for infectious diseases and SCIT for a long time, there is growing concern about chronic alum exposure during SCIT, especially in the paediatric setting.
  • Some reports have linked alum overexposure to systemic adverse events including renal failure and various neurodegenerative disease such as Alzheimer’s. Therefore, there is a growing need to replace alum with a different adjuvant in SCIT.
  • alum is known for inducing a mixed Th1/Th2/Treg immune response.
  • SCIT aims to counteract Th2-skewed immune responses, and this is achieved by prolonged exposure to alum- adsorbed allergens.
  • the Th2 stimulation by alum results in a transient increase in IgE which may contribute to adverse events.
  • small particles ⁇ 600 nm
  • nanoparticles ⁇ 200 nm are also transported to the lymph nodes and taken up by lymph node resident DCs. Therefore, attention has focused on various types of nanoparticles (NPs) to replace micron-sized alum in allergy vaccine delivery systems.
  • APCs antigen presenting cells
  • DCs dendritic cells
  • MHCII major histocompatibility complex II
  • costimulatory molecules such as CD80 and CD86. 8
  • co-stimulatory molecules are presented on the DC surface and can interact with CD28 on CD4 + T-cells, thereby stimulating T- cells to expand and differentiate. Differentiation of T-cells has a major impact on the induced immune response.
  • Th1 cells While differentiation into T helper 1 (Th1) cells allows the induction of type I cytokines like interferon y (IFN-y) and tumour necrosis factor a (TNF-a), leading to potentiation of the cellular immunity, differentiation into Th2 cells primarily leads to induction of interleukin 4 (IL- 4), IL-5 and IL-13, which strengthen the humoral arm of the immune response.
  • Adjuvants can influence the differentiation of the immune response towards Th1 or Th2, depending on the physicochemical properties of the adjuvant nanoparticles, such as size, shape, surface charge, rigidity and particle composition.
  • ELPs Elastin-like polypeptides
  • VPGXG pentapeptide VPGXG in which guest residue X can be any amino acid.
  • ELP-based micelles comprise amphiphilic ELPs of which the hydrophobic block contains a different guest residue from the hydrophilic block. These polypeptide diblock copolymers self-assemble into spherical micelles above the critical micelle temperature (CMT). ELP assembly is induced by hydrophobic collapse of the hydrophobic ELP block, forming the micelle core with the hydrophilic blocks pointing outwards in solution.
  • CMT critical micelle temperature
  • ELP assembly is induced by hydrophobic collapse of the hydrophobic ELP block, forming the micelle core with the hydrophilic blocks pointing outwards in solution.
  • ELP- based micelles are a promising platform for drug delivery due to their biocompatibility, facile design regarding physicochemical properties 1321-23 and simple modification with cargo. However, the adjuvant properties of ELP micelles have not
  • the invention is based on the surprising finding that ELP based micelles comprising an immunomodulator can act as adjuvants which show improved properties in comparison to alum (particularly due to their ability to induce a less T helper 2 (Th2)- and more Th1 -skewed immune response).
  • Th2 T helper 2
  • the ELPs provide an adjuvant that has an improved cellular mediated response and reduced humoral response.
  • the ELP based micelles have also been found to reduce IgE and increase IgG, demonstrating that ELP based micelles are prime candidates for use in allergy immunotherapy, particularly as an alternative adjuvant to alum for subcutaneous allergy immunotherapy.
  • a composition comprising a micelle, wherein the micelle comprises one or more elastin-like polypeptides (ELP) and one or more immunomodulators.
  • ELP elastin-like polypeptides
  • the composition can be used to modulate an immune response in a subject.
  • the one or more ELPs are amphiphilic.
  • the ELPs comprise at least one hydrophobic block and at least one hydrophilic block.
  • the one or more ELPs comprise amphiphilic copolymers.
  • the one or more ELPs comprise amphiphilic diblock copolymers. The use of amphiphilic ELPs provides a composition wherein the ELPs are configured to self-assemble into the micelles.
  • At least one of the one or more ELPs comprise the sequence (VPGX hb G)n(VPGX h PG) n (SEQ ID NO: 1) or (VPGX h PG) n (VPGX hb G) n (SEQ ID NO: 2) wherein X h P is any hydrophilic amino acid residue and X hb is any hydrophobic amino acid residue.
  • X hp is serine.
  • X hb is isoleucine.
  • At least one of the one or more ELPs comprise the sequence MX 1 (VPGX hb G) 4 8(VPGX h PG) 4 8X 2 (SEQ ID NO: 3), wherein X 1 and X 2 are each independently selected from any amino acid.
  • at least one of the one or more ELPs comprise the sequence MG(VPGX hb G) n (VPGX h PG) n Y (SEQ ID NO: 4).
  • at least one of the one or more ELPs comprise the sequence (VPGIG)n(VPGSG) n (SEQ ID. NO: 5).
  • At least one of the one or more ELPs comprise the sequence (VPGIG)48 (VPGSG)4s (SEQ ID NO: 6). In certain embodiments, at least one of the one or more ELPs comprise the sequence of MG(VPGIG)48(VPGSG)4sY (SEQ ID. NO: 7). In certain embodiments, the ELP comprises the sequence MG(VPGIG)4s(VPGSG)48 (SEQ ID NO: 32).
  • the ELPs may facilitate induction of a Th1 -skewed immune response in a subject in response to an immunomodulator.
  • the ELPs may facilitate switching from a Th2- to a Th1-biased immune response to an immunomodulator.
  • the immunomodulator is covalently bound to the ELP. In certain embodiments, the immunomodulator is covalently bound to the ELP to form a ELP- immunomodulator fusion protein. In certain embodiments, the immunomodulator is covalently bound to the ELP by chemical conjugation.
  • the immunomodulator is covalently bound to the ELP so that the immunomodulator is located on the external surface of the micelle. In certain embodiments, the immunomodulator is covalently bound to a hydrophilic block of an ELP. In certain embodiments, the immunomodulator is covalently bound to the C-terminal of an ELP comprising a C-terminal hydrophilic block.
  • the ELP-immunomodulator fusion protein comprises the sequence “(VPGX hb G)n(VPGX hp G) n -immunomodulator” or “immunomodulator-(VPGX h PG) n (VPGX hb G) n ”. In certain embodiments, the ELP-immunomodulator fusion protein comprises the sequence “(VPGIG)48(VPGSG)48-immunomodulator”.
  • the ELP-immunomodulator fusion protein comprises the sequence “MG(VPGIG)48(VPGSG)48-immunomodulator” or “immunomodulator-MG(VPGIG)48(VPGSG)48”.
  • composition comprising a micelle comprising one or more elastin-like polypeptides (ELP), wherein the immunomodulator is covalently bound to an ELP to form an ELP-immunomodulator fusion protein.
  • ELP elastin-like polypeptides
  • Binding of an immunomodulator to a hydrophilic block of an amphiphilic ELP provides a micelle that has an immunomodulator presented on the corona (external surface) of the micelle allowing for recognition and binding of the immunomodulator by immune cells or immune system molecules.
  • the use of covalent attachment may also provide a more stably bound immunomodulator.
  • the immunomodulator is non-covalently bound to the ELP.
  • the immunomodulator is non-covalently bound to the ELP so that the immunomodulator is located on the external surface of the micelle.
  • the immunomodulator is non-covalently bound to a hydrophilic block of an ELP.
  • the immunomodulator is non-covalently bound to the C-terminal of an ELP comprising a C-terminal hydrophilic block.
  • the immunomodulator is non-covalently bound to the ELP via an intermediary binding molecule.
  • the use of non-covalent bonding of an immunomodulator may allow for tuneable release of an immunomodulator in response to stimuli. For example, in response to pH, temperature, or enzymatic processing.
  • the immunomodulator comprises at least one epitope selected from the group consisting of: an allergenic epitope, a viral epitope, a bacterial epitope, a parasitic epitope, a disease-associated epitope, and a tumour-associated epitope.
  • the immunomodulator comprises an allergenic epitope.
  • the immunomodulator comprises an allergenic epitope from, or derived from, a Type I allergen.
  • the immunomodulator comprises an allergenic epitope from, or derived from, Bet v 1.
  • the immunomodulator covalently bound to the ELP comprises the sequence:
  • the ELP-immunomodulator may comprise the sequence MG(VPGIG)48(VPGSG)48- Bet v 1 .
  • the ELPs is covalently bound to a first coiled coil forming peptide.
  • the first coiled coil peptide is an intermediary binding molecule (e.g. peptide E).
  • the first coiled coil peptide is an immunomodulator (e.g. peptide K).
  • the first coiled coil peptide is an intermediary binding molecule and an immunomodulator (e.g., peptide K).
  • the immunomodulator is covalently bound to a cognate coiled coil forming peptide.
  • the immunomodulator may comprise a further coiled coil forming peptide configured to form a coiled coil (complex) with the first coiled coil forming peptide that is covalently bound to the ELP.
  • the coiled coil complex is stable, ensuring colocalization of the immunomodulator and micelle, but may dissociate at low pH, thus enabling endosomal escape.
  • the first coiled coil forming peptide comprises peptide K and/or peptide E.
  • the first coiled coil forming peptide comprises peptide K and the cognate coiled coil forming peptide comprises peptide E.
  • the first coiled coil forming peptide comprises peptide E and the cognate coiled coil forming peptide comprises peptide K.
  • a composition comprising a micelle, wherein the micelle comprises: a) one or more elastin-like polypeptides (ELP) covalently bound to a first coiled coil forming peptide; and b) one or more immunomodulators covalently bound to a cognate coiled coil forming peptide, wherein the immunomodulator is non-covalently bound to the ELP via the coiled coil forming peptides.
  • ELP elastin-like polypeptides
  • composition comprising a micelle, wherein the micelle comprises a peptide K covalently bound to elastin-like polypeptide (ELP) and an immunomodulator covalently bound to a further ELP.
  • ELP elastin-like polypeptide
  • the first coiled coil forming peptide is covalently bound to the ELP so that the first coiled coil forming peptide is located on the external surface of the micelle. In certain embodiments, the first coiled coil forming peptide is covalently bound to a hydrophilic block of an ELP. In certain embodiments, the first coiled coil forming peptide is covalently bound to the C- terminal of an ELP comprising a C-terminal hydrophilic block.
  • the ELP covalently bound to peptide K comprises the sequence “MG(VPGIG) 4 8(VPGSG) 4 8YWSGGG(KIAALKE)n” (SEQ ID NO: 21) wherein n is an integer between 2 and 5. In certain embodiments, the ELP covalently bound to peptide K comprises the sequence “MG(VPGIG) 4 8(VPGSG) 4 8YWSGGG(KIAALKE) 4 ” (SEQ ID NO: 9). In certain embodiments, the ELP covalently bound to peptide K comprises the sequence “MG(VPGIG) 48 (VPGSG) 48 YWSGGG(EIAALEK)n” (SEQ ID NO: 22) wherein n is an integer between 2 and 5. In certain embodiments, the ELP covalently bound to peptide E comprises the sequence “MG(VPGIG) 48 (VPGSG) 48 YWSGGG(EIAALEK) 4 ” (SEQ ID NO: 10).
  • the presence of coiled coil domains may increase the composition’s ability to induce an immune response due to the tendency of coiled coil domains to interact with cellular membranes.
  • the first coiled coil forming peptide may be peptide K.
  • Peptide K may interact with and/or destabilize membranes, such as cell membranes of antigen presenting cells. Peptide K may interact with membranes, burying the hydrophobic face of the helix in the bilayer while the lysine side chains “snorkel” towards the polar/nonpolar interface.
  • the micelle comprises an average hydrodynamic diameter of from about 30 to about 70 nm.
  • the micelle comprises an average polydispersity index of from about 0.001 to about 0.25.
  • the micelle comprises an average zeta potential of from about -15 mV to about 15 mV.
  • the micelles in the composition are monodispersed.
  • the micelles are spherical.
  • Adjuvants such as the micelles described herein, can influence the differentiation of the immune response towards Th1 or Th2, depending on the physicochemical properties of the adjuvant, such as nanoparticle size, shape, surface charge, and rigidity. For example, nanoscale spherical particles are best suited to induce Th 1 -biased responses.
  • a pharmaceutical formulation comprising a composition as described herein, further comprising a pharmaceutically acceptable carrier, excipient, diluent and/or adjuvant.
  • a micelle comprising one or more ELPs as described herein for delivery of an immunomodulator as described herein.
  • a micelle comprising one or more ELPs as described herein for forming of an immunogenic vaccine.
  • a micelle comprising one or more ELPs as described herein as an adjuvant.
  • a pharmaceutical formulation as described for use in preventing and/or treating an allergic disease in another aspect of the invention, there is provided a pharmaceutical formulation as described for use in preventing and/or treating an allergic disease.
  • pharmaceutical formulation is for use in preventing an allergic disease.
  • a method of preventing and/or treating an allergic disease comprising administering to a subject a therapeutically effective amount of a pharmaceutical formulation described herein
  • the immunomodulator is an allergenic epitope as described herein.
  • the pharmaceutical formulation is for use in allergy immunotherapy.
  • the allergy immunotherapy is subcutaneous immunotherapy.
  • the immunomodulator is an epitope from or derived Bet v 1 as described herein.
  • a method of preventing and/or treating cancer comprising administering to a subject a therapeutically effective amount of a pharmaceutical formulation as described herein.
  • the immunomodulator is a tumour-associated epitope.
  • the pharmaceutical formulation comprises a micelle comprising: a) one or more elastin-like polypeptides (ELP) covalently bound to a first coiled coil forming peptide; and b) an immunomodulator covalently bound to a cognate coiled coil forming peptide, wherein the immunomodulator is non-covalently bound to the ELP via the coiled coil forming peptides.
  • ELP elastin-like polypeptides
  • the pharmaceutical formulation comprises a micelle, wherein the micelle comprises a peptide K covalently bound to elastin-like polypeptide (ELP) and an immunomodulator covalently bound to a further ELP.
  • ELP elastin-like polypeptide
  • a pharmaceutical formulation as described herein for use in preventing and/or treating an infectious disease.
  • a method of preventing and/or treating an infectious disease comprising administering to a subject a therapeutically effective amount of a pharmaceutical formulation as described herein.
  • the pharmaceutical formulation is a vaccine. In certain embodiments, the pharmaceutical formulation is for use in a method of vaccination of a subject.
  • the immunomodulator is a pathogen associated epitope. In certain embodiments, the immunomodulator is selected from the group of a viral epitope, a bacterial epitope, or a parasitic epitope.
  • the pharmaceutical formulation comprises a micelle comprising: a) one or more elastin-like polypeptides (ELP) covalently bound to a first coiled coil forming peptide; and b) one or more immunomodulators covalently bound to a cognate coiled coil forming peptide, wherein the immunomodulator is non-covalently bound to the ELP via the coiled coil forming peptides.
  • ELP elastin-like polypeptides
  • the pharmaceutical formulation comprises a micelle, wherein the micelle comprises a peptide K covalently bound to elastin-like polypeptide (ELP) and an immunomodulator covalently bound to a further ELP.
  • ELP elastin-like polypeptide
  • a method for inducing an immune response specific for an immunomodulator in a subject comprising administering to a subject a therapeutically effective amount of a pharmaceutical formulation as described herein.
  • Figure 1 shows micellar formulations displaying OVA323 used in this study. All formulations contain ELP and the covalent and hybrid micelles also contain ELP-OVA323, whereas the coiled coil micelles contain peptide E-OVA323 and ELP-K. ELP to OVA323 epitope ratio and ELP to peptide K ratio are 10:1. Cartoon is not to scale;
  • FIG. 2 shows LC-MS spectrum of peptide E. Chromatogram is shown in the top and middle panels. Observed mass (bottom): 1271.84; calculated mass ([M+2H + ] 2+ ): 1272.97;
  • Figure 3 shows LC-MS spectrum of peptide K. Chromatogram is shown in the top and middle panels. Observed mass (bottom): 1281.77; calculated mass ([M+2H + ] 2+ ): 1283.07;
  • FIG. 4 shows LC-MS spectrum of E-OVA323. Chromatogram is shown in the top and middle panels. Observed mass (bottom): 1432.89 and 1074.89; calculated mass ([M+3H + ] 3+ and [M+4H + ] 4+ ): 1434.28 and 1075.96; Figure 5 shows LC-MS spectrum of E-OVA323-TMR. Chromatogram is shown in the top and middle panels. Observed mass (bottom): 1647.32 and 1235.46; calculated mass ([M+3H + ] 3+ and [M+4H + ] 4+ ): 1648.18 and 1236.39;
  • FIG. 6 shows RP-HPLC analysis of ELP-E (top), ELP-K (middle) and ELP-OVA323 (bottom). Gradient of 0 to 90% acetonitrile in water containing 0.1% TFA. Traces were normalized to match the peak height of ELP;
  • FIG. 7 shows expression, purification and identification of ELP-E (top), ELP-K (middle) and ELP-OVA323 (bottom).
  • Samples were analyzed on 10% SDS polyacrylamide gels with Coomassie Blue (ELP-E and ELP-K) or copper chloride (ELP-OVA323) staining.
  • ELP-E, ELP-K and ELP-OVA323 are clearly visible at 43 kDa, 43 kDa and 40 kDa, respectively.
  • the black arrow marks the location of the purified polypeptide.
  • Lys lysate; C1-5 refer to samples taken after 1-5 cycles of ITC.
  • Figure 8 shows inverse transition behaviour of ELP-E and ELP-K compared to ELP.
  • [polypeptide] 10 pM; SLS measured in 10 mM PB pH 7.8 as a function of temperature;
  • FIG. 9 shows CMC determination of ELP-E (top left) and ELP-OVA323 (bottom left) and CAC determination of ELP-K (top right). Measured in 10 mM PB pH 7.8 at 37 °C. Count rate was measured by SLS as a function of [ELP-E], [ELP-K] or [ELP-OVA323], Count rates were normalized to the count rate of the buffer. Gray data represent samples that did not contain particles according to the autocorrelation functions (see Figure 10); black data represent the samples for which the autocorrelation functions had a sigmoidal shape. The CMC of ELP-E was determined at 0.11 pM and the CAC of ELP-K as 56 nM by calculating the intercept of the trend lines;
  • Figure 12 shows size distributions of ELP/ELP-K micelles in the absence (top) and presence (bottom) of equimolar amounts of peptide
  • Z-average values are included in Table 3;
  • Figure 14 shows colocalization of FITC-ELP and E-OVA323-TMR of coiled coil micelles.
  • Figure 16 shows inverse transition behavior of ELP-OVA323 compared to ELP.
  • [polypeptide] 10 pM
  • DLS measured in 10 mM PB pH 7.8 as a function of temperature.
  • the CMT was determined as the temperature value above which the count rate is stable: ⁇ 28 °C for both polypeptides;
  • the medium magnifications were used to analyze the average height of the imaged micelles (g): 23 nm (covalent), 15 nm (coiled coil) and 11 nm (hybrid).
  • Micelles are indicated with green arrows and clustered micelles are indicated with white arrows.
  • Figure 22 shows coiled coil association efficiency on the ELP micelle surface.
  • [polypeptide] 20 pM
  • [E-OVA-TMR] 2 pM
  • separation of unbound E-OVA323 from micelle in the presence (coiled coil) or absence (ELP/E-OVA323-TMR) of ELP-K was performed at 37 °C.
  • the fluorescent signals of the resulting samples were normalized to the value of the coiled coil sample.
  • E- OVA323-TMR concentrations in the relevant range are linearly associated with the fluorescent signal;
  • Figure 23 shows BMDC uptake of FITC-labelled formulations with and without ELP-K.
  • BMDCs were incubated for 4 hours with fluorescently labelled micelles or as a negative control with medium only. OVA323 concentrations were (from left to right) 270, 90, 30, 10, 3.3 and 1.1 nM. The BMDCs were subsequently analyzed with flow cytometry;
  • Figure 24 shows BMDC uptake of FITC-labelled formulations with and without ELP-K.
  • [OVA323] 270 nM (top left), 90 nM (top right), 30 nM (bottom left) and 10 nM (bottom right).
  • BMDCs were incubated for 4 hours with fluorescently labelled micelles or with medium only as a negative control. The BMDCs were subsequently analysed with flow cytometry. Significant differences between BMDC uptake percentages of samples with ELP-K and samples without ELP-K were determined using a one-way ANOVA with a Tukey’s multiple comparison test.
  • FIG. 25 shows BMDC uptake of free E-OVA323-TMR and coiled coil micelles containing both E-OVA323-TMR and FITC-ELP.
  • FIG. 26 shows ELP-K-dependent uptake of ELP micelles into BMDCs.
  • BMDCs were incubated for 4 hours with fluorescently labelled peptide or micelles containing 90 nM OVA323 (900 nM polypeptide). The BMDCs were subsequently washed thoroughly with medium to remove excess particles and imaged on a confocal microscope.
  • Various images were taken from each sample: (a) bright field image, (b) blue channel (cell nuclei), (c) green channel (ELP), (d) red channel (E- OVA323), (e) overlay of all fluorescent channels, (f) complete overlay.
  • Figure 27 shows maturation of BMDCs following exposure to micelles.
  • BMDCs were incubated for 4 hours with micelles or with E-OVA323 peptide or medium only as negative controls.
  • OVA323 concentrations were (from left to right) 270, 90, 30, 10, 3.3 and 1.1 nM and the plain ELP micelles were added in the same polypeptide concentration range as the other samples.
  • the BMDCs were subsequently analysed with flow cytometry;
  • Figure 28 shows proliferation of OT-II cells in vitro.
  • BMDCs were incubated for 4 hours with micelles, peptide or with medium only.
  • OVA323 concentrations were (from left to right) 270, 90, 30, 10, 3.3 and 1.1 nM.
  • the BMDCs were subsequently exposed for 3 days to OT-II cells containing fluorescent dye. The decrease of fluorescent signal in the OT-II population was then analysed with flow cytometry.
  • As negative controls BMDCs were pulsed with medium, plain ELP micelles or ELP/ELP-K micelles (containing 10% ELP-K).
  • ELP micelles mixed with free OVA323 peptide (“ELP/OVA323”) was included as a positive control.
  • the polypeptide concentrations of ELP and ELP-K and ELP/OVA323 was 2.7 pM, matching the highest polypeptide concentration of the covalent, coiled coil and hybrid groups;
  • Figure 29 shows proliferation of OT-II cells in vitro.
  • [OVA323] 270 nM (top), 90 nM (middle) and 30 nM (bottom).
  • BMDCs were incubated for 4 hours with micelles, peptide or with medium only. The BMDCs were subsequently exposed for 3 days to OT-II cells containing fluorescent dye. The decrease of fluorescent signal in the OT-II population was then analysed with flow cytometry;
  • Figure 33 shows expression, purification and identification of ELP-Bet v 1 .
  • the samples were analysed on 10% SDS polyacrylamide gels with Coomassie Blue staining.
  • ELP-Bet v 1 is clearly visible at around 60 kDa. In both gels, the black arrow marks the location of the purified protein, top)
  • Expression and purification by immune-affinity chromatography Lanes marked with - and + are before and after adding IPTG; Lys is lysate; FT is flow through; W1-3 refer to wash fractions; E1-4 refer to elution fractions, bottom left) Further purification by ITC.
  • P is pellet; S is supernatant; hot spin is the centrifugation step at 22 °C; cold spin is the centrifugation step at 4 °C. bottom right);
  • Figure 34 shows RP-HPLC analysis of ELP-Bet v 1. Gradient of 0 to 90% acetonitrile in water containing 0.1 % TFA. Trace was normalized to match the peak height of ELP;
  • FIG 35 shows endotoxin level determination using mTLR4 HEK reporter cell assay.
  • ELP contained 0.38 EU LPS/mg and ELP/ELP-Bet v 1 contained 0.11 EU LPS/mg
  • Figure 36 shows Inverse transition behaviour of ELP-Bet v 1 and ELP/ELP-Bet v 1 compared to ELP.
  • [polypeptide] 10 pM; SLS measured in 10 mM PB pH 7.8 as a function of temperature. The CMT was determined as the temperature value above which the count rate is stable: ⁇ 22 °C (ELP), 24 °C (ELP/ELP-Bet v 1) and ⁇ 28 °C (ELP-Bet v 1);
  • FIG. 37 shows CMC determination of ELP-Bet v 1 (left) and 9:1 ELP/ELP-Bet v 1 (right). Measured in 10 mM PB pH 7.8 at 37 °C. Count rate was measured by SLS as a function of [ELP- Bet v 1] or [ELP/ELP-Bet v 1], Count rates were normalized to the count rate of the buffer. Gray data points represent samples that did not contain particles according to the autocorrelation functions; black data points represent the samples for which the autocorrelation functions had a sigmoidal shape. The CMC was determined to be 1.1 pM (ELP-Bet v 1) and 0.22 pM (ELP/ELP- Bet v 1) respectively by calculating the intercept of the trend lines;
  • Figure 38 shows TEM image of ELP/ELP-Bet v 1 particles. Grid was prepared with 37 °C 20 pM polypeptide solution in water and stained with 1% uranyl acetate;
  • Figure 40 shows rapid micellization of ELP and ELP/ELP-Bet v 1 upon dilution of cold sample into 37 °C TB.
  • Figure 41 shows IgE binding capacities of Bet v 1 -displaying micelles.
  • the samples were diluted in 10 mM PB, 280 mM sucrose pH 7.4 to various concentrations and incubated with a diluted serum pool of 36 birch pollen allergic patients. Then, unbound IgE was captured by immobilized Bet v 1 and subsequently measured on a Phadia 250 device;
  • Figure 42 shows Circular dichroism (CD) analysis of ELP-Bet v 1. Each sample was measured at 25 °C (solid lines) and 37 °C (dashed lines);
  • Figure 43 shows mediator release of patients 1-10.
  • Rat basophil cells transfected with human IgE receptor were sensitized with human sera from birch pollen allergic patients. The cells were then stimulated with the respective antigen (Bet v l , plain ELP micelles mixed with Bet v l “ELP/Bet v 1”, ELP/ELP-Bet v 1 and ELP-Bet v 1) in concentrations ranging from 80 pg/mL to 0.024 fg/mL.
  • the release of mediator p-hexosaminidase was measured using the fluorogenic substrate 4-methyl umbelliferyl-N-acetyl-beta-D-glucosaminide. The level of mediator release in the absence of antigen is shown with a dashed line;
  • Figure 44 shows hypoallergenicity of ELP/ELP-Bet v 1 and ELP-Bet v 1.
  • Rat basophil cells transfected with human IgE receptor were sensitized with human sera from birch pollen allergic patients. The cells were then stimulated with the respective antigen (Bet v 1 , plain ELP micelles mixed with Bet v 1 “ELP/Bet v 1”, ELP/ELP-Bet v 1 and ELP-Bet v 1) in concentrations ranging from 80 pg/mL to 0.024 fg/mL.
  • the release of mediator p-hexosaminidase was measured using the fluorogenic substrate, 4-methyl umbelliferyl-N-acetyl-beta-D-glucosaminide.
  • Figure 45 shows immunization protocol. Mice were immunized with 36 pg Bet v 1on day 0 by subcutaneous injection followed by booster immunizations on days 7 and 14 (red arrows). Blood samples were taken for serum immunoglobulin analyses on days -1 , 6, 13 and 20. The mice received intranasal BPE challenges to further boost immunoglobulin production on days 28, 29 and 30. At day 31 the mice were sacrificed;
  • Figure 46 shows Serum immunoglobulin levels in immunized mice of pilot study. Bet v 1 specific lgG1 (a) and lgG2a (b) at different time points, c) Bet v 1 specific IgE levels at day 31.
  • the dotted lines represent average endpoint lgG1 , lgG2a and IgE levels of a buffer control group from other immunogenicity experiments to indicate the background signal;
  • Figure 47 shows Cytokine expression in re-stimulated lymph node cultures from pilot study. Expression of IL-4 (a), IL-5 (b), IL-10 (c), IL-13 (d), IFN-y (e) and IL-17 (f) in lung draining lymph nodes, stimulated ex vivo with Bet v 1 . * p ⁇ 0.05, ** p ⁇ 0.01
  • Figure 48 shows Serum immunoglobulin levels in immunized mice. Bet v 1 specific lgG1 (a) and lgG2a (b) at different time points, c) Bet v 1 specific IgE levels at day 31 ;
  • Figure 49 shows Cytokine expression in re-stimulated lymph node cultures. Expression of IL-4 (a), IL-5 (b), IL-10 (c), IL-13 (d), IFN-y (e) and IL-17 (f) in lung draining lymph nodes, stimulated ex vivo with Bet v 1 . * p ⁇ 0.05, ** p ⁇ 0.01 ;
  • compositions provided herein include micelles that include elastin-like peptides.
  • Elastin-like- polypeptides are a genetically engineered polypeptide with unique phase behavior (see for e.g. S. R. MacEwan, et al., Biopolymers 94(1) (2010) 60-77) which promotes recombinant expression, protein purification and self-assembly of nanostructures (see for e.g. A. Chilkoti, et al., Advanced Drug Delivery Reviews 54 (2002) 1093-1111).
  • ELPs have potential advantages over chemically synthesized polymers. First, because they are biosynthesized from a genetically encoded template, ELPs can be made with precise molecular weight.
  • ELP biosynthesis produces very complex amino acid sequences with nearly perfect reproducibility. This enables very precise selection of the location of attachment of additional moieties, such as an immunomodulator. Thus an additional moiety can be selectively placed on the corona of an ELP micelle, buried in the core of an ELP micelle, or dispersed equally throughout the polymer.
  • ELP can self-assemble into multivalent nanoparticles.
  • ELP are designed from native amino acid sequences found extensively in the human body they are biodegradable, biocompatible, and tolerated by the immune system.
  • ELPs undergo an inverse phase transition temperature, T t , above which they phase separate into structures such as micelles.
  • ELPs are artificial polypeptides composed of repeated pentapeptide sequences, (VPGXG) n (SEQ ID. NO: 11) derived from human tropoelastin, where X is a “guest residue” which is any amino acid.
  • This peptide motif displays rapid and reversible de-mixing from aqueous solutions above a transition temperature, T t .
  • T t transition temperature
  • ELPs adopt a highly water soluble random coil conformation; however, above T t , they separate from solution, coalescing into a second aqueous phase.
  • the T t of ELPs can be tuned by choosing the guest residue and ELP chain length as well as fusion peptides at the design level (see for e.g.
  • ELP phase is both biocompatible and highly specific for ELPs or ELP fusion proteins, even in complex biological mixtures.
  • Genetically engineered ELPs are monodispersed, biodegradable, and non-toxic. Throughout this description, ELPs are identified by the single letter amino acid code of the guest residue followed by the number of repeat units, n. N may be any number, for example n may be from 1 to 100.
  • I48S48 represents a diblock copolymer of ELP comprising 48 isoleucine (I) pentamers (VPGIG)48 (SEQ ID. NO: 12) at the amino terminus and 48 serine (S) pentamers (VPGSG)48, (SEQ ID. NO: 13) at the carboxy terminus.
  • VPGIG isoleucine
  • S serine
  • VPGSG 48 serine
  • VPGSG 48, (SEQ ID. NO: 13) at the carboxy terminus.
  • a “diblock” refers to an ELP with two blocks or structural units of repeated polypeptide sequence.
  • the diblock (VPGIG)4S (VPGSG)48 (SEQ ID. NO: 6) comprises 48 repeated units of a polypeptide having the sequence VPGIG (SEQ ID. NO: 14) and 48 repeated units of a polypeptide having the sequence VPGSG (SEQ ID NO: 15).
  • the ELPs include polymeric or oligomeric repeats of the pentapeptide VPGXG (SEQ ID NO: 16), where the guest residue X is any amino acid.
  • X may be a naturally occurring or non-naturally occurring amino acid.
  • the guest residue X may be a non-classical (non-genetically encoded) amino acid.
  • non-classical amino acids include: D-isomers of the common amino acids, 2, 4-diaminobutyric acid, a-amino isobutyric acid, A-aminobutyric acid, Abu, 2-amino butyric acid, y-Abu, c-Ahx, 6- amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t- butylalanine, phenylglycine, cyclohexylalanine, p-alanine, fluoro-amino acids, designer amino acids such as p-methyl amino acids, C a-methyl amino acids
  • Selection of X is independent in each ELP structural unit (e.g., for each structural unit defined herein having a guest residue X).
  • X may be independently selected for each structural unit as an amino acid having a positively charged side chain, an amino acid having a negatively charged side chain, or an amino acid having a neutral side chain, including in some examples, a hydrophobic side chain.
  • the structural units, or in some cases polymeric or oligomeric repeats, of the ELP sequences may further include (e.g. at the N- or C- terminus of each structural unit) one or more amino acid residues that do not eliminate the overall effect of the molecule, that is, in imparting certain improvements to the immunomodulator component as described.
  • such one or more amino acids also do not eliminate or substantially affect the phase transition properties of the ELP component (relative to the deletion of such one or more amino acids).
  • the ELP may have a sequence MX 1 (VPGX hb G)48(VPGX hp G)4sX 2 (SEQ ID NO: 3), wherein X 1 and X 2 are each any amino acid and wherein X hp is any hydrophilic amino acid residue and X hb is any hydrophobic amino acid residue.
  • X hp is serine.
  • X hb is isoleucine.
  • the ELP diblock polymer may have the sequence MG(VPGX hb G) n (VPGX hp G) n Y (SEQ ID NO: 4).
  • the methionine residue at the N-terminal of the ELP may not be included. For example, when the ELP is synthetically synthesised.
  • the T t of the ELP component can be modified by varying ELP chain length, as the T t generally increases with decreasing MW.
  • the hydrophobicity scale developed by Urry et al. (WO9632406, which is hereby incorporated by reference in its entirety) is preferred for predicting the approximate T t of a specific ELP sequence.
  • ELP component length can be kept relatively small, while maintaining a target T t , by incorporating a larger fraction of hydrophobic guest residues (e.g., amino acid residues having hydrophobic side chains) in the ELP sequence.
  • T t of the ELP is affected by the identity and hydrophobicity of the guest residue, X
  • additional properties of the molecule may also be affected. Such properties include, but are not limited to solubility, bioavailability, persistence, and half-life of the molecule.
  • the ELP is a diblock copolymer having a first ELP structural unit (i.e. a first block) that includes a guest residue (X) that is a hydrophobic amino acid residue and a second ELP structural unit (i.e. second block) that includes a guest residue (X) that is a hydrophilic amino acid residue.
  • the ELP component includes a first hydrophobic unit and second hydrophilic unit thus providing an amphiphilic ELP molecule.
  • Hydrophobic amino acid refers to an amino acid having a side chain that is uncharged at physiological pH and that is repelled by aqueous solution.
  • examples of genetically encoded hydrophobic amino acids are glycine, alanine, isoleucine, leucine, valine proline, phenylalanine, methionine, and tryptophan.
  • An example of a non-genetically-encoded hydrophobic amino acid is t-BuA.
  • Hydrophobic amino acids include amino acids having aromatic or nonpolar side chains.
  • Hydrophilic amino acids include amino acids having acidic, basic or polar side chains. "Hydrophilic amino acid” refers to an amino acid having a side chain that is attracted by aqueous solution. Examples of genetically encoded hydrophilic amino acids are, serine, arginine, lysine, glutamine, aspartic acid, glutamic acid, asparagine, and histidine. Examples of non-genetically- encoded hydrophilic amino acids are Cit and hCys.
  • the ELP may comprise the sequence (VPGX hb G) n (VPGX h PG) n (SEQ ID NO: 1) or (VPGX h PG) n (VPGX hb G) n (SEQ ID NO: 2) wherein X hp is any hydrophilic amino acid residue and X hb is any hydrophobic amino acid residue.
  • X hp is serine.
  • X hb is isoleucine.
  • Amphiphilic ELPs self-assemble into spherical micelles above the critical micelle temperature (CMT) and/or at a critical micelle concentration (CMC). ELP assembly is induced by hydrophobic collapse of the hydrophobic ELP block, forming the micelle core with the hydrophilic blocks pointing outwards in solution.
  • the ELP includes a sequence of (VPGIG)n(VPGSG) n (SEQ ID NO: 5).
  • the ELP has a sequence of MG(VPGIG)48(VPGSG) 48 Y (SEQ ID NO:7).
  • ELPs that make up the micelle may be covalently or non-covalently bound to additional moieties as described herein. Binding of immunomodulators and other specific additional moieties is described below in more detail.
  • additional moieties such as an immunomodulator, coiled coil forming peptide as described herein or any other additional moieties, may be bound so that they are configured to be located on the external surface of the micelle.
  • additional moieties (other than immunomodulators or intermediary linking molecules) may be configured to be located inside the micelle, for example, in the core of the micelle.
  • the additional moieties may be bound to the hydrophilic block.
  • the additional moiety may be bound to the C-terminal of the ELP.
  • the additional moiety may be bound to the N-terminal of the ELP.
  • the ELP may be covalently bound to the additional moiety by recombinant fusion methods, for example, by providing an ELP additional moiety fusion protein.
  • a recombinantly-produced ELP fusion protein may include the ELP and the additional moiety associated with one another by genetic fusion.
  • the fusion protein may be generated by translation of a polynucleotide encoding the therapeutic component cloned in-frame with the ELP (or vice versa).
  • the ELP and the additional moieties can be fused using a linker peptide of various lengths to provide greater physical separation and allow more spatial mobility between the fused portions, and thus maximize the accessibility of the additional moieties, for instance, for binding to its cognate receptor.
  • the linker peptide may consist of amino acids that are flexible or rigid.
  • a flexible linker may include amino acids having relatively small side chains, and which may be hydrophilic.
  • the flexible linker may contain a stretch of glycine and/or serine residues.
  • More rigid linkers may contain, for example, more sterically hindering amino acid side chains, such as (without limitation) tyrosine or histidine.
  • the linker may be less than about 50, 40, 30, 20, 10, or 5 amino acid residues.
  • the linker can be covalently linked to and between an ELP and an additional moiety, for example, via recombinant fusion.
  • the linker or peptide spacer may be protease-cleavable or non-cleavable.
  • cleavable peptide spacers include, without limitation, a peptide sequence recognized by proteases (in vitro or in vivo) of varying type, such as Tev, thrombin, factor Xa, plasmin (blood proteases), metalloproteases, cathepsins (e.g., GFLG, etc.), and proteases found in other corporeal compartments.
  • the fusion protein may be inactive, less active, or less potent as a fusion, which is then activated upon cleavage of the spacer in vivo.
  • a non-cleavable spacer may be employed.
  • the non-cleavable spacer may be of any suitable type known in the art.
  • the ELP may be covalently bound to an additional moiety via chemical conjugation.
  • the conjugates can be made by chemically coupling an ELP to an additional moiety by any number of methods well known in the art (See e.g. Nilsson et al., 2005, Ann Rev Biophys Bio Structure 34: 91-118).
  • the chemical conjugate can be formed by covalently linking the additional moiety to the ELP, directly or through a short or long linker moiety, through one or more functional groups on the additional moiety, e. g., amine, carboxyl, phenyl, thiol or hydroxyl groups, to form a covalent conjugate.
  • Various conventional linkers can be used, e.
  • a micelle that includes ELP covalently bound to an additional moiety.
  • the additional moiety may be conjugated via click chemistry.
  • Click chemistry refers to a chemical approach to conjugation introduced by Sharpless in 2001 and describes chemistry tailored to generate substances quickly and reliably by joining units together. See, e.g., Kolb, Finn and Sharpless Angewandte Chemie International Edition 2001 40, 2004-2021; Evans, Australian Journal of Chemistry 2007 60, 384-395).
  • Exemplary coupling reactions include, but are not limited to, formation of esters, thioesters, amides (e.g., such as peptide coupling) from activated acids or acyl halides; nucleophilic displacement reactions (e.g., such as nucleophilic displacement of a halide or ring opening of strained ring systems); azide-alkyne Huisgen cycloaddition; thiol-yne addition; imine formation; Michael additions (e.g., maleimide addition reactions); and Diels-Alder reactions (e.g., tetrazine [4+2] cycloaddition).
  • nucleophilic displacement reactions e.g., such as nucleophilic displacement of a halide or ring opening of strained ring systems
  • azide-alkyne Huisgen cycloaddition thiol-yne addition
  • imine formation Michael additions (e.g., maleimide addition reactions)
  • click chemistry reactions and click-chemistry handles can be found in, e.g., Kolb, H. C.; Finn, M. G. and Sharpless, K. B. Angew. Chem. Int. Ed. 2001 , 40, 2004-2021. Kolb, H. C. and Sharless, K. B. Drug Disc. Today, 2003, 8, 112-1137; Rostovtsev, V. V.; Green L. G.; Fokin, V. V. and Shrapless, K. B. Angew. Chem. Int. Ed. 2002, 41 , 2596-2599; Tomoe, C. W.; Christensen, C. and Meldal, M. J. Org. Chem. 2002, 67, 3057-3064.
  • the micelle may include ELPs non-covalently bound to an additional moiety.
  • the ELP may be directly or indirectly (i.e. via an intermediary binding molecule) bound to an additional moiety via hydrogen bonding, ionic bonding, or Van der Waals forces.
  • the ELP may be bound to an immunomodulator as described herein via a intermediatory moiety such as a coiled coil forming peptide as described herein.
  • the intermediatory moiety may be covalently bound to the ELP as described herein and mediate non-covalent binding to an additional moiety such as an immunomodulator that includes a cognate binding partner for the intermediatory moiety as described herein.
  • the ELPs may also be associated with a detectable label that allows for the visual detection of in vivo uptake of the ELPs.
  • Suitable labels include, for example, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, a detectable label sold under ALEXA-FLUOR®, stilbene, Lucifer Yellow, CASCADE BLUE®, and TEXAS RED®.
  • Other suitable optical dyes are described in Haugland, Richard P. (1996) MOLECULAR PROBESTM Handbook (which is a Guide to Fluorescent Probes and Labeling Technologies).
  • the ELPs described may be crosslinked.
  • Crosslinked ELPs may be formed by producing a dimer of an amphiphilic ELP as described herein.
  • Dimeric amphiphilic ELPs include two ELP molecules joined via a linker between the terminal of the hydrophobic block of each ELP.
  • Crosslinked ELPs may provide an altered CMT and/or CMC.
  • Methods of crosslinking may include chemically crosslinking ELPs. Any suitable agent that is capable of binding two amine groups (i.e. amine to amine crosslinkers may be used).
  • the ELPs may be crosslinked by reaction with agents such as bis(sulfosuccinimidyl)suberate (BS3), disuccinimidyl suberate (DSS), or N-hydroxysuccinimide esters (NHS esters).
  • the ELP such as an amphiphilic ELPs described herein may include one or more cysteine residues, for example at the hydrophobic terminus. This may allow for binding of two ELP molecules together via a disulphide bond (disulphide bridge).
  • crosslinked ELPs may be produced by recombinantly expressing dimeric amphiphilic ELPs. Such dimeric ELPs may include a protein linker expressed between each hydrophobic terminal. It will be understood that in the case that the ELP has a hydrophilic block at the N-terminal of the ELP, the ELPs in a dimer may be linked via the N-terminals of the ELPs.
  • the ELPs described herein may include modifications. Such modifications include acetylation, carboxylation, glycosylation, phosphorylation, lipidation and/or acylation. Numerous methods of modifying proteins are known in the art. For example, modifications may be added to ELPs in vivo by host cell post-translational modification systems or may be added in vitro by chemical or enzymatic based methods.
  • the ELPs described herein may be configured to self-assemble into micelles.
  • the micelles may include a mixture of ELPs as described herein.
  • the micelles described herein may include ELPs (without any additional moieties bound) and one or more ELPs each with one or more independently selected additional moiety attached.
  • ELPs and other recombinant proteins described herein can be prepared by expressing polynucleotides encoding the polypeptide sequences described herein in an appropriate host cell, i.e., a prokaryotic or eukaryotic host cell This can be accomplished by methods of recombinant DNA technology known to those skilled in the art. It is known to those skilled in the art that modifications can be made to any peptide to provide it with altered properties. Polypeptides described herein can be modified to include unnatural amino acids.
  • the peptides may comprise D-amino acids, a combination of D- and L-amino acids, and various “designer” amino acids (e.g., p-methyl amino acids, C-a-methyl amino acids, and N-a-methyl amino acids, etc.) to convey special properties to peptides. Additionally, by assigning specific amino acids at specific coupling steps, peptides with a-helices, turns, p sheets, a-turns, and cyclic peptides can be generated.
  • designer amino acids e.g., p-methyl amino acids, C-a-methyl amino acids, and N-a-methyl amino acids, etc.
  • the ELPs can be expressed and purified from a suitable host cell system.
  • suitable host cells include prokaryotic and eukaryotic cells, which include, but are not limited to bacterial cells, yeast cells, insect cells, animal cells, mammalian cells, murine cells, rat cells, sheep cells, simian cells and human cells.
  • Examples of bacterial cells include Escherichia coli, Salmonella enterica and Streptococcus gordonii.
  • the host cell is E. coli.
  • the cells can be purchased from a commercial vendor such as the American Type Culture Collection (ATCC, Rockville Md., USA) or cultured from an isolate using methods known in the art.
  • suitable eukaryotic cells include, but are not limited to 293T HEK cells, as well as the hamster cell line BHK-21 ; the murine cell lines designated NIH3T3, NSO, C127, the simian cell lines COS, Vero; and the human cell lines HeLa, PER.C6 (commercially available from Crucell) U-937 and Hep G2.
  • a non-limiting example of insect cells include Spodoptera frugiperda.
  • yeast useful for expression include, but are not limited to Saccharomyces, Schizosaccharomyces, Hansenula, Candida, Torulopsis, Yarrowia, or Pichia. See e.g., U.S. Pat. Nos. 4,812,405; 4,818,700; 4,929,555; 5,736,383; 5,955,349; 5,888,768 and 6,258,559.
  • the phase transition behaviour of the ELPs allows for easy purification.
  • the ELPs may be purified from host cells using methods known to those skilled in the art. These techniques involve the crude fractionation of the cellular milieu to polypeptide and non-polypeptide fractions. Having separated the polypeptide from other proteins, the polypeptide of interest may be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suited to the preparation of a pure peptide or polypeptide are filtration, ion-exchange chromatography, exclusion chromatography, polyacrylamide gel electrophoresis, affinity chromatography, or isoelectric focusing.
  • a particularly efficient method of purifying peptides is fast protein liquid chromatography or even HPLC.
  • protein purification may also be aided by the thermal transition properties of the ELP domain as described in U.S. Pat. No. 6,852,834.
  • purified will refer to a protein or peptide composition that has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity. Where the term “substantially purified” is used, this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the proteins in the composition.
  • the micelles provided herein include at least one immunomodulator.
  • Immunomodulator refers to a substance capable of altering (e.g., inhibiting, decreasing, increasing, enhancing or stimulating) the working of any component of the innate, humoral and/or cellular immune system of a mammal.
  • the immunomodulator may include at least one epitope.
  • An epitope also known as antigenic determinant, is the part of an antigen that is recognized by the immune system, for example by antibodies, B cells, or T cells.
  • the epitope is the molecular region of an antigen which is bound by the antibody, B cell or T cell.
  • epitopes are usually non-self-proteins, sequences derived from the host that can be recognized (as in the case of autoimmune diseases) are also epitopes.
  • Epitope includes any determinant capable of specific binding to an immunoglobulin or otherwise interacting with an immune system cell or molecule.
  • Epitopes generally consist of chemically active surface groupings of molecules such as amino acids or carbohydrate or sugar side chains and can have specific three-dimensional structural characteristics, as well as specific charge characteristics.
  • An epitope may be "linear” or “conformational.”
  • the epitope may be of any chemical nature, including without limitation, peptides, carbohydrates, lipids, glycopeptides and glycolipids.
  • the epitope may be at least substantially the same as a naturally occurring epitope. It may be identical to a naturally occurring epitope, or a modified form of a naturally occurring epitope.
  • linear epitope refers to an epitope with all of the points of interaction between the protein and the interacting molecule (such as an antibody) occur linearly along the primary amino acid sequence of the protein (continuous).
  • formational epitope refers to an epitope in which discontinuous amino acids that come together in three-dimensional conformation.
  • a conformational epitope In a conformational epitope, the points of interaction occur across amino acid residues on the protein that are separated from one another.
  • binding molecules such as antibodies
  • binding molecules for a particular target antigen will preferentially recognize an epitope on the target antigen in a complex mixture of proteins and/or macromolecules.
  • Regions of a given polypeptide that include an epitope can be identified using any number of epitope mapping techniques, well known in the art. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, New Jersey.
  • linear epitopes may be determined by concurrently synthesizing large numbers of peptides on solid supports, the peptides corresponding to portions of the protein molecule, and reacting the peptides with antibodies while the peptides are still attached to the supports.
  • Such techniques are known in the art and described in, e.g., U.S. Patent No. 4,708,871 ; Geysen et al., (1984) Proc. Natl. Acad. Sci. USA 8:3998-4002; Geysen et al., (1985) Proc. Natl. Acad. Sci. USA 82:78-182; Geysen et al., (1986) Mol. Immunol. 23:709-715.
  • conformational epitopes are readily identified by determining spatial conformation of amino acids such as by, e.g., hydrogen/deuterium exchange, x-ray crystallography and two-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols, supra.
  • Antigenic regions of proteins can also be identified using standard antigenicity and hydropathy plots, such as those calculated using, e.g., the Omiga version 1.0 software program available from the Oxford Molecular Group. This computer program employs the Hopp/Woods method, Hopp et al., (1981) Proc. Natl. Acad. Sci USA 78:3824-3828; for determining antigenicity profiles, and the Kyte- Doolittle technique, Kyte et al., (1982) J. Mol. Biol. 157:105-132; for hydropathy plots.
  • the epitope may be referred to by the source that it has been derived from/from which it originates.
  • the epitope may be selected from one or more of: an allergenic epitope, a viral epitope, a bacterial epitope, parasitic epitope, disease associated epitope, and a tumour associated epitope.
  • an “allergenic epitope” refers to any antigenic determinant/component part of (or derived from) an allergen (the antigen) that is capable of specific binding to an immunoglobulin or otherwise interacting with an immune system molecule or cell (e.g. T cell or B cell).
  • an allergenic epitope may be a linear or conformational epitope that is a (small) part of the allergen.
  • the epitope may be present within a longer sequence (for example, the a larger part, or all of the allergen may be present).
  • a “viral epitope” refers to any antigenic determinant/component part of (or derived from) a virus (the antigen) that is capable of specific binding to an immunoglobulin or otherwise interacting with an immune system molecule or cell (e.g. T cell or B cell).
  • a viral epitope may be a linear or conformational epitope that is a (small) part of a viral protein for example.
  • the epitope may be present within a longer sequence (for example, the a larger part, or all of a viral protein may be present).
  • a “bacterial epitope” refers to any antigenic determinant/component part of (or derived from) bacteria (the antigen) that is capable of specific binding to an immunoglobulin or otherwise interacting with an immune system molecule or cell (e.g. T cell or B cell).
  • a bacterial epitope may be a linear or conformational epitope that is a (small) part of a bacterial protein for example.
  • the epitope may be present within a longer sequence (for example, a larger part, or all of a bacterial protein may be present).
  • tumour-associated epitope can be an epitope from a tumour associated antigen (TAA).
  • TAA tumour associated antigen
  • the epitope may be an epitope derived from a naturally occurring antigen or epitope.
  • a derived epitope may differ from a naturally occurring epitope by 1 , 2, 3, 4, or more amino acid residues, as compared to the native epitope.
  • These modifications as compared to a naturally occurring epitope may independently be selected from: a substitution of an amino acid as compared to the original sequence; a deletion of an amino acid residue as compared to the original sequence; and introduction of an additional amino acid residue as compared to the original sequence.
  • the epitope may be recognised and bound by a B cell, immunoglobulin or antibody, for example.
  • Epitopes that are bound by B cells are typically at least five amino acids, more often at least six amino acids, still more often at least seven or eight amino acids in length, that may be continuous (“linear”) or discontinuous (“conformational”) (the latter being formed by the folding of a protein to bring non-contiguous parts of the primary amino acid sequence into physical proximity).
  • B-cells may also bind to carbohydrate epitopes.
  • the epitope may be recognised and bound by a T cell (via a T cell receptor for example). Epitopes that are recognised by T cell receptors are typically presented to the T cell receptor in the context of class I or class II MHC molecules.
  • the class I epitopes are usually 8 to 15, more often 9-11 amino acids in length.
  • the class II epitopes are usually 5-24 (a 24 mer is the longest peptide which can fit in the Class II groove), more often 8-24 amino acids.
  • Carbohydrate epitopes (as small as a single sugar unit (e.g., Tn)) may also be recognised by T cells. They are preferably no larger than five sugars. Many such epitopes are known.
  • the epitope is derived from or part of an allergenic antigen.
  • the immunomodulator may comprise an allergenic epitope.
  • the immunomodulator may comprise part of an allergen (which includes an allergenic epitope).
  • the immunomodulator may be an allergen (which includes an allergenic epitope).
  • Allergenic epitopes refer to an epitope derived from an antigen that causes an allergic reaction (i.e. and allergen).
  • allergen encompasses "allergen extracts” and "allergenic epitopes”. Examples of allergens include but are not limited to pollens; house dust and dust mites; animal allergens; mold and fungus; insect bodies and insect venom; feathers; food; and drugs (e.g., penicillin).
  • the allergen from which the epitope is derived may be a Type I allergen. In some examples, the allergen may be a type II allergen.
  • Type I allergens are allergens that illicit a Type I allergic reaction.
  • Type I allergic reaction hypersensitivity
  • IgE immunoglobulin E
  • Pollen allergens include for example, grass pollen, tree pollen (e.g. birch, alder, hazel, olive, cypress, cedar, plane tree) and weed pollen (e.g. ragweed, mugwort, pellitory, Russian thistle, plantain, goosefoot).
  • tree pollen e.g. birch, alder, hazel, olive, cypress, cedar, plane tree
  • weed pollen e.g. ragweed, mugwort, pellitory, Russian thistle, plantain, goosefoot.
  • the allergen is birch pollen allergen.
  • the allergenic epitope is derived from or is a Bet v protein, for example, Bet v 1 , Bet v 2, Bet v 3, Bet v 4, Bet v 5, Bet v 6, or Bet v 7.
  • the allergenic epitope includes a Bet v 1 epitope, is derived from or is Bet v 1 .
  • exemplary pollen allergens that the epitope may be (derived) from include Amb a 1 family of pectate lyases (e.g., UniProt accession numbers P27759, P27760 , P27761 , P27762 from Ambrosia artemisiifolia (short ragweed)); the defensin-like Art v 1 family (e.g., from mugwort and feverfew, e.g., UniProt ace. no.
  • Amb a 1 family of pectate lyases e.g., UniProt accession numbers P27759, P27760 , P27761 , P27762 from Ambrosia artemisiifolia (short ragweed)
  • the defensin-like Art v 1 family e.g., from mugwort and feverfew, e.g., UniProt ace. no.
  • Dust mite allergens that the epitope may be (derived) from include, for example, dust mite protein Der f 1‘, Der f 2, Der p 1 , Der p 2, Der p 5, Der p 7, dust mite protein Der p 10, Der p 11 , Der p 20, Der p 21 , Der p 23, Bio t 5, Bio 1 10, Bio 1 21 , from Dermatophagoides pteronyssinus, Dermatophagoides farinae, and Blomia tropicalis, respectively.
  • Animal allergens may occur in dander, feathers, hair, saliva, and excretions (e.g., urine) from animals such as cat, dog, camel, chinchilla, cow, deer, gerbil, goat, guinea pig, hamster, hog, horse, mohair, monkey, mouse, rabbit, sheep.
  • excretions e.g., urine
  • animals such as cat, dog, camel, chinchilla, cow, deer, gerbil, goat, guinea pig, hamster, hog, horse, mohair, monkey, mouse, rabbit, sheep.
  • domesticated animals such as cats (Fells domesticus) and dogs (Canis lupus familiaris) are common sources of allergy.
  • Fel d 1 UniProt acc. no. P30438 (chain 1); UniProt acc. no. P30440 (chain 2)
  • Fel d 3 and Fel d 4 are major cat allergens.
  • Can f 1 UniProt
  • mice e.g., Mus musculus
  • rats e.g., Rattus norvegicus
  • rabbits e.g. European rabbit [Oryctolagus cuniculus]
  • Identified allergens include, e.g., Mus m 1 , Rat n 1 , and Ory c 1 , in these species, respectively.
  • Fungi e.g., fungal spores or fragments (e.g., hyphal fragments)
  • Alternaria e.g., Alternaria alternata (Altemaria rot fungus)
  • Cladosporium e.g., Cladosporium herbarum, Cladosporium cladosporioides
  • Aspergillus e.g., Aspergillus fumigatus, Aspergillus niger
  • Fusarium Penicillium are exemplary allergenic fungi of interest.
  • an allergen the epitope is derived from comprises a protein found in or produced by Alternaria, Cladosporium, Aspergillus, Fusarium, Penicillium, or other fungus.
  • an allergen the epitope is derived from can comprise an Alt a, Asp a, Asp n, Cla or Pen protein.
  • Cockroach allergens are significant causes of allergy in many areas of the world.
  • Cockroach species include, for example, Blattella germanica (German cockroach) and Periplaneta americana (American cockroach), and Blatta orientalis (Oriental cockroach)
  • Cockroach allergens include, for example, Bia g 1 , Bia g 2, Bia g 5, Bia g 5, Bia g 6, Bia g 7, and Bia g 8 (from B. germanica) and Per a 1 , Per a 3, Per a 6, Per a 7, Per a 9, and Per a 10 (from P. Americana).
  • an allergen comprises an epitope derived from a Bia g, Per a, or Bia o allergen.
  • Ant, moths, fleas, flies e.g., house fly, horse fly, mayfly
  • mosquitos are also sources of allergens.
  • an allergen the epitope may be derived from is a cockroach, ant, moth, flea, fly, or mosquito protein.
  • Insect venoms (e.g., from insects of the order Plymenoptera, e.g., bees, hornets, or wasps) that are potential causes of severe allergic reactions include venoms from European Hornet (Vespa crabr ), Honey Bee (Apis mellifera), Hornet (Dolichovespula spp.), Paper Wasp (Polistes spp.), Yellow Jacket (Vespula spp.), White (Bald)-faced Hornet (Dolichovespula maculata), Yellow Hornet (Dolichovespula arena ria).
  • European Hornet Vespa crabr
  • Honey Bee Apis mellifera
  • Hornet Dolichovespula spp.
  • Paper Wasp (Polistes spp.)
  • Yellow Jacket Vespula spp.
  • White (Bald)-faced Hornet Dolichovespula maculata
  • Yellow Hornet Dolich
  • an allergen the epitope may be derived from is a venom (or extract or component thereof) of a bee, wasp, or hornet.
  • an allergen the epitope may be derived from can comprise an Api, Doi, or Ves protein.
  • Food allergens that the epitope may be (derived) from include, but are not limited to proteins in legumes such as peanuts, peas, lentils and beans, as well as the legume-related plant lupin, tree nuts such as almond, cashew, walnut, Brazil nut, filbert/hazelnut, pecan, pistachio, beechnut, butternut, chestnut, chinquapin nut, coconut, ginkgo nut, lychee nut, macadamia nut, nangai nut and pine nut, egg, fish, shellfish such as crab, crawfish, lobster, shrimp and prawns, molluscs such as clams, oysters, mussels and scallops, milk, soy, wheat, gluten, corn, meat such as beef, pork, mutton and chicken, gelatine, sulphite, seeds such as sesame, sunflower and poppy seeds, and spices such as coriander, garlic and mustard, fruits, vegetables such as celery, and rice.
  • pollens can be collected from the respective plants, which may be cultivated or in the wild.
  • Fungal extracts can be produced from pure culture mycelial mats or allergens can be isolated from culture medium.
  • Rusts and smuts can be obtained from natural growths.
  • Epithelial extracts can be produced from the hide, hair, or feathers containing the natural dander, or from separated dander.
  • Insect and mite extracts can be produced from the whole body of the insects or mite, respectively.
  • venom or venom-containing organs can be isolated or a whole-body extract can be used.
  • House dust can he made from various dusts ordinarily found in the home (e.g., upholstery dust, mattress dust, or general dust accumulating on surfaces). Other dusts (e.g., grain dust, wood dust, cotton dust) can be collected from the appropriate location. Food extracts can be prepared from the edible portions of the respective foods, e.g., freshly obtained foods.
  • source allergen material e.g., pollen, insect, dander
  • source allergen material can be subjected initially to pulverization, drying, defatting (by extraction using organic solvent), or other steps as appropriate for the particular allergen.
  • Centrifugation can be used, e.g., to separate solid or particulate matter.
  • Resulting material can be incubated in an aqueous medium (e.g., water or suitable buffered solution, e.g., ammonium bicarbonate, phosphate buffered saline, etc.) for a suitable period of time to at least partly solubilize proteins.
  • Crude extract can be processed using, e.g., dialysis, filtration, fractionation, chromatography, etc.
  • Extracts of allergens specifically processed for safe use in human immunotherapy are available commercially.
  • GREER Laboratories Tnc. Allergy and Immunotherapy division publishes a brochure entitled “Human Allergy Products and Sendees” available on-line at the company website currently at: http://www.greerlabs.com/files/catalogs/HumanAllergyCatalog.pdf.
  • GREER also publishes a brochure entitled “Source Materials Products and Sendees” available online at the company website currently at -192017200098 06Jan 2017: http://www.greerlabs.com/files/catalogs/SourceMateiialsCatalog.pdf, which details available allergens that can be used as raw materials for production of allergen extracts or more highly purified allergen protein preparations. Both publications are incorporated herein by reference.
  • the epitope is derived from or part of a viral antigen.
  • the immunomodulator may comprise a viral epitope.
  • the immunomodulator may comprise part of a viral protein (which includes a viral epitope).
  • the immunomodulator may be a viral protein (which includes a viral epitope).
  • the epitope is derived from or part of a viral antigen, such as an epitope derived from an infectious virus.
  • Retroviridae e.g., human immunodeficiency viruses, such as HIV-1 (also referred to as HTLV-III, LAV or HTLV- lll/LAV, or HLV-III; and other isolates, such as HIV-LP; Picornaviridae (e.g., polio viruses, hepatitis A virus; enteroviruses, human coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g., strains that cause gastroenteritis); Togaviridae (e.g., equine encephalitis viruses, rubella viruses); Flaviridae (e.g., dengue viruses, encephalitis viruses, yellow fever viruses); Coronaviridae (e.g., coronaviruses); Rhabdoviridae (e.g., vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g., e
  • the epitope is derived from or part of a bacterial antigen.
  • the immunomodulator may comprise a bacterial epitope.
  • the immunomodulator may comprise part of a bacterial protein (which includes a bacterial epitope).
  • the immunomodulator may be a bacterial protein (which includes a bacterial epitope).
  • the epitope is derived from or part of a bacterial antigen, such as an epitope derived from an infectious bacteria.
  • infectious bacteria examples include: Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia, Mycobaderia sps (e.g. M. tuberculosis, M. avium, M. intracellulare, M. kansaii, M.
  • Streptococcus pyogenes Group A Streptococcus
  • Streptococcus agalactiae Group B Streptococcus
  • Streptococcus viridans group
  • Streptococcus faecalis Streptococcus bovis
  • Streptococcus anaerobic sps.
  • Streptococcus pneumoniae pathogenic Campylobacter spp., Enterococcus spp., Haemophilus influenzae, Bacillus antracis, Corynebaderium diphtheriae, Corynebacterium spp., Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridium tetani,
  • the epitope is derived from or part of a parasitic antigen.
  • the immunomodulator may comprise a parasitic epitope.
  • the immunomodulator may comprise part of a parasitic protein (which includes a parasitic epitope).
  • the immunomodulator may be a parasitic protein (which includes a parasitic epitope).
  • the epitope is derived from or part of a parasitic antigen, such as a protein from Hookworm (Necator americanus), Scabies mite (Sarcoptes scabiei var. hominis), Roundworm (Ascaris lumbricoides), Flatworm blood fluke (Schistosoma mansoni, S. haematobium, S. japonicum), Tapeworm (Taenia solium), Pinworm (Enterobius vermicularis), Toxoplasma gondii, Giardia lamblia or Entamoeba histolytica.
  • the epitope is a disease-associated epitope.
  • An epitope may be indirectly associated with a disease if the epitope is of an antigen which is specifically produced or overproduced by infected cells of the subject, or which is specifically produced or overproduced by other cells of the subject in specific, but non-immunological, response to the disease, e.g., an angiogenic factor which is overexpressed by nearby cells as a result of regulatory substances secreted by a tumour.
  • the term “disease-associated epitope” also includes any non-naturally occurring epitope which is sufficiently similar to an epitope naturally associated with the disease in question so that antibodies or T cells which recognize the natural disease epitope also recognize the similar nonnatural epitope.
  • An epitope may be said to be directly associated with a particular tumour if it is an intracellular, surface or secreted antigen that is present on/in said tumour. It need not be present on all cells of the tumour type in question, or on all cells of a particular tumour, or throughout the entire life of the tumour. It need not be specific to the tumour in question.
  • An epitope may be said to be “tumour-associated” in general if it is so associated with any tumour (cancer, neoplasm).
  • the tumour-associated epitope may be a tumor-associated antigen (TAA), or derived from a TAA.
  • TAA tumor-associated antigen
  • the epitope is derived from or part of a TAA.
  • the immunomodulator may comprise a TAA epitope.
  • the immunomodulator may comprise part of a TAA (which includes a tumour-associated epitope).
  • the immunomodulator may be a TAA (which includes a tumour-associated epitope).
  • Tumours may be of mesenchymal or epithelial origin. Cancers include cancers of the colon, rectum, cervix, breast, lung, stomach, uterus, skin, mouth, tung, lips, larynx, kidney, bladder, prostate, brain, and blood cells.
  • the epitope is more frequently associated with that tumour that with other tumours, or with normal cells.
  • there should be a statistically significant (p 0.05) difference between its frequency of occurrence in association with the tumour in question, and its frequency of occurrence in association with (a) normal cells of the type from which the tumour is derived, and (b) at least one other type of tumour.
  • An epitope may be said to be “tumour-specific” in general if it is associated more frequently with tumours (of any or all types) than with normal cells. It need not be associated with all tumours.
  • tumour specific epitope also includes any non-naturally occurring epitope which is sufficiently similar to a naturally occurring epitope specific to the tumour in question (or as appropriate, specific to tumours in general) so that antibodies or T cells stimulated by the similar epitope will be essentially as specific as cytotoxic T lymphocytes stimulated by the natural epitope.
  • tumour-versus-normal specificity is more important than tumour-versus-tumour specificity as (depending on the route of administration and the particular normal tissue affected) higher specificity generally leads to fewer adverse effects.
  • Tumour-versus-tumour specificity is more important in diagnostic as opposed to therapeutic uses.
  • Tumour-associated epitopes include, but are not limited to, peptide epitopes such as those of mutant p53, the point mutated Ras oncogene gene product, her 2/neu, c/erb2, and the MLIC1 core protein, and carbohydrate epitopes such as sialyl Tn (STn), TF, Tn, CA 125, sialyl Le x , sialyl Le a and P97.
  • peptide epitopes such as those of mutant p53, the point mutated Ras oncogene gene product, her 2/neu, c/erb2, and the MLIC1 core protein
  • carbohydrate epitopes such as sialyl Tn (STn), TF, Tn, CA 125, sialyl Le x , sialyl Le a and P97.
  • Carbohydrate epitopes are also of interest.
  • any of three types of tumour-associated carbohydrate epitopes which are highly expressed in common human cancers may be presented. These particularly include the lacto series type 1 and type 2 chains, cancer associated ganglio chains, and neutral glycosphingolipids.
  • lacto series Type 1 and Type 2 chains are as follows: Lewis a, dimeric Lewis a, Lewis b, Lewis b/Lewis a, Lewis x, Lewis, y, Lewis a/Lewis x.
  • cancer- associated ganglio chains are as follows: GM3. GD3, GM2, GM4, GD2, GM1 , GD-1a, GD-1b.
  • Neutral sphingolipids include globotriose, globotetraose, globopentaose, isoglobotriose, isoglobotetraose, mucotriose, mucotetraose, lactotriose, lactotetraose, neolactotetraose, gangliotriose, gangliotetraose, galabiose, and 9-O-acetyl-GD3.
  • Numerous antigens of clinical significance bear carbohydrate determinants.
  • One group of such antigens comprises the tumour- associated mucins (Roussel, et al., Biochimie 70, 1471 , 1988).
  • mucins are glycoproteins found in saliva, gastric juices, etc., that form viscous solutions and act as lubricants or protectants on external and internal surfaces of the body. Mucins are typically of high molecular weight (often >1 ,000,000 Dalton) and extensively glycosylated. The glycan chains of mucins are O-linked (to serine or threonine residues) and may amount to more than 80% of the molecular mass of the glycoprotein.
  • Mucins are produced by ductal epithelial cells and by tumours of the same origin, and may be secreted, or cell-bound as integral membrane proteins (Burchell, et al., Cancer Res., 47, 5476, 1987; Jerome, et al., Cancer Res., 51 , 2908, 1991).
  • the immunomodulator may be a peptide K molecule as described herein.
  • Peptide K is a coiled coil forming peptide as described herein, but as shown by the examples provided herein peptide K may act as an immunomodulator.
  • Peptide K binds parallel to membranes and due to cationic and amphiphilic properties peptide K may have cell penetrating peptide (CPP) activity.
  • CPP cell penetrating peptide
  • coiled coil domains of peptide K may interact with scavenger receptors from cells such as dendritic cells. Both membrane binding and CPP characteristics potentially increase the uptake of coiled coils and conjugates thereof into cells.
  • peptide K may act to alter immune response in a subject as well as acting as an intermediary binding molecule as described herein.
  • the micelles provided herein include ELP and an immunomodulator. As described above, additional moieties, such as immunomodulators may be attached to an ELP using a variety of methods.
  • the immunomodulator is attached to the ELP so that the immunomodulator is displayed on the outside of a micelle (i.e. the corona of the micelle). This allows for the immunomodulator and any epitopes thereof to be accessible to binding molecules of the immune system and thus lead to alterations in immune response.
  • the immunomodulator is bound to a hydrophilic block of an amphiphilic ELP.
  • the immunomodulator may be bound to the N-terminal of the ELP.
  • the immunomodulator may be bound to the C-terminal of the ELP.
  • the immunomodulator may be bound to the C-terminal carboxylic group of the ELP.
  • the orientation of the immunomodulator may be selected so that once attached, epitopes of the immunomodulator are accessible to immune system binding molecules.
  • the immunomodulator is a peptide or polypeptide the immunomodulator may be attached via the N- terminal, C-terminal or via sidechains of intrachain amino acids of the peptide or polypeptide.
  • an immunomodulator may be attached to the ELP molecule covalently.
  • an immunomodulator as described herein may be bound or attached to an ELP by recombinantly expressing the ELP and immunomodulator as a fusion protein.
  • the immunomodulator may be covalently attached an ELP via chemical methods as described herein.
  • ELP-immunomodulator fusion proteins As such, provided herein are ELP-immunomodulator fusion proteins.
  • nucleic acids encoding such fusion proteins may be provided herein.
  • the immunomodulator may be Bet v 1 and the ELP may be I48S48.
  • the Bet v 1 may be bound to the C-terminal ELP via the N-terminal amide group or C-terminal carboxylic group of the Bet v 1 peptide.
  • the ELP fusion protein may have a sequence of:
  • the immunomodulator is non-covalently bound to the ELP molecule.
  • Non- covalent binding strategies are well known such as specific ligand- and protein-binding methods such as biotin-avidin interaction, antigen-antibody interaction, Ni-NTA-hexahistidine interaction, aptamer-protein binding, and specific-sequence DNA and DNA-binding protein interactions.
  • Non- covalent attachment may allow for controllable reversible binding of an immunomodulator to the ELP.
  • the immunomodulator is bound via an intermediary binding molecule that may be covalently bound to the ELP molecule and a cognate of the intermediary binding molecule.
  • cognate binding partner and “cognate” refer to a second molecule, which specifically interacts with the intermediary binding molecule.
  • the “intermediary binding molecule” and “cognate binding partner” comprise a binding pair.
  • multiple intermediary binding molecules have the same cognate binding partner, and in some examples, multiple cognate binding partners bind the same intermediary binding molecule.
  • the ELP may be bound to a peptide that is configured to form a coiled coil motif with its cognate partner, the cognate partner being bound to the immunomodulator, thus providing a system that allows for the immunomodulator to be non-covalently attached to the ELP (i.e. via the intermediary binding molecule).
  • a coiled coil motif or coiled coil refers to a peptide/protein sequence usually with a contiguous pattern of hydrophobic residues spaced 3 and 4 residues apart, which assembles (folds) to form a multimeric bundle of helices.
  • the sequences, structures, and interactions of coiled-coils have been studied extensively and well documented in for example, Mason J.M., Arndt K.M. Coiled coil domains: Stability, specificity, and biological implications. Chembiochem. 2004;5:170-176. doi: 10.1002/cbic.200300781 ; Lupas A.N., Gruber M. The structure of a-helical coiled coils. Adv. Protein Chem.
  • each helix has a periodicity of seven (a heptad repeat), with anywhere from two (in designed coiled coils) to 200 of these repeats in a protein.
  • the helices are amphipathic, being encoded by a relatively straightforward sequence repeat of hydrophobic (H) and polar (P)residues, (HPPHPPP)n.
  • This repeat is usually denoted (a-b-c-d-e-f-g)n in one helix, and (a’-b’-c’-d’-e’-f’-g’)n in the other.
  • a helical diagram of a typical dimeric coiled coil shows that hydrophobic amino acid residues are placed in the positions of a and d, while polar residues are in the positions of e and g.
  • the hydrophobic-polar patterns of amino acid residues at the interface between coiled-coils directly influence the interaction between the helices.
  • the number of helices of a coiled coil (e.g., dimeric, trimeric, tetrameric or multimeric) is decided by packing in the hydrophobic core at the positions of a and d.
  • the ELP may be bound to a first coiled coil forming peptide
  • the immunomodulator may be bound to a second coiled coil forming peptide that is configured to form a coiled coil (complex) with the first coiled coil forming peptide
  • a micelle may include ELPs each having a different first coiled coil forming peptide allowing for multiple immunomodulators, each being bound to a respective cognate partner for each first coiled coil forming peptide.
  • the first coiled coil forming peptide may be multivalent (i.e. may be able to form a trimer, tetramer, or pentamer etc.) and configured to form a coiled coil with multiple cognate partners, thus allowing binding of multiple immunomodulators to a single ELP including the first coiled coil forming peptide.
  • Coiled coil forming peptides can form a homomeric coiled coil (i.e., wherein the first coiled coil forming peptide and any further coiled coil forming peptides are the same) or a heteromeric coiled coil (i.e. wherein the first coiled coil forming peptide and any further coiled coil forming peptides are different).
  • the coiled coil formed by the first and any further coiled coil forming peptides may be parallel or anti-parallel.
  • a knobs insert into a’-d’-g’- d’ holes, but in antiparallel coiled-coils, the hole consists of residues at e’-a’-d’-a’ positions (prime indicates positions on the opposing a-helix).
  • the first coiled coil peptide (i.e. , covalently bound to an ELP) may be a peptide comprising the sequence (KIAALKE)n (SEQ ID NO: 17) referred to herein as peptide K.
  • the cognate partner of peptide K may be a peptide comprising the sequence (EIAALEK)n (SEQ ID NO: 18) referred to herein as peptide E.
  • peptide E has the sequence “Ac-YG(EIAALEK)3-NH2” (SEQ ID NO: 19). In some examples, peptide K has the sequence “Ac-(KIAALKE)3GW-NH2” (SEQ ID NO: 20).
  • an ELP covalently bound to peptide K has the sequence: “MG(VPGIG)48(VPGSG)48YWSGGG(KIAALKE) 4 ” (SEQ ID NO: 9).
  • an ELP covalently bound to peptide E has the sequence: “MG(VPGIG)48(VPGSG)48YWSGGG(EIAALEK) 4 ” (SEQ ID NO: 10).
  • the ELP comprises peptide E and the immunomodulator comprises peptide K. In other examples, the ELP comprises peptide K and the immunomodulator comprises peptide E.
  • coiled coil peptide systems include GCN4 pLI, Leucine zipper peptide, JR2KC/JR2E, KV/KI/EV/EI, SVLP (SEQ ID NO: 23), SAGE (SEQ ID NO: 24), SAPN (SEQ ID NO: 25),CCE/CCK, and Helix A and Helix B.
  • Further examples of coiled coil peptide systems include those described in Lapenta, Fabio, et al. "Coiled coil protein origami: from modular design principles towards biotechnological applications.” Chemical Society Reviews 47.10 (2016): 3530-3542.
  • Coiled coil peptides may have a number of advantages such as being responsive to external stimuli, allowing controllable or tuneable release of moieties linked via such coiled coil peptides. For example, binding of the coiled coil peptide to its cognate peptide may be modulated by pH, temperature, and/or enzymatic processing.
  • compositions described herein include micelles that comprise ELPs as described herein.
  • the micelles may include a mixture of ELPs described herein.
  • the micelles may include ELPs (i.e. not bound to an immunomodulator or intermediary binding molecule as described herein), one or more ELPs covalently bound to an immunomodulator, one or more ELPs bound non-covalently bound to an immunomodulator, and/or one or more ELPs covalently bound to a coiled coil peptide (e.g. wherein the immunomodulator does not comprise a cognate of the coiled coil forming peptide).
  • Amphiphilic ELPs are capable of self-assembling into micelles when heated to the CMT of the ELPs or mixture thereof.
  • the CMT of ELPs or mixtures thereof may be determined by known methods.
  • the ELPs described herein may be configured to form micelles at a desired temperature.
  • the ELPs may be designed via selection of the number of repeat structural units and/or the guest residue in each unit to have a desired CMT.
  • the formation of micelles may also be altered by the amounts of different ELPs included in a micelle (i.e. , by the mixture of ELPs used).
  • the CMT may be understood as the temperature that the ELPs self-assemble into micelles.
  • the CMT of ELPs may be determined by any methods known in the art, for example by dynamic light scattering methods.
  • the CMT of ELPs described herein or mixture thereof may be at least 15°C. In some examples the CMT of ELPs described herein or mixture thereof may be at least 16°C. In some examples the CMT of ELPs described herein or mixture thereof may be at least 17°C. In some examples the CMT of ELPs described herein or mixture thereof may be from 15°C to 40°C. In some examples, the CMT of the ELPs described herein or mixture thereof may be from 16°C to 37°C. In some examples, the CMT of the ELPs described herein or mixture thereof may be from 16°C to 27°C. In some examples, the CMT of the ELPs described herein, or mixture thereof may be about 16°C to 26°C.
  • the CMT of the ELPs described herein or mixture thereof may be about 16°C, 17°C, 18°C, 19°C, 20°C, 21 °C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 21°C, 32°C, 33°C, 34°C, 35°C, 36°C, or 37°C.
  • the ELPs may be designed or selected so that micelles are formed and stabilised in a subject (e.g., a human).
  • the CMT may be less than 37°C, less than 36°C, less than 35°C, less than 34°C, less than 32°C, less than 31 °C, less than 30°C, less than 29°C, less than 28°C, less than 27°C, less than 26°C, or less than 25°C.
  • the ELPs described herein may be designed to have a CMT that allows for micelle formation in a subject (e.g., a human).
  • the ELPs described herein may also be designed or selected to have a CMC that is below a predetermined threshold. For example, a CMC below a known immunogenicity concentration or below a concentration that is toxic to a subject. CMC may be determined by known methods, such as static light scattering methods.
  • the ELPs or mixtures thereof may have a CMC of at least 0.004 pM.
  • the ELPs or mixture thereof may have a CMC from 0.05 to 1.2 pM.
  • the ELPs or mixture thereof may have a CMC from 0.1 to 1.1 pM.
  • the ELPs or mixture thereof may have a CMC less than 110 pM.
  • the micelles formed by the ELPs or mixture of ELPs described herein may have an average hydrodynamic diameter in the nanoscale. That is to say that the micelles may have an average hydrodynamic diameter of less than 1000 nm.
  • the micelles may have an average hydrodynamic diameter of less than 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 55, 50, 45, 40, 35, or 30 nm.
  • the micelles have average hydrodynamic diameter of from 10 nm to 1000 nm.
  • the micelles have average hydrodynamic diameter of from 10 nm to 100 nm.
  • the micelles have average hydrodynamic diameter of from 10 nm to 70 nm.
  • the micelles have average hydrodynamic diameter of from 30 nm to 70 nm. In some examples, the micelles have average hydrodynamic diameter of from 40 nm to 70 nm. In some examples, the micelles have average hydrodynamic diameter of from 50 nm to 60 nm.
  • the micelles formed by the ELPs or mixture of ELPs described herein may have an average polydispersity index from 0.001 to 0.5.
  • Polydispersity index refers to the ratio of “weight average molecular weight” to “number average molecular weight” for a particular polymer.
  • the polydispersity index represents the distribution of individual molecular weights within the polymer sample.
  • the ELPs may have an average polydispersity index from 0.001 to 0.2.
  • the ELPs may have an average polydispersity index less than 0.25.
  • the micelles may be monodispersed. Monodispersed refers to a composition including micelles that all have substantially same molecular weight and are not aggregated.
  • the micelles may be any shape.
  • the micelles may be substantially spherical.
  • the micelles formed by the ELPs or mixture of ELPs described herein may have an average zeta potential from -15 mV to 15 mV. In some examples, the ELPs may have an average zeta potential from -15 mV to -2 mV.
  • Zero potential refers to measured electrical potential of a colloidal particle in aqueous environment, measured with an instrument such as a Zetasizer 3000 using Laser Doppler microelectrophoresis under the conditions specified.
  • the zeta potential describes the potential at the boundary between bulk solution and the region of hydrodynamic shear or diffuse layer.
  • electrokinetic potential because it is the potential of the particles which acts outwardly and is responsible for the particle's electrokinetic behaviour.
  • concentration of a specific ELP described herein in a micelle may be from 1 to 100% of the total amount of ELP in a micelle.
  • a micelle comprising 10% of an ELP bound to an immunomodulator consists of at least 10% of ELPs bound to an immunomodulator and 90% or less ELPs not bound to immunomodulators (i.e. ELPs not bound to the immunomodulator, any other immunomodulator or any other additional moiety (such as an intermediary binding molecule).
  • the amount of an ELP bound to an immunomodulator that is present in a micelle is from 5 % to 100 % of the total amount of ELP in the micelle. In some examples, the amount is from 20 % to 100 % of the total amount of ELP in the micelle. In some examples, the amount is from 5 % to 50 % of the total amount of ELP in the micelle. In some examples, the amount is from 5 % to 20 % of the total amount of ELP in the micelle.
  • a micelle that includes 10% ELP bound to an immunomodulator and 90% of other ELPs not bound to the same or any immunomodulator.
  • a micelle may have 10% of ELP covalently bound to an immunomodulator and 90% of ELP comprising (VPGX hb G) n (VPGX hp G) n (SEQ ID NO: 1) or (VPGX hp G) n (VPGX hb G) n (SEQ ID NO: 2) not bound to an immunomodulator.
  • the ELP bound to the immunomodulator comprises ((VPGX hb G)n(VPGX h PG) n -immunomodulator) or (immunomodulator-(VPGX h PG) n (VPGX hb G) n ).
  • the ELP covalently bound to the immunomodulator and the ELP not bound to the immunomodulator is the ELP I48S48.
  • a micelle that includes 20% ELP bound to an immunomodulator and 80% of other ELPs not bound to an immunomodulator, for example, 20% of ELP covalently bound to an immunomodulator and 80% of an ELP comprising (VPGX hb G) n (VPGX h PG) n (SEQ ID NO: 1) or (VPGX h PG) n (VPGX hb G) n (SEQ ID NO: 2) not bound to an immunomodulator.
  • a micelle that includes 30% ELP bound to an immunomodulator and 70% of other ELPs not bound to an immunomodulator, for example, 30% of ELP covalently bound to an immunomodulator and 70% of an ELP comprising (VPGX hb G) n (VPGX h PG) n (SEQ ID NO:1) or (VPGX hp G) n (VPGX hb G) n (SEQ ID NO: 2) not bound to an immunomodulator.
  • a micelle that includes 40% ELP bound to an immunomodulator and 60% of other ELPs not bound to an immunomodulator, for example, 40% of ELP covalently bound to an immunomodulator and 60% of an ELP comprising (VPGX hb G) n (VPGX hp G) n (SEQ ID NO: 1) or (VPGX hp G)n(VPGX hb G) n (SEQ ID NO: 2) not bound to an immunomodulator.
  • a micelle that includes 50% ELP bound to an immunomodulator and 50% of other ELPs not bound to an immunomodulator, for example, 50% of ELP covalently bound to an immunomodulator and 50% of an ELP comprising (VPGX hb G) n (VPGX hp G) n (SEQ ID NO: 1) or (VPGX hp G)n(VPGX hb G) n (SEQ ID NO: 2) not bound to an immunomodulator.
  • a micelle that includes 60% ELP bound to an immunomodulator and 40% of other ELPs not bound to an immunomodulator, for example, 60% of ELP covalently bound to an immunomodulator and 40% of an ELP comprising (VPGX hb G) n (VPGX hp G) n (SEQ ID NO: 1) or (VPGX hp G)n(VPGX hb G) n (SEQ ID NO: 2) not bound to an immunomodulator.
  • a micelle that includes 70% ELP bound to an immunomodulator and 30% of other ELPs not bound to an immunomodulator, for example, 70% of ELP covalently bound to an immunomodulator and 30% of an ELP comprising (VPGX hb G) n (VPGX hp G) n (SEQ ID NO: 1) or (VPGX hp G)n(VPGX hb G) n (SEQ ID NO: 2) not bound to an immunomodulator.
  • a micelle that includes 80% ELP bound to an immunomodulator and 20% of other ELPs not bound to an immunomodulator, for example, 80% of ELP covalently bound to an immunomodulator and 20% of an ELP comprising (VPGX hb G) n (VPGX hp G) n (SEQ ID NO: 1) or (VPGX hp G)n(VPGX hb G) n (SEQ ID NO: 2) not bound to an immunomodulator.
  • a micelle that includes 90% ELP bound to an immunomodulator and 10% of other ELPs not bound to an immunomodulator, for example, 90% of ELP covalently bound to an immunomodulator and 10% of an ELP comprising (VPGX hb G) n (VPGX hp G) n (SEQ ID NO: 1) or (VPGX hp G)n(VPGX hb G) n (SEQ ID NO: 2) not bound to an immunomodulator.
  • a micelle that includes 100% ELP bound to an immunomodulator, for example, 100% of an ELP comprising (VPGX hb G) n (VPGX hp G) n (SEQ ID NO: 1) or (VPGX hp G) n (VPGX hb G) n (SEQ ID NO: 2) covalently bound to an immunomodulator.
  • the ELP bound to the immunomodulator comprises “(VPGX hb G) n (VPGX hp G) n -immunomodulator” or “immunomodulator-(VPGX hp G)n(VPGX hb G) n ”.
  • the ELP covalently bound to the immunomodulator and the ELP not bound to the immunomodulator is the ELP I48S48.
  • the micelle may comprise or consist of the ELPs “(VPGIG)4s(VPGSG)48- immunomodulator” and the ELP I48S48. This may be referred to herein as a coiled coil micelle (i.e. a micelle that includes an ELP species covalently bound to an immunomodulator).
  • the ELP is covalently bound to an intermediary binding molecule, which allows for non-covalent binding of an immunomodulator with a cognate partner.
  • a micelle that includes 5% ELP covalently bound to an intermediary binding molecule and 95% of ELPs not bound to an immunomodulator or intermediary binding molecule, for example, 5% of ELP covalently bound to an immunomodulator and 95% of an ELP comprising (VPGX hb G) n (VPGX hp G) n (SEQ ID NO: 1) or (VPGX hp G) n (VPGX hb G) n (SEQ ID NO: 2) not bound to an immunomodulator or intermediary binding molecule.
  • a micelle that includes 10% ELP bound to an intermediary binding molecule and 90% of ELPs not bound to an immunomodulator or intermediary binding molecule, for example, 10% of ELP covalently bound to an immunomodulator and 90% of an ELP comprising (VPGX hb G)n(VPGX hp G) n (SEQ ID NO: 1) or (VPGX hp G) n (VPGX hb G) n (SEQ ID NO: 2) not bound to an immunomodulator or intermediary binding molecule.
  • a micelle that includes 20% ELP bound to an intermediary binding molecule and 80% of ELPs not bound to an immunomodulator or intermediary binding molecule, for example, 20% of ELP covalently bound to an immunomodulator and 80% of an ELP comprising (VPGX hb G) n (VPGX hp G) n (SEQ ID NO: 1) or (VPGX hp G) n (VPGX hb G) n (SEQ ID NO: 2) not bound to an immunomodulator or intermediary binding molecule.
  • a micelle that includes 30% ELP bound to an intermediary binding molecule and 70% of other ELPs not bound to an immunomodulator or intermediary binding molecule, for example, 30% of ELP covalently bound to an immunomodulator and 70% of an ELP comprising (VPGX hb G) n (VPGX hp G) n (SEQ ID NO: 1) or (VPGX hp G)n(VPGX hb G) n (SEQ ID NO: 2) not bound to an immunomodulator or intermediary binding molecule.
  • a micelle that includes 40% ELP bound to an intermediary binding molecule and 60% of other ELPs not bound to an immunomodulator or intermediary binding molecule, for example, 40% of ELP covalently bound to an immunomodulator and 60% of an ELP comprising (VPGX hb G) n (VPGX hp G) n (SEQ ID NO: 1) or (VPGX hp G)n(VPGX hb G) n (SEQ ID NO: 2) not bound to an immunomodulator or intermediary binding molecule.
  • a micelle that includes 50% ELP bound to an intermediary binding molecule and 50% of other ELPs not bound to an immunomodulator or intermediary binding molecule, for example, 50% of ELP covalently bound to an immunomodulator and 50% of an ELP comprising (VPGX hb G) n (VPGX hp G) n (SEQ ID NO: 1) or (VPGX hp G)n(VPGX hb G) n (SEQ ID NO: 2) not bound to an immunomodulator or intermediary binding molecule.
  • a micelle that includes 60% ELP bound to an intermediary binding molecule and 40% of other ELPs not bound to an immunomodulator or intermediary binding molecule, for example, 60% of ELP covalently bound to an immunomodulator and 40% of an ELP comprising (VPGX hb G) n (VPGX hp G) n (SEQ ID NO: 1) or (VPGX hp G)n(VPGX hb G) n (SEQ ID NO: 2) not bound to an immunomodulator or intermediary binding molecule.
  • a micelle that includes 70% ELP bound to an intermediary binding molecule and 30% of other ELPs not bound to an immunomodulator or intermediary binding molecule, for example, 70% of ELP covalently bound to an immunomodulator and 30% of an ELP comprising (VPGX hb G) n (VPGX hp G) n (SEQ ID NO: 1) or (VPGX hp G)n(VPGX hb G) n (SEQ ID NO: 2) not bound to an immunomodulator or intermediary binding molecule.
  • a micelle that includes 80% ELP bound to an intermediary binding molecule and 20% of other ELPs not bound to an immunomodulator or intermediary binding molecule, for example, 80% of ELP covalently bound to an immunomodulator and 20% of an ELP comprising (VPGX hb G) n (VPGX hp G) n (SEQ ID NO: 1) or (VPGX hp G)n(VPGX hb G) n (SEQ ID NO: 2) not bound to an immunomodulator or intermediary binding molecule.
  • a micelle that includes 90% ELP bound to an intermediary binding molecule and 10% of other ELPs not bound to an immunomodulator or intermediary binding molecule, for example, 90% of ELP covalently bound to an immunomodulator and 10% of an ELP comprising (VPGX hb G) n (VPGX hp G) n (SEQ ID NO: 1) or (VPGX hp G)n(VPGX hb G) n (SEQ ID NO: 2) not bound to an immunomodulator or intermediary binding molecule.
  • a micelle that includes 100% ELP bound to an intermediary binding molecule, for example, 100% of ELP covalently bound to an intermediary binding molecule such as 100% of an ELP comprising (VPGX hb G) n (VPGX hp G) n (SEQ ID NO: 1) or (VPGX hp G)n(VPGX hb G) n (SEQ ID NO: 2) covalently bound to an intermediary binding molecule.
  • the ELP bound to the intermediary binding molecule comprises “(VPGX hb G)n(VPGX hp G) n -intermediary binding molecule” or “intermediary binding molecule- (VPGX hp G)n(VPGX hb G) n ”.
  • the ELP covalently bound to the intermediary binding molecule and the ELP not bound to the intermediary binding molecule is the ELP I48S48.
  • the micelle may comprise or consist of the ELPs “MG(VPGIG)48(VPGSG)48YWSGGG(KIAALKE) 4 ” (SEQ ID NO: 9) and the ELP l 48 S48.
  • the micelle may comprise or consist of the ELPs “MG(VPGIG)48(VPGSG)48YWSGGG(EIAALEK) 4 ” (SEQ ID NO: 10) and the ELP l 48 S48.
  • This may be referred to herein as a coiled coil micelle (i.e. , a micelle that includes an ELP species covalently bound to an intermediary binding molecule to allow for non-covalent binding of an immunomodulator that comprises a cognate of intermediary binding molecule).
  • a micelle that includes 10% ELP bound to a first immunomodulator, 10% ELP bound to a second immunomodulator and/or an intermediary binding molecule, and 80% of ELPs not bound to an immunomodulator or intermediary binding molecule, for example, 10% of ELP covalently bound to a first immunomodulator, 10% of ELP covalently bound to peptide K and 80% of an ELP comprising (VPGX hb G) n (VPGX hp G) n (SEQ ID NO: 1) or (VPGX hp G) n (VPGX hb G) n (SEQ ID NO: 2) not bound to an immunomodulator or intermediary binding molecule.
  • the ELP covalently bound to the first immunomodulator, the ELP covalently bound to the second immunomodulator ad/or intermediary binding molecule and the ELP not bound to the intermediary binding molecule is the ELP I48S48.
  • the micelle may comprise or consist of the ELPs: “(VPGIG)4s(VPGSG)48- immunomodulator”; “MG(VPGIG) 4 8(VPGSG) 4 8YWSGGG(KIAALKE) 4 ” (SEQ ID NO: 9); and the ELP I48S48-
  • the concentration of each ELP in a micelle be described by the ratio of each ELP in the micelle.
  • a micelle may have a ratio of ELP not bound to an immunomodulator or intermediary binding molecule described herein to ELP bound to an immunomodulator or intermediary binding molecule as described herein of 9:1 .
  • the composition includes 9 parts of an ELP comprising (VPGX hb G) n (VPGX hp G) n (SEQ ID NO: 1) or (VPGX hp G) n (VPGX hb G) n (SEQ ID NO: 2) not bound to an immunomodulator or intermediary binding molecule to 1 part ELP comprising “(VPGX hb G) n (VPGX hp G) n -immunomodulator”, “immunomodulator- (VPGX hp G)n(VPGX hb G) n ”, “(VPGX hb G)n(VPGX hp G) n -intermediary binding” or “intermediary binding-(VPGX hp G)n(VPGX hb G) n ”.
  • the ELP covalently bound to an immunomodulator may be “(VPGIG)48(VPGSG)48-immunomodulator”.
  • the ELP covalently bound to an intermediary binding molecule may be “(VPGIG)48(VPGSG)48-intermediary binding molecule”.
  • the ELP not bound to an immunomodulator, or intermediary binding molecule may be the ELP I48S48.
  • a micelle may have a ratio of ELP not bound to an immunomodulator or intermediary binding molecule described herein: ELP bound to an immunomodulator: and ELP bound to a second immunomodulator or intermediary binding molecule of 8: 1 : 1.
  • the composition includes 8 parts of an ELP comprising (VPGX hb G) n (VPGX hp G) n (SEQ ID NO: 1) or (VPGX hp G)n(VPGX hb G) n (SEQ ID NO: 2) not bound to an immunomodulator or intermediary binding molecule to 1 part ELP comprising “(VPGX hb G) n (VPGX hp G) n -immunomodulator”, or “immunomodulator-(VPGX hp G)n(VPGX hb G) n ”, and 1 part ELP comprising “(VPGX hb G)n(VPGX hp G) n -intermediary binding” or “intermediary binding- (VPGX hp G)n(VPGX hb G) n ”.
  • the ELP covalently bound to an immunomodulator may be “(VPGIG)48(VPGSG)48-immunomodulator”.
  • the ELP covalently bound to an intermediary binding molecule may be “(VPGIG)48(VPGSG)48-intermediary binding molecule”.
  • the ELP not bound to an immunomodulator, or intermediary binding molecule may be the ELP I48S48. This may be referred to herein as a hybrid micelle (i.e.
  • a micelle that includes two species of ELPs covalently bound to different immunomodulators or one ELP species covalently bound to an immunomodulator and one ELP bound to an intermediary binding molecule such as peptide K [which may also be considered an immunomodulatory molecule]).
  • the amphiphilic ELPs described herein are mixed at an appropriate ratio in a buffer and heated to a temperature above the CMT.
  • the ELPs in buffer may be heated to a temperature of at least 16°C.
  • the ELPs in buffer are heated to a temperature of at least 20°C.
  • the ELPs in buffer are heated to a temperature of at least 30°C.
  • the ELPs in buffer are heated to a temperature of at least 37°C.
  • the buffer may be any suitable buffer.
  • the buffer may be physiologically acceptable buffer such as phosphate buffered saline or phosphate buffer.
  • physiologically acceptable buffer such as phosphate buffered saline or phosphate buffer.
  • Other suitable buffers are well known in the art.
  • the micelles may be formed so that additional agents are encapsulated within the core of the micelle.
  • agents may be hydrophobic and as such when the ELPs self-assemble, the agents are automatically incorporated into the core of the micelles.
  • additional agents that may be incorporated into the micelles may include hydrophobic drugs or hydrophobic immunomodulatory molecules that may for example be used in immunotherapy such as allergen immunotherapy such as anti-histamines, anti-lgE monoclonal antibodies such as omalizumab, or steroids.
  • the micelles may comprise vitamin D3 encapsulated within the core of the micelle.
  • additional agents may be attached to the hydrophobic block of the ELP so that when the ELPs self-assemble the additional agents are incorporated into the core of the micelle.
  • the buffer may include an immunomodulator bound to an intermediary binding molecule that is the cognate partner of an intermediary binding molecule bound to ELPs of the micelle.
  • an immunomodulator covalently bound to peptide K may be added to the buffer prior to heating and formation of micelles.
  • an immunomodulator covalently bound to peptide E may be added to the buffer prior to heating and formation of micelles.
  • the amount of immunomodulator bound to a cognate intermediary binding molecule may be equal to the amount of ELP bound to an intermediary binding molecule. For example, it may be an equimolar amount.
  • the immunomodulator bound to a cognate intermediary binding molecule may be added to the buffer at a ratio of 1 :1 to the amount of ELP bound to an intermediary binding molecule.
  • a micelle includes one part of ELPs covalently bound to peptide K
  • the same amount of an immunomodulator covalently bound to peptide E may be added to the buffer and vice versa.
  • a free cognate intermediary binding molecule may be added to the buffer. This may help reduce homodimerization of intermediary binding molecules bound to ELPs which may lead to aggregation of micelles.
  • the micelle includes ELPs covalently bound to peptide K
  • free peptide E may be added to the buffer. This may help prevent peptide K molecules binding to other peptide K molecules thus preventing aggregation of micelles.
  • the free cognate intermediary binding partner may be added at an equimolar amount to the amount of intermediary binding molecule bound to ELPs.
  • a micelle when a micelle includes more than 20% ELPs bound to an intermediary binding molecule, such as peptide K, a free cognate intermediary binding molecule may be added to the buffer. In some examples, when a micelle includes more than 25% ELPs bound to an intermediary binding molecule, such as peptide K, a free cognate intermediary binding molecule may be added to the buffer. In some examples, when a micelle includes more than 30% ELPs bound to an intermediary binding molecule, such as peptide K, a free cognate intermediary binding molecule may be added to the buffer.
  • ELPs and micelles formed therefrom have a number of advantages over common adjuvants such as alum.
  • adjuvant refers to a substance that modulates the immunogenicity of an antigen.
  • ELPs as described herein and micelles formed therefrom may be for use as adjuvants for vaccines. ELPs as described herein, and micelles formed therefrom may lead to a strong cellular immune response in comparison to known adjuvants such as alum. In some examples, the ELPs as described herein, and micelles formed therefrom provide a reduced Th2 (or a lower Th2 skewed) response in comparison to known adjuvants such as alum.
  • the use of the ELPs as described herein and micelles formed therefrom may be particular advantageous for vaccination against intracellular pathogens such viruses (e.g., CMV HIV), bacteria (e.g., Listeria, Mycobacteria, Salmonella (e.g., S. typhi) enteropathogenic Escherichia coli (EPEC), enterohaemorrhagic Escherichia coli (EHEC), Yersinia, Shigella, Chlamydia, Chlamydophila, Staphylococcus, Legionella), protozoa (e.g., Taxoplasma), fungi, and intracellular parasites (e.g., Plasmodium (e.g., P. vivax, P. falciparum, P. ovale, and P. malariae). Furthermore, the ELPs as described herein, and micelles formed therefrom may reduce humoral response against an immunogenic immunomodulator.
  • viruses e.g., CMV HIV
  • bacteria
  • the increase in uptake of antigens (immunomodulators) bound to the ELPs as described herein and micelles formed therefrom may increase antigen presenting cell maturation and/or increase T cell expansion and differentiation.
  • the ELPs as described herein, and micelles formed therefrom may increase differentiation of T cells to Th1 cells.
  • the ELPs described herein, and micelles formed therefrom may be used for delivery of an immunomodulator to a subject.
  • the ELPs described herein, and micelles formed therefrom may be used for delivery of an immunomodulator to specific cell types, for example delivery of an immunomodulator to antigen presenting cells such as dendritic cells.
  • the ELPs described herein, and micelles formed therefrom may be used as adjuvants for cancer immunotherapy.
  • the ELPs described herein, and micelles formed therefrom may be used as adjuvants for allergen immunotherapy. It is shown that when an allergenic immunomodulator is used, the ELPs described herein, and micelles formed therefrom may decrease production of I g E in response to the allergen. In addition, they may increase IgG production and lead to earlier IgG production in comparison to known adjuvants such as alum. In addition, the use of the ELPs described herein and micelles formed therefrom may reduce the overall T cell response to the allergen in comparison to known adjuvants such as alum. Furthermore, the ELPs described herein, and micelles formed therefrom may reduce cellular mediated immune response to the allergen.
  • the ELPs described herein, and micelles formed therefrom may be used for forming immunogenic vaccines.
  • compositions for modulating an immune response for example, modulating an immune response in a subject such as a human.
  • modulating altering
  • regulating are used to interchangeably to refer to any change in cells of the immune system or the activity of such cells.
  • control includes increasing or decreasing the number of various cell types, increasing or decreasing the activity of these cells, or stimulating or suppressing the immune system, which may be caused by other possible changes in the immune system.
  • compositions provided herein may modulate certain parts of the immune response differently.
  • the compositions provided herein may increase cell-mediated immune response and/or reduce humoral immune response.
  • the compositions provided herein may increase cell-mediated immune response and/or increase humoral immune response.
  • the compositions provided herein may reduce cell-mediated immune response and/or reduce humoral immune response.
  • Immuno response refers to a response by a cell of the immune system of a subject (for example of a human), such as a B cell, T cell (CD4 + or CD8 +), regulatory T cell, antigen-presenting cell, dendritic cell, monocyte, macrophage, NKT cell, NK cell, basophil, eosinophil, or neutrophil, to a stimulus.
  • the response may be for a particular antigen (an “antigen specific response”), and may be a response by a CD4 T cell, CD8 T cell , or B cell via their antigen-specific receptor.
  • an immune response is a T cell response, such as a CD4+ response or a CD8+ response.
  • Such responses by these cells can include, for example, cytotoxicity, proliferation, cytokine or chemokine production, trafficking, or phagocytosis, and can be dependent on the nature of the immune cell undergoing the response.
  • exemplary immune responses include humoral immune responses (e.g., production of antigenspecific antibodies) and cell-mediated immune responses (e.g., production of antigen-specific T cells).
  • Assays for assessing an immune response are known in the art and may comprise in vivo assays, such as assays to measure antibody responses and delayed type hypersensitivity responses. For example, an assay to measure antibody responses primarily may measure B-cell function as well as B-cell/T-cell interactions.
  • antibody titers in the blood may be compared following an antigenic challenge.
  • In vitro assays may comprise determining the ability of cells to divide, or to provide help for other cells to divide, or to release lymphokines and other factors, express markers of activation, and lyse target cells.
  • Lymphocytes in mice and man can be compared in in vitro assays.
  • the lymphocytes from similar sources such as peripheral blood cells, splenocytes, or lymph node cells, are compared. It is possible, however, to compare lymphocytes from different sources as in the non-limiting example of peripheral blood cells in humans and splenocytes in mice.
  • cells may be purified (e.g., B-cells, T-cells, and macrophages) or left in their natural state (e.g., splenocytes or lymph node cells).
  • the cells can be tested in vitro for their ability to proliferate using mitogens or specific antigens.
  • the ability of cells to divide in the presence of specific antigens can be determined using a mixed lymphocyte reaction (MLR) assay.
  • MLR mixed lymphocyte reaction
  • Supernatant from the cultured cells can be tested to quantitate the ability of the cells to secrete specific lymphokines.
  • the cells can be removed from culture and tested for their ability to express activation antigens. This can be done by any method that is suitable as in the non-limiting example of using antibodies or ligands which bind to the activation antigen as well as probes that bind the RNA coding for the activation antigen.
  • Humoral immune response refers to the stimulation of antibody production.
  • Humoral immune response also refers to the accessory proteins and events that accompany antibody production, including T helper cell activation and cytokine production, affinity maturation, and memory cell generation.
  • lymphocytes such as the defence provided by sensitized T cell lymphocytes when they directly lyse cells expressing foreign antigens and secrete cytokines (e.g., IFN-gamma.), which can modulate macrophage and natural killer (NK) cell effector functions and augment T cell expansion and differentiation.
  • cytokines e.g., IFN-gamma.
  • the compositions of the invention may lead to an altered T cell response.
  • the compositions may alter T helper 1 cell (Th1) response.
  • the compositions provided herein may alter Th2 cell response.
  • T helper (Th) cells provide helper functions to other cells of the immune system, especially the antigen-presenting cells (APCs) such as macrophages, dendritic cells, and B cells and are important for their activation and maturation.
  • APCs antigen-presenting cells
  • CD4+ Th cells including Th1 , Th2, Th17, and T regulatory cells, each activated by a specific set of cytokines and transcription factors and characterized by the cytokines they secrete and effector functions they perform.
  • Th1 cells derive from the alpha:beta lineage of T cells and recognize antigens presented by major histocompatibility complex (MHC) class I or II molecules. Th1 cells play important roles in the identification and eradication of intracellular pathogens such as viruses and bacteria, including Mycobacterium tuberculosis, Mycobacterium leprae, and Leishmania. These pathogens typically reside in phagocytic vesicles within cells such as macrophages and often evade intracellular killing by preventing lysosomal fusion. Th1 cells help to activate macrophages against these pathogens and overcome these microbial evasion strategies.
  • MHC major histocompatibility complex
  • Th2 cells which are the other major subset of CD4+ T cells, help to recognize extracellular pathogens such as helminths and parasites and activate B cell-mediated antibody responses.
  • the two cytokines that play a critical role in Th1 differentiation are Interferon Gamma (IFNy) and IL-12.
  • IFNy Interferon Gamma
  • IL-12 secreted by APCs upon T cell engagement drives differentiation into Th1 effector cells through the activation of STAT4 transcription factor.
  • STAT4 induces IFNy production, which is another driver towards Th1 differentiation by activating transcription factors STAT1 and Tbet.
  • Tbet is considered the master regulator of Th1 differentiation since it induces more expression of IFNy, leading to a positive feedback loop to strengthen the Th1 response.
  • Th1 cells are in cell-mediated immunity and inflammation, including the activation of cytolytic and other effector functions of other immune cells such as macrophages, B cells, and CD8+ cytotoxic T lymphocytes (CTLs).
  • CTLs cytotoxic T lymphocytes
  • CD40 Ligand CD40L
  • Effector Th1 cells also secrete copious amounts of IFNy that, besides further expanding the Th1 population, also activate cytolytic activities of macrophages through the induction of more than 200 target genes.
  • cytotoxic activity in macrophages is key to killing intracellular pathogens such as viruses, intracellular bacteria, and protozoa.
  • a classic example of Th1-dependent immune response is during Mycobacteria infection. Mycobacteria escape lysosomal fusion within macrophages; however, peptides derived from these pathogens displayed by MHC Class II on the surface of infected macrophages lead to activation of Th1 responses, which turns on the cytolytic properties of macrophages.
  • Th1 responses play crucial roles in activating CD8+ cytotoxic T lymphocytes (CTLs) to target and destroy tumours, in addition to leading to increasing CTL survival and memory.
  • CTLs cytotoxic T lymphocytes
  • CD40 also activates class switching in B cells to produce lgG2a antibodies.
  • Th2 cells mediate the activation and maintenance of the humoral, or antibody-mediated, immune response against extracellular parasites, bacteria, allergens, and toxins. Th2 cells mediate these functions by producing various cytokines such as IL-4, IL-5, IL-6, IL-9, and IL-13. These cytokines are responsible for a strong antibody production, eosinophil activation, and inhibition of several macrophage functions, thus providing phagocyte-independent protective responses. These cytokines also counteract the Th1 responses that allow for the Th2 responsiveness to IL-4. Functionally, Th2 cytokines have effects on many cell types in the body as the cytokine receptors are widely expressed on numerous cell types.
  • Th2 cells stimulate and recruit specialized subsets of immune cells, such as eosinophils and basophils, to the site of infection or in response to allergens or toxins leading to tissue eosinophilia and mast cell hyperplasia. They induce mucus production, goblet cell metaplasia, and airway hyper-responsiveness. Th2 cells also control the regulation of B cell class-switching to IgE. Because of their influence on the production of antibodies and allergic responses, over activation of Th2 cells appears to be responsible for the exacerbation of allergies (Type-1 , immediate hypersensitivity reactions), autoimmune reactions such as chronic graft-versus host disease, progressive systemic sclerosis, and systemic lupus erythematosus.
  • Type-1 immediate hypersensitivity reactions
  • autoimmune reactions such as chronic graft-versus host disease, progressive systemic sclerosis, and systemic lupus erythematosus.
  • Th2 cells are also known to be responsible for the development of asthma and other allergic inflammatory diseases. Interestingly, Th2 cells also produce the growth factor amphiregulin and IL-24 which have anti-tumor effects.
  • the surrounding cytokine environment drives polarization of naive T cells into specific cell lineages, where the Th2 cell type is specifically triggered by interleukin (I L)-4 and IL-2.
  • Th cells migrate to sites of inflammation in the periphery, where they reencounter their cognate antigen and secrete effector cytokines, thus driving an antigen-specific immune response.
  • IL-5, IL-9, and IL-13 are secreted by Th2 cells only once they have reached inflamed tissue sites.
  • the cytokine milieu in the surrounding inflammatory microenvironment drives different Th2 effector activities.
  • Th2 memory cell subsets that carry out different effector roles depending on the cytokine influence.
  • Th2 cells that produce high levels of IL-5 have been shown to contribute to the pathogenesis of allergic asthma, while cells that produce IL-5, IL-17, and IFN- y, in addition to IL-4 and IL-13, have been identified as noncanonical memory Th2 cells that drive chronic allergic inflammatory diseases.
  • Memory Th2 cells can be divided into at least four distinct subpopulations based on the levels of expressed chemokine (C-X-C motif) receptor 3 (CXCR3) and CD62L. All four subpopulations characteristically produce large amounts of IL-4 and IL-13 in response to antigenic re-stimulation. However, only the CXCR3
  • CXCR3 C-X-C motif receptor 3
  • CD62L CD62L
  • ILC are immune cells derived from common lymphoid progenitors and are grouped based on their cytokine and transcription factor profiles, where ILC2 function in part to regulate Th2 phenotype.
  • IL-33 can induce a strong Th2 immunity and eosinophilic inflammation by amplifying the Th2 cytokine response in lung and intestine tissue, IL-33 has little effect on the Th2 effector subset and instead mediates inflammation via memorytype Tpath cells.
  • Exposure to an allergen may initiate a Th2 response in the airway by stimulating epithelial and endothelial cells to produce IL-33, thereby driving IL-5 production by Th2 memory cells and exacerbating the eosinophilic inflammation.
  • Th2 cells producing IL-4, IL-5, and IL-13 are implicated in a number of inflammatory diseases, including rhinitis, rhino-conjunctivitis, asthma, chronic rhinosinusitis, atopic dermatitis, food allergy and eosinophilic gastrointestinal disorders such as ulcerative colitis.
  • Th2 cytokines drive pathogenesis of human asthma and murine allergic airway inflammation models by promoting eosinophilic infiltration, increased mucus production through goblet cell metaplasia, and tissue fibrosis.
  • Th2 cell-derived IL-4, IL-5, and IL-13 contribute to B cell proliferation and isotype class switching from immunoglobulin lgG1 to IgE, a key antibody involved in parasitic helminth infection and allergic diseases associated with Th2 cells.
  • Th2 cells have also been shown to induce the alternate activation (M2) macrophage phenotype, which mediates resolution of the inflammatory stage and initiates tissue repair in wound healing processes. Effector Th2 cells mediate immune responses to parasitic helminth infections, venoms, certain bacterial infections, and promote wound healing by inducing alternate activation (M2) macrophage phenotype.
  • Methods of studying, identifying and/or detecting Th cells include but are not limited cytokine profiling, cytokine ELISAs, and flow cytometry.
  • the effects on T cell response in a subject may differ.
  • the effects of the compositions provided herein may be a change in immune response, such as an increase or decrease, in comparison to a composition comprising alum.
  • the change may be in comparison to a subject that has not been administered a composition of the invention.
  • the compositions may be for use as a vaccine.
  • immunogenic compositions in some examples, provided herein are immunogenic compositions.
  • the compositions are vaccine compositions.
  • immunogenic composition and “immunological composition” and “immunogenic or immunological composition” refer to compositions that elicit an immune response against an antigen or immunogen after administration into a subject.
  • vaccine and “vaccine composition” refers to compositions that induce a protective immune response against the antigen of interest, or which efficaciously protects against the antigen; for instance, after administration to the subject, elicits a protective immune response against the targeted antigen or immunogen.
  • compositions provided herein may be for use as a vaccine and increase immunogenicity of an antigen or immunogen of interest.
  • the compositions may lead to an increased uptake of immunogen.
  • the compositions provided herein may increase T-cell proliferation in comparison to free immunogen.
  • the compositions may increase dendritic cell maturation.
  • compositions provided herein may be for use in treating an allergic disease and the compositions may provide an increased humoral response and a weak Th2 skewing effect in comparison to a composition comprising alum.
  • the compositions may provide a decreased overall T helper cell response.
  • the compositions provided herein may increase levels of IL10. In some examples, the compositions provided herein may increase in IFN-y. In some examples, the compositions provided herein may decrease Th2 response. For example, the compositions provided herein may decrease levels of IgE. In some examples, the compositions provided herein may increase levels of IgG, such as levels of lgG2a and/or lgG4 in a human subject (or IgG 1 in a murine subject). In some examples, the compositions provided herein may cause earlier production of IgG antibodies earlier (i.e., sooner after administration). In some examples, the compositions provided herein may reduce IgE crosslinking. In some examples, the compositions provided herein may decrease IL-4 levels. In some examples, the compositions provided herein may decrease IL-13 levels.
  • Th1 cells secrete interferongamma and other cytokines which trigger B cells to produce IgG antibodies.
  • a critical cytokine produced by Th2 cells is IL-4, which drives B cells to produce IgE.
  • Th1 and Th2 responses are mutually exclusive since Th1 cells suppress the induction of Th2 cells and vice versa.
  • antigens that trigger a strong Th1 response simultaneously suppress the development of Th2 responses and hence the production of IgE antibodies.
  • compositions provided herein may reduce the release of histamines in a subject in response to an allergen.
  • compositions provided herein may include additional agents.
  • the compositions may include vitamins.
  • the compositions may include vitamin A and/or vitamin D3.
  • compositions may include vitamin A.
  • compositions may include vitamin D.
  • compositions may include vitamin D3.
  • the compositions may also include cognate intermediary binding molecules. These may help to reduce aggregation of micelles. For example, when micelles include ELPs bound to peptide K, the compositions may also include free peptide E. In some examples, when micelles include at least 20% ELPs bound to peptide K, the compositions may also include free peptide E. In some examples, when micelles include at least 25% ELPs bound to peptide K, the compositions may also include free peptide E. In some examples, when micelles include at least 30% ELPs bound to peptide K, the compositions may also include free peptide E.
  • compositions provided herein may be provided as part of a pharmaceutical formulation.
  • such formulations may be administered to a human subject in need thereof (as described elsewhere herein).
  • a pharmaceutical formulation and the compositions described herein may comprise a composition or micelle as described herein along with a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier.
  • compositions and formulations may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, supplementary immune potentiating agents such as adjuvants and cytokines and optionally other therapeutic agents or compounds.
  • pharmaceutically acceptable refers to a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the selected micelle without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical formulation in which it is contained.
  • Excipients are natural or synthetic substances formulated alongside an active ingredient (e.g. a micelle or composition as provided herein), included for the purpose of bulking-up the formulation or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating drug absorption or solubility. Excipients can also be useful in the manufacturing process, to aid in the handling of the active substance concerned such as by facilitating powder flowability or non-stick properties, in addition to aiding in vitro stability such as prevention of denaturation over the expected shelf life. Pharmaceutically acceptable excipients are well known in the art. A suitable excipient is therefore easily identifiable by one of ordinary skill in the art. By way of example, suitable pharmaceutically acceptable excipients include water, saline, aqueous dextrose, glycerol, ethanol, and the like.
  • Adjuvants are pharmacological and/or immunological agents that modify the effect of other agents in a formulation.
  • Pharmaceutically acceptable adjuvants are well known in the art. A suitable adjuvant is therefore easily identifiable by one of ordinary skill in the art.
  • a pharmaceutical formulation may comprise an adjuvant selected from the group consisting of: AS03; AddaS03; AS04; MF59; AddaVax; Poly l:C; R848; Cpg; virus-like particles; virosomes; MPL; and flagellin protein.
  • a suitable adjuvant may be an agonist of Toll-like receptors (TLRs).
  • TLRs Toll-like receptors
  • a suitable adjuvant may be an agonist of a TLR selected from the group consisting of: TLR3; TLR4; TLR7; TLR8; and TLR9.
  • Polyinosic:polycytidylic acid (also referred to as Poly I :C) is suitable example of an adjuvant of this sort that is an agonist of TLR3.
  • MLP is an example of an adjuvant that is an agonist of TLR4.
  • R848 an imidazoquinoline
  • CpG oligodeoxynucleotides (also referred to as CpG, or CpG ODNs) constitute an example of an adjuvant that is an agonist of TLR9.
  • Diluents are diluting agents. Pharmaceutically acceptable diluents are well known in the art. A suitable diluent is therefore easily identifiable by one of ordinary skill in the art.
  • Carriers are non-toxic to recipients at the dosages and concentrations employed and are compatible with other ingredients of the formulation.
  • carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.
  • Pharmaceutically acceptable carriers are well known in the art. A suitable carrier is therefore easily identifiable by one of ordinary skill in the art.
  • compositions and formulations provided herein may be for use as a medicament.
  • compositions and formulations described herein may be for use in preventing and/or treating cancer.
  • compositions provided herein may help to induce effective tumour-reactive T-cell responses to a tumour.
  • the immunomodulator may include a tumour- associated epitope.
  • the compositions and formulations described herein may effectively help generate a population of immune cells, in particular of CD8 + effector T cells (also known as cytotoxic T lymphocytes (CTLs)).
  • CTLs cytotoxic T lymphocytes
  • the immune cells induced by administration of the compositions or formulations described herein may be reactive to the epitope, or epitopes, presented on the micelles. These immune cells are then primed for the killing of cancer cells that present the same or similar epitopes.
  • Such compositions and formulations for use in treating cancer may be referred to as “cancer vaccines” or “cancer immunotherapy vaccines”.
  • the medical uses and methods of treating cancer may include administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical formulation or composition as described herein.
  • Tumours may be of mesenchymal or epithelial origin. Cancers include cancers of the colon, rectum, cervix, breast, lung, stomach, uterus, skin, mouth, tung, lips, larynx, kidney, bladder, prostate, brain, and blood cells. The medical uses and methods of treatment described herein may be used in the treatment of solid tumours.
  • a cancer to be treated by a medical use or method of treatment described herein may be a solid tumour selected from but not limited to the group consisting of: pancreatic ductal adenocarcinoma; pancreatic cancer; breast cancer; melanoma; non-small cell lung cancer; small cell lung cancer; nasopharyngeal cancer; hepatocellular cancer; colorectal cancer; oesophageal cancer; gastric cancer; anal cancer; small intestine cancer; mesothelioma; kidney cancer; renal cell carcinoma; bladder cancer; prostate cancer; ovarian cancer; vulval cancer; cervical cancer; penile cancer; uveal melanoma; retinoblastoma; sarcoma; osteosarcoma; glioblastoma; adrenocortical carcinoma; neuroblastoma; Wilms tumour; endometrial cancer; and thyroid cancer.
  • treatment and “ treating” should be taken as encompassing therapy undertaken in order to prevent, slow down, or reduce an undesired physiological change or disorder, such as the growth, development or spread of cancer.
  • beneficial or desired results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized state of disease (which is to say, disease that is not worsening), delay or slowing of disease progression, de-staging the tumour (e.g., changing from borderline resectable to amendable for surgical resection), amelioration or palliation of the disease state, and remission (either partial or total).
  • Treatment may bring about prolonged survival as compared to expected survival if not receiving treatment.
  • treatment may provide a patient with an improved standard of life as compared to that which would be expected if not receiving treatment.
  • the medical uses and methods of treatment described herein may be of particular benefit in preventing the growth, progression or metastasis of tumours in subjects receiving treatment.
  • the medical uses and methods of treatment described herein may be of particular benefit in preventing growth, or reducing size, of such tumours. It will be appreciated that these effects are able to beneficially reduce a subject’s tumour burden.
  • the medical use and methods of treating cancer provided herein may include administration of at least additional therapeutic agent.
  • the additional therapeutic agent may be an agent currently used for prevention or treatment of cancer (i.e. , an anti-cancer agent).
  • anti-cancer agents include, but are not limited to, antibodies, antibody fragments, conjugates, drugs, cytotoxic agents, proapoptotic agents, toxins, nucleases (including DNAses and RNAses), hormones, immunomodulators, chelators, boron compounds, photoactive agents or dyes, radioisotopes or radionuclides, oligonucleotides, interference RNA, peptides, anti- angiogenic agents, chemotherapeutic agents, cytokines, chemokines, prodrugs, enzymes, binding proteins or peptides or combinations thereof.
  • chemotherapeutic drugs include vinca alkaloids, anthracyclines, epidophyllotoxins, taxanes, antimetabolites, tyrosine kinase inhibitors, alkylating agents, antibiotics, Cox-2 inhibitors, antimitotics, antiangiogenic and proapoptotic agents, doxorubicin, methotrexate, taxol, other camptothecins, and others from these and other classes of anticancer agents, and the like.
  • cancer chemotherapeutic drugs include nitrogen mustards, alkyl sulfonates, nitrosoureas, triazenes, folic acid analogs, pyrimidine analogs, purine analogs, platinum coordination complexes, hormones, and the like.
  • Suitable chemotherapeutic agents are described in REMINGTON'S PHARMACEUTICAL SCIENCES, 19th Ed. (Mack Publishing Co. 1995), and in GOODMAN AND GILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, 7th Ed. (MacMillan Publishing Co. 1985), as well as revised editions of these publications.
  • Other suitable chemotherapeutic agents, such as experimental drugs are known to those of skill in the art.
  • Exemplary drugs include, but are not limited to, 5-fluorouracil, afatinib, aplidin, azaribine, anastrozole, anthracyclines, axitinib, AVL-101 , AVL-291 , bendamustine, bleomycin, bortezomib, bosutinib, bryostatin-1 , busulfan, calicheamycin, camptothecin, carboplatin, 10- hydroxycamptothecin, carmustine, Celebrex, chlorambucil, cisplatin (CDDP), Cox-2 inhibitors, irinotecan (CPT-1 1), SN-38, carboplatin, cladribine, camptothecans, crizotinib, cyclophosphamide, cytarabine, dacarbazine, dasatinib, dinaciclib, docetaxel, dactinomycin, daunorubicin,
  • compositions and formulations described herein may be for use in preventing and/or treating an allergic disease.
  • Allergic disease refers to a condition caused by hypersensitivity of the immune system to typically harmless substances in the environment. Allergic diseases include, but are not limited to, asthma, hypersensitivity lung diseases, rhinitis, rhino-conjunctivitis, rhinosinusitis, atopic eczema, contact dermatitis, allergic conjunctivitis (intermittent and persistent), vernal conjunctivitis (hay fever), atopic keratoconjunctivitis, giant papillary conjunctivitis, urticaria (hives), angioedema, hypersensitivity pneumonitis, eosinophilic bronchitis, vasculitis, hypersensitivity vasculitis, antineutrophil cytoplasmic antibody (ANCA) associated vasculitis, Wegner's granulomatosis, Churg Strauss vasculitis, microscopic polyangiitis, temporal arteritis, celiac disease, mastocytosis, and anaphylococc
  • Allergic reaction refers to the body's response to an allergen.
  • An allergic reaction can be localized to one area (skin that came into contact with allergen) or generalized. Allergic reactions may include, but are not limited to, rash, itching, hives, swelling, difficulty breathing, wheezing, angioedema, difficulty swallowing, nasal congestion, runny nose, shortness of breath, nausea, stomach cramps, abdominal pain, vomiting and/or low blood pressure.
  • Subjects in need of treatment may identified or diagnosed as having an allergic disease by any suitable method known in the art. For example, diagnosis made through any one or more of clinical history, detection of specific IgE molecules in the blood stream (i.e., via blood tests), skin prick testing, patch test methods, challenge test methods or elimination methods (such as elimination diets).
  • the medical uses and methods of preventing and/or treating an allergic disease may include administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical formulation or composition as described herein.
  • compositions and formulations described herein that include an immunomodulator that includes an allergenic epitope may be used in medical uses and method for treating allergy. It will be apparent that in the majority of cases that the allergenic epitope is an epitope from or derived from an allergen that causes allergy symptoms or allergic reaction in the subject.
  • compositions and formulations described herein that include an immunomodulator that includes an allergenic epitope may provide an increased level of IgG and/or decreased levels of IgE in a subject.
  • the use of micelles as described herein may increase the amount of allergenic epitope required to produce a detectable IgE response.
  • use of micelles as described herein may lead to a weaker pro-inflammatory response as well as a reduced Th2-skewed response to the allergenic epitope.
  • compositions and formulations described herein that include an immunomodulator that includes an allergenic epitope may be for use in methods of allergy immunotherapy (AIT).
  • AIT allergy immunotherapy
  • SCIT subcutaneous allergy immunotherapy
  • Compositions and formulations for use in AIT and/or SCIT may be referred to as allergy vaccines.
  • AIT comprises administering an allergen to the patient in order to treat an allergy to that allergen of the patient, i.e., reducing current or future immune response, such as an allergenspecific IgE response and/or histamine release by mastocytes and/or granulocytes induced by the allergen, and/or manifestation of clinical symptoms of allergy.
  • Immunotherapy is conventionally carried out by administering repeatedly a mono-dose or incremental doses of an allergen to a patient in need thereof, thereby resulting in an adaptive immune response of the patient who becomes desensitised to the allergen.
  • AIT or SCIT increasing doses of the allergen or allergenic epitope are administered, followed by a maintenance dose for several years, with the goal of inducing immunological changes leading to symptom amelioration while on therapy, as well as sustained desensitization off AIT or SCIT (immune tolerance).
  • subjects receive increasing doses of the allergen or allergenic epitope at weekly intervals over several weeks to months, under tightly monitored medical supervision. The gradual dose escalation enables tolerability to therapy and mitigates risk of severe hypersensitivity reactions related to allergen administration.
  • the dosing regimen for AIT or SCIT includes a cluster dosing regimen.
  • the cluster dosing regimen may include an up-titration regimen followed by a maintenance regimen.
  • the up- titration regimen may include administering increasing doses of the antigen or antigenic epitope over a period of at least 4 weeks or from 1 to 24 weeks (e.g., over a period of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, or 24 weeks, e.g., from 4-10 weeks, from 4-8 weeks, from 6-12 weeks, from 6-10 weeks, from 6-8 weeks, from 8-12 weeks, or from 8-10 weeks).
  • the up-titration regimen be over a period of more than 24 week.
  • the maintenance regimen may include administering one or more maintenance doses of the allergen or allergenic epitope at the highest dose administered during the up-titration regimen.
  • the maintenance regimen includes administering maintenance doses every 1 to 4 weeks for at least 8 weeks (e.g., for at least s weeks, 10 weeks, 12 weeks, 14 weeks, 16 weeks, or longer).
  • the up-titration regimen may include up-titrating from a dose of 1 bioequivalent allergy unit (BAU) to a dose of at least about 4,000 BAU (e.g., over a period of 4, 5, 6, 7, 8, 9, 10, 11 , or weeks) and the maintenance regimen may include administering one or more maintenance doses at least about 4,000 BAU.
  • BAU bioequivalent allergy unit
  • prevent refers to preventing development of allergy, an allergic reaction or an allergic condition.
  • the term, as used herein, also includes reducing or abrogating allergen sensitization to prevent an allergic reaction.
  • “Response” of a subject to treatment indicates that the subject manifests a reduction in the clinical symptoms.
  • Clinical symptoms may be assessed over the course of treatment, i.e. symptoms before treatment may be compared to symptoms during and after treatment. Alternatively, a reduction in symptoms may be determined by comparison to a baseline level established before treatment.
  • Concerning allergy this approach is particularly useful where, for example, immunotherapy is carried out in subjects not currently experiencing symptoms, as may be the case for seasonal grass pollen allergy sufferers, who may be treated before the pollen season.
  • Symptoms may be assessed by standard methods, such as patient self-assessment or recording of the amount of medication required.
  • the degree of a subject’s response to treatment may be assessed by measuring the degree of reduction of severity in symptoms.
  • the medical use and methods of treating allergic disease provided herein may include administration of at least additional therapeutic agent.
  • the additional therapeutic agent is selected from the group consisting of: a steroid, an antihistamine, a decongestant, and an anti-lgE agent.
  • the additional therapeutic agent is a steroid (e.g., a corticosteroid, such as an inhaled corticosteroid (ICS)).
  • the additional therapeutic agent is an antihistamine (e.g., loratadine, fexofenadine, cetirizine, diphenhydramine, promethazine, carbinoxamine, desloratadine, hydroxyzine, levocetirizine, triprolidine, brompheniramine, or chlorpheniramine).
  • the additional therapeutic agent is a decongestant (e.g., pseudoephedrine or phenylephrine).
  • the additional therapeutic agent is an anti-lgE agent (e.g., omalizumab).
  • compositions and formulations described herein may be for use in preventing and/or treating an infectious disease.
  • the medical uses and methods of preventing and/or treating an infectious disease may include administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical formulation or composition as described herein.
  • infectious disease refers to a disease which results from an infection.
  • An infection is a condition caused by the invasion of an organism by a foreign agent (i.e., infectious agent).
  • infectious agents include, but are not limited to, bacteria, fungi, viruses, viroids, nematodes (e.g., parasites such as roundworms and pinworms), anthropods (e.g., mites, fleas, lice, ticks), and macroparasites (e.g., tapeworms).
  • Common infectious diseases include bacterial and viral infections.
  • compositions and formulations described herein when for use in methods of treating infectious disease may include an epitope or antigen from or derived from the pathogen causing the infectious disease.
  • compositions and formulations described herein may be particularly useful in prevention or treatment of infectious diseases caused by intracellular pathogens.
  • viruses e.g., CMV HIV
  • bacteria e.g., Listeria, Mycobacteria, Salmonella (e.g., S. typhi) enteropathogenic Escherichia coli (EPEC), enterohaemorrhagic Escherichia coli (EHEC), Yersinia, Shigella, Chlamydia, Chlamydophila, Staphylococcus, Legionella
  • protozoa e.g., Taxoplasma
  • fungi e.g., Plasmodium (e.g., P.
  • compositions and formulations described herein may reduce humoral response against an immunogenic immunomodulator including epitopes derived from such intracellular pathogens and increase cellular mediated response.
  • the methods of preventing and/or treating infectious disease is a method of vaccination.
  • Vaccination refers to the administration of a composition or formulation as described herein intended to generate an immune response, for example to a disease-causing pathogen.
  • Vaccination can be administered before, during, and/or after exposure to a disease-causing pathogen, and in some examples, before, during, and/or shortly after exposure to the agent.
  • vaccination includes multiple administrations, appropriately spaced in time, of a composition or formulation as described herein.
  • Treatment in relation to infectious diseases refers to any administration of a composition or formulation as described herein that partially or completely alleviates, ameliorates, relieves, inhibits, delays onset of, reduces severity of and/or reduces incidence of one or more symptoms or features of an infectious disease or the predisposition toward the disease.
  • Such treatment may be of a subject who does not exhibit signs of the relevant disease, and/or of a subject who exhibits only early signs of the disease. Alternatively or additionally, such treatment may be of a subject who exhibits one or more established signs of the relevant disease.
  • the term “treating” in reference to infectious diseases refers to the vaccination of a subject.
  • Prevention refers to a delay of onset of an infectious disease. Prevention may be considered complete when onset of an infectious disease, disorder has been delayed for a predefined period of time.
  • treatment generally are taken to include an intervention performed with the intention of preventing the development or altering the pathology of a condition, disorder or symptom (e.g., an allergic disease, infectious disease, etc). Accordingly, “treatment” refers to both therapeutic treatment and prophylactic or preventative measures (such as vaccination), wherein the object is to prevent or slow down (lessen) the targeted condition, disorder or symptom. “Treatment” therefore encompasses a reduction, slowing or inhibition disease symptoms, for example at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% when compared to before treatment.
  • the term “subject” generally refers to an individual, e.g., a human, having or at risk of having a specified condition, disorder or symptom.
  • the subject may be a patient i.e., a subject in need of treatment in accordance with the invention.
  • the subject may have received treatment for the condition, disorder or symptom.
  • the subject has not been treated prior to treatment in accordance with the present invention.
  • the compositions and formulations described herein generally can be administered to the subject by any conventional route, including injection or by gradual infusion over time.
  • the administration may, for example, by intramuscular, intravascular, intracavity, intracerebral, intralesional, rectal, subcutaneous, intradermal, epidural, intrathecal, percutaneous administration.
  • compositions, formulations, methods of treatment, and medical uses described herein may provide micelles as described herein to a recipient via any suitable route of administration.
  • compositions and formulations can be administered via any desired route of administration.
  • Compositions and formulations, or medical uses may make use of a route of administration selected from the group consisting of: intravenous (iv) administration; subcutaneous (sc) administration; intramuscular (im) administration; intradermal (id) administration; sublingual (si) administration; and intranasal administration.
  • compositions of the invention for use with the desired route of administration.
  • compositions and formulations described herein are for administration in an effective amount.
  • An “effective amount” is an amount that alone, or together with further doses, produces the desired (therapeutic or non-therapeutic) response.
  • the effective amount to be used will depend, for example, upon the therapeutic (or non-therapeutic) objectives, the route of administration, and the condition of the patient/subject.
  • the suitable dosage of a compositions or formulations of the invention for a given patient/subject will be determined by the attending physician (or person administering the composition), taking into consideration various factors known to modify the action of the compositions or formulations of the invention for example severity and type of disease, body weight, sex, diet, time and route of administration, other medications and other relevant clinical factors.
  • the dosages and schedules may be varied according to the particular condition, disorder or symptom the overall condition of the patient/subject. Effective dosages may be determined by either in vitro or in vivo methods.
  • nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context they are used by those of skill in the art.
  • the elastin-like polypeptide (ELP) diblock copolymer micelles were studied as an adjuvant for vaccine delivery.
  • the micelles can be readily functionalized with peptide or protein antigens by standard recombinant techniques.
  • the resulting micelles are decorated covalently with antigens at the periphery, similar to spike proteins on viruses.
  • the requirement for intracellular processing of covalently conjugated antigens can negatively influence the desired immune response.
  • coiled coil-mediated conjugation is reversible and can release the potential protein.
  • the coiled coil complex is stable, ensuring colocalization, but dissociates at low pH, enabling endosomal escape.
  • Coiled coil-mediated association of antigens to adjuvants is of interest for three reasons. The first is to study the relation between the strength of antigen attachment to the adjuvant and the induced immune response by examining the difference between covalent and non-covalent attachment strategies. Furthermore, each strategy has implications on internal processing and the resulting ability to present the OVA323 epitopes on MHCII. Lastly, the presence of coiled coil domains may increase the immune response due to its tendency to interact with cellular membranes.
  • the peptide antigen “OVA323” (amino acids 323-339 from ovalbumin) was chosen as a model antigen to study the immunogenicity of ELP adjuvants.
  • Three differently functionalized micelles, called “covalent”, “coiled coil” and “hybrid” were designed ( Figure 1), referring to the conjugation strategy of OVA323 to ELP. Since the coiled coil and covalent micelles differ both in the strength of attachment and in the presence of peptide K on the micelle corona, the hybrid micelles were designed to compare the strength of attachment with the coiled coil micelles and the presence of ELP-K with the covalent micelles.
  • the amount of antigen cargo per micelle can be controlled by changing the ratio of plain ELP to functionalized ELP, enabling optimization of the desired immune response.
  • the covalent, coiled coil and hybrid formulations are compared to non-functionalized micelles (“ELP”) and fully functionalized micelles (“ELP-OVA323”).
  • Ultrapure water was obtained using a Milli-Q® system.
  • ELP-E, ELP-K and ELP-OVA323 were constructed from the pET52b- ELP plasmid described below.
  • pET52-ELP-E, pET52-ELP-K, and pET52-ELP-OVA323 were constructed by cloning the respective DNA fragments (BaseClear, Leiden, the Netherlands) into the Acc651 and Notl sites.
  • a pET25 expression vector coding for ELP was provided by the MacKay laboratory.13
  • the Xbal- Acul (blunted by T4 DNA polymerase treatment) DNA fragment comprising the ELP sequence was recloned into Xbal-Smal digested pET52b (+) and maintained in E.coli XL10-Gold.
  • pET52b-ELP plasmid Xbal and BseRI sites are available for inserting DNA fragments upstream, and Acc65l, BamHI, BsrGI, Sall, Eagl, Notl, Sacl, and Avril sites are available for inserting DNA fragments downstream of the ELP coding sequence.
  • ELP, ELP-E, ELP-K and ELP-OVA323 were expressed and purified as described for ELP below.
  • ELP (MG(VPGIG)48(VPGSG)4SY (SEQ ID NO: 7) was expressed by transforming the plasmids into E. coli BL21(DE3) cells using the heat shock and calcium methods. 35
  • the cell culture was grown in LB medium containing ampicillin (250 pg/mL) at 37 °C. A starter culture of 10 mL was added to 1 L of medium and the cells were cultured until an OD600 of ⁇ 0.5. The cultures were cooled to 18 °C and induced overnight with 0.05 mM IPTG. The cells were harvested, washed with 0.9% NaCI solution and the cell pellet was frozen at -80 °C.
  • Cells were lysed in PB containing 1 mM pefablock (Roche Diagnostics), 1 mg/mL lysozyme (Thermo Scientific)), 2 mM MgCh, 25 u/mL benzonase (250 u/pL; Sigma-Aldrich) in a total volume of 10 mL.
  • the mixture was incubated at 4 °C for approximately 45 minutes and sonicated with a 13 mm probe on ice at 25% amplitude for 5 minutes in 5 second intervals.
  • the solids were removed from the lysate by centrifugation at 4 °C (37000 rpm, 228783 ref) for 30 minutes.
  • ELP was purified by inverse transition cycling (ITC). NaCI was added to the lysate to reach a concentration of 4 M. After incubation for 30 minutes at room temperature the mixture was centrifuged (10000 rpm, 17100 ref) for 30 minutes. The pellet was suspended in cold PB and incubated for at least 30 minutes at 4 °C. After centrifugation (10000 rpm, 17100 ref) for 30 minutes at 4 °C, the soluble ELP was collected by decanting the supernatant. 4 additional cycles of ITC were applied, using 3 M NaCI for the incubation at room temperature. The final supernatant was dialyzed against PB. The ELP concentration was determined using UV-Vis spectroscopy. ELP was frozen in liquid N2 and then stored at -20 °C.
  • ITC inverse transition cycling
  • Peptide E was synthesized on a Biotage Syro I fully automated parallel peptide synthesizer using standard Fmoc chemistry.
  • Rink amide resin with a loading of 0.55 mmol/g (Sigma-Aldrich) was used as a support.
  • Coupling reactions were performed with 0.5 M HCTU (Novabiochem) in DMF (Biosolve), 2 M DIPEA (Carl Roth) in a 1 :1 mixture of NMP (Biosolve) and DMF and 0.5 M of the Fmoc-protected amino acid (Novabiochem) in DMF. Deprotection steps were carried out with 40% piperidine (Biosolve) in DMF. All solutions contained 1 g/L LiCI (Sigma-Aldrich).
  • amino acids were coupled to the peptide using double coupling steps.
  • the N- terminus was acetylated with 0.5 M acetic anhydride (Biosolve) and 0.125 M DIPEA in NMP for 2 hours.
  • the peptide was cleaved from the resin using a mixture of 2.5% triisopropylsilane (Sigma- Aldrich), 2.5% water and 95% TFA (Biosolve) and subsequently precipitated in cold diethyl ether (Honeywell). The precipitate was collected by centrifugation, dissolved in water and lyophilized.
  • Peptide K, E-OVA323 and E-OVA323-GC were synthesized on a CEM Liberty Blue automated peptide synthesizer using similar methods as described above.
  • Peptides were purified using preparative reverse phase HPLC on a Shimadzu system consisting of two LC-8A pumps and an SPD-10AVP LIV-VIS detector and equipped with a Kinetex Evo C18 column.
  • the purity of the fractions was confirmed by LC-MS. Pure fractions were combined and lyophilized to obtain the peptide as a powder.
  • LC-MS was measured on a Thermo Scientific TSQ access MAX device for mass detection combined with an Ultimate 3000 liquid chromatography system. Liquid chromatography was performed using a gradient of 0 to 90% acetonitrile in water containing 0.1 % TFA on a 50 x 4.6 mm Phenomenex Gemini 3 pm C18 column.
  • Samples for SDS-PAGE were mixed with reducing Laemmli sample buffer and, without heating first, loaded on a 10% SDS poly acrylamide gel. Electrophoreses was performed at 200 V. The gels were stained with 0.5 M CuCh for 15 minutes and washed with water for 3 x 5 minutes. The stained gels were photographed on a black background. Fluorescently labeled polypeptides were visualized on gels using a Thypoon FLA 9500 Fluorescent Image Analyzer Scanner (GE Healthcare). The percentage of dye attached to the polypeptide with respect to the free dye was determined using Fiji Imaged software.
  • the count rates were normalized to the count rate of PB and plotted as a function of log [ELP], The shape of the autocorrelation functions was used to determine whether particles were detected.
  • One trendline was fitted through the data points for which particles were detected and another through those of non-particulate ELPs.
  • the CMC was determined as the concentration corresponding to the intercept of these trendlines.
  • the DLS measurements for the heating/cooling cycles were performed on a 0 °C sample that had been placed in the 37 °C cell of the instrument. Measurements were started immediately without incubation time at 37 °C. The measurement duration was 1 second at a ⁇ 5 second interval with an attenuator of 8.
  • a 20 pM solution of ELP in milliQ, milliQ and 1 % uranyl acetate (each 10 pL) were placed on a piece of parafilm and incubated at 37 °C for 2 minutes.
  • a preheated copper grid was subsequently placed on top of the ELP, milliQ and staining droplets, every time removing excess liquid by blotting with filter paper.
  • the grid was incubated at 37 °C for 1-2 hours and imaged on a JEOL TEM 1010 electron microscope with an accelerating voltage of 100 kV.
  • Samples for AFM were prepared by drop-casting 20 pL of 37 °C 2 pM ELP or ELP dimer on a silicon oxide wafer (Siegert Wafer) with a 285 nm thermal oxide layer or on a mica disc (V1 grade; Muscovite). The samples were dried at 37 °C for 30 minutes. AFM images were recorded using a JPK NanoWizard Ultra Speed microscope and the obtained data was processed using the JPK SPM Data Processing software. All experiments were performed using a silicon probe (Olympus, OMCL-AC160TS) with a nominal resonance frequency of 300 kHz. The images were all scanned and recorded (with a resolution of 512x512 pixels) in intermittent contact mode in air at room temperature. Mass spectrometry
  • ELP-E The mass of ELP-E, ELP-K and ELP-OVA323 was determined as described for ELP below.
  • Electro-spray ionization was used via Nano-spray source with ESI emitters (New Objectives) fused silica tubing 360 pm OD x 25 pm ID tapered to 5 ⁇ 0.5 pm (5 nL/cm void volume).
  • the following settings in positive resolution mode were used: source temperature of 80 °C, capillary voltage 4.5 kV, nano flow gas of 0.25 bar, purge gas 250 L/h, trap gas flow 2.0 mL/min, cone gas 100 L/h, sampling cone 25 V, source offset 25, trap CE 32 V, scan time 3.0 seconds, mass range 400-2400 m/z.
  • Lock mass acquiring was performed with a mixture of Leu Enk (556.2771) and Glu Fib (785.84265), lockspray voltage 3.5 kV, Glufib fragmentation was used as calibrant.
  • Masslynx software was used for acquisition and Ent3 software for polymer envelope signal deconvolution.
  • the MaxEnt 1 software was used for mass deconvolution of the charge state envelopes.
  • CD spectra were measured on a JASCO J-815 CD spectrometer connected to a Peltier temperature controller using a quartz cuvette with a path length of 1 mm.
  • the reported spectra are averages of 10 spectra that were measured in quick succession, using a range of 190 to 260 nm with 1 nm intervals and a bandwidth of 1 nm.
  • the polypeptide concentration was 25 pM.
  • the sample was incubated at 37 °C for 90 seconds prior to starting the measurement. The measured ellipticity values were corrected for the polypeptide concentration and the number of amino acid residues.
  • E-OVA323-TMR The efficiency of coiled coil association of E-OVA323 to the 10% ELP/ELP-K was studied using E-OVA323-TMR.
  • 175 pL of each sample was incubated at 37 °C for 5 minutes.
  • unbound E- OVA323-TMR was removed using a centrifugal filter unit (Amicon Ultra-0.5 mL) at 10000 ref and 37 °C for 5 minutes.
  • the resulting solutions were washed five times with 175 pL 37 °C 10 mM PB pH 7.8 using the same centrifugation protocol.
  • the samples were diluted with 300 pL PB and the fluorescence was measured using fluorescence spectroscopy.
  • Fluorescence spectroscopy Fluorescence spectra were measured on an FS920 steady state spectrometer from Edinburgh Instruments using an excitation of 541 nm (slit size of 5 nm) and emission of 572 nm (slit size of 1 nm). A series of E-OVA323-TMR concentrations ranging from 62.5 nM to 2 pM was measured to confirm the linear correlation between fluorescent signal and TMR concentration.
  • BMDCs were cultured as described in literature. 37 Bone marrow was taken from the hind legs of wild-type C57BL/6 or TIM4-/- mice. The bone marrow cells were suspended using a 70 pm cell strainer (Greiner Bio-One). The cells were incubated for 10 days at 37 °C and 5% CO2 in Iscove’s Modified Dulbecco’s Medium (IMDM; Lonza) containing 2 mM L-glutamine, 8% (v/v) FCS, 100 U/rnL penicillin/streptomycin (Lonza), 50 pM p-mercaptoethanol (Sigma) and 20 ng/mL GM- CSF (PeproTech). The medium was refreshed every 2-3 days. For the first 9 days, the cells were cultured in 95 mm Petri dishes (Greiner Bio-One) and for the last day in 96 well F-bottom plates (Greiner Bio-One).
  • IMDM Modified Dulbecco’s Medium
  • Fluorescently labeled micelles in PB were diluted 10 times with medium and added to the DCs in 96 well plates (for flow cytometry analysis) and an S well plate (Ibidi GmbH; for confocal microscopy). Hoechst dye (final concentration of 0.01 mg/mL) was added to each of the wells of the 8 well plate. Each well contained 50000 cells in 200 pL. For the negative control BMDCs were treated with medium only. The cells were incubated at 37 °C with 5% CO2 for 4 hours. The cells attached to the bottom of the 8 well plate were washed with medium ten times and imaged by confocal microscopy.
  • the cells in the 96 well plates were isolated by centrifugation and transferred with 100 pL 4 mM EDTA to U-bottom 96 well plates. Excess EDTA was removed by centrifugation and the cells were labeled with live/dead APC-Cy7, CD11cPeCy7, and CD86APC (Invitrogen) in FACS buffer (PBS containing 1 % FCS and 2 mM EDTA) for 20 minutes at 4 °C. Next, the cells were spun down, washed with FACS buffer, spun down again, resuspended in FACS buffer and analyzed by flow cytometry.
  • FACS buffer PBS containing 1 % FCS and 2 mM EDTA
  • T-cells were obtained as described in literature. 38 Spleens from donor mice and were mashed with a syringe plunger and suspended in PBS through a 70 pm cell strainer. The buffer was removed by centrifugation and the red blood cells were lysed for 1 minute with 0.15 M NH4CI, 1 mM KHCO3, 0.1 mM Na2EDTA (pH 7.3).
  • CD4 + T-cells were isolated by negative selection using sheep-anti-rat IgG Dynabeads (Dynal, Invitrogen) and an excess amount of anti-B220 (RA3-6B2), anti-CD11 b (M1/70), anti-MHCH (M5/114) and anti-CD8 (YTS169) mAb in Magnetic-activated cell sorting (MACS; Miltenyi Biotec) buffer.
  • the cells were spun down and resuspended in 1 pM carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes) in PBS. Next, the cells were labeled for 10 minutes at 37 °C and 5% CO2 before removing excess CFSE by centrifugation. A suspension of 250000 cells/mL in medium was prepared and stored at 4 °C.
  • OVA323 Invivogen
  • E-OVA323 in PB Various micelle solutions and peptide solutions of OVA323 (Invivogen) or E-OVA323 in PB were diluted 10 times with medium and added to the DCs in the 96 well plates. Each well contained 10000 cells. OVA323 concentrations were 270 nM, 90 nM, 30 nM, 10 nM, 3.3 nM and 1.1 nM, which corresponds to total polypeptide concentrations of 2.7 pM, 900 nM, 300 nM, 100 nM, 33 nM and 11 nM, respectively.
  • the control samples (ELP, ELP/ELP-K and ELP/OVA323) were only included at a total polypeptide concentration of 2.7 pM.
  • BMDCs were treated with medium only. Cells were incubated at 37 °C with 5% CO2 for 4 hours, spun down and mixed with 50000 T-cells suspended in medium. Next, the cells were incubated for 3 days at 37 °C with 5% CO2. After centrifugation the cells were resuspended in FACS buffer containing CD4efluor, Thy1.2PeCy7, CD25APC, CD67Pe and live/dead APC-Cy7 (Invitrogen). After incubation at 4 °C for 20 minutes, the buffer and excess antibodies were removed by centrifugation and the cells were taken up in FACS buffer. The cells were analyzed by flow cytometry.
  • BMDCs were visualized on an SP8 LIGHTNING Confocal Microscope using a 63x lens.
  • the colocalization percentage was determined using Imaged software by setting the lower and upper thresholds at 15 and 255, respectively and subsequently running the 3D MultiColoc plugin of 3D Image suite.
  • the resulting endotoxin levels in the cell assays were at least 40 times lower than the acceptable concentration of endotoxins in commercial subunit vaccines.
  • the endotoxin levels were determined using HEK-blue TLR4 reporter cells (InvivoGen) expressing either the human (ELP) or murine (ELP-K and ELP-OVA323) TLR4-MD2 receptor complex. Suspensions of 25 thousand cells in 100 pL were mixed with 100 pL polypeptide or LPS samples at various concentrations in a 96 well plate (Greiner Bio-One). The plate was incubated at 37 °C overnight, resulting in the activation of the TLR4 pathway, which was detected by secretion of embryonic alkaline phosphatase (SEAP).
  • SEAP embryonic alkaline phosphatase
  • the SEAP levels were measured with a QUANTI-Blue assay. First, 20 pL of the cell supernatants were incubated with 180 pL QUANTI-Blue solution (rep-qb1 , InvivoGen) for 2 hours at 37 °C. Next, the optical density (OD) at 650 nm was measured with a Tecan i- control 1.7.1.12 plate reader. The endotoxin levels in the covalent, coiled coil and hybrid micelles were ⁇ 5 Ell/rng.
  • ELP consists of a hydrophobic block at the N-terminus and a hydrophilic block at the C-terminus, facilitating assembly into well-defined micelles.
  • 31 ELP-E, ELP-K and ELP-OVA323 were also expressed in Escherichia Coli (E. Coli) and purified with five cycles of inverse transition cycling (ITC). The purity of samples at various stages was studied by SDS PAGE and Coomassie Blue (ELP-E and ELP-K) or copper chloride (ELP-OVA323) staining (Figure 7). Copper chloride staining was used, because ELP-OVA323 does not contain enough positively charged amino acids required for efficient Coomassie Blue staining.
  • ELP-E and ELP- OVA323 were purified more efficiently in the first two cycles than ELP-K.
  • the non-ELP moieties may have some affinity for E. coli proteins, encapsulating these into the ELP aggregates and thereby complicating the purification. Conjugation of peptide K might be most problematic due to the amphiphilic character, causing ELP-K to bind various proteins.
  • an impurity of approximately 70 kDa was especially difficult to remove.
  • ELP- E and ELP-K and ELP-OVA323 were at least 95% pure after five cycles of ITC, as confirmed by HPLC (Figure 7).
  • ELP-E and ELP-K were studied with SLS measurements to analyze the effect of extending ELPs with a coiled coil peptide on the self-assembly in solution.
  • the ELPs were dissolved in phosphate buffer (PB) and light scattering was recorded as a function of temperature.
  • PB phosphate buffer
  • the onset of ELP-E assembly occurred at a higher temperature as compared to ELP, while ELP-K assembly was already observed at a lower temperature ( Figure 8).
  • the CMT is defined as the temperature at which the polypeptide starts to aggregate, resulting in an increase in scattering.
  • Extension of ELP with peptide E resulted in a 4 °C increase in CMT (22 vs. 26 °C).
  • the scattering intensity plateau is higher for ELP-E than ELP. This can be due to either an increased number of micelles formed by ELP-E or the formation of larger micelles.
  • DLS revealed no difference in size for ELP and ELP-E micelles (Table 2), thus the increased scattering of ELP-E at the plateau can be contributed to an increase in particle number.
  • ELP-K exhibited a different inverse transition behavior than ELP and ELP-E as ELP-K tends to assemble into large aggregates.
  • the critical aggregation temperature (CAT) of ELP-K was ⁇ 16 °C ( Figure 8).
  • Peptide K is prone to homodimerization and is also known to interact with membranes, burying the hydrophobic face of the helix in the bilayer while the lysine side chains “snorkel” towards the polar/nonpolar interface. 25 Therefore it is likely that peptide K also has an affinity for the hydrophobic domain of ELP micelles. This might result in intra-micellar and inter-micellar interactions between peptide K and the ELP core inducing severe aggregation.
  • CMC critical micelle concentration
  • CAC critical aggregation concentration
  • ELP micelles Before using ELP micelles as an adjuvant, micelle formation was studied as a function of ELP to coiled coil peptide ratio. For this, ELP and ELP-E or ELP-K were mixed in the absence and presence of the complementary water-soluble peptide K or E ( Figure 11 and 12).
  • Table 3 Composition and size of coiled coil displaying ELP micelles
  • ELP/ELP-K (9:1) mixtures formed stable micelles at 37 °C with a hydrodynamic diameter comparable to plain ELP micelles (54 nm and 61 nm, respectively).
  • ELP-K apparently interacts with neighbouring micelles, possibly due to homocoiling 25 or the general affinity of peptide K for hydrophobic structures.
  • ELP/ELP-K (9:1) micelles were somewhat larger and more polydisperse as compared to ELP/ELP-E or plain ELP micelles (Table 2).
  • Transmission electron microscopy (TEM) revealed the formation of spherical micelles with comparable size distributions for all formulations ( Figure 15).
  • the zeta potential was near neutral for all formulations due to the absence of charged amino acids in ELP, the major component in these assemblies.
  • ELP-E micelles had a more negative zeta potential (-10.9 mV), because peptide E contains multiple glutamates.
  • ELP-OVA323 was comparable to ELP (0.10 pM vs. 0.15 pM; Figures 9 and 10). Likewise, size and surface charge of ELP-OVA323 micelles are identical to ELP micelles (Table 4). Thus, covalent conjugation of the OVA323 epitope to ELP did not alter its self-assembly behavior.
  • Covalent, coiled coil and hybrid micelles were prepared by mixing the individual polypeptide and peptide solutions in the correct ratio and heating the resulting formulations to 37 °C.
  • the resulting assemblies were analyzed with DLS and zeta potential measurements revealing comparable size and surface charge of these micelles, albeit with a higher polydispersity compared to ELP or ELP-OVA323 micelles (Table 4).
  • the surface charge of each micelle formulation was near neutral, which is expected as each formulation contains non-charged ELP as the main component.
  • the morphology of the micelles was investigated with AFM (Figure 17; and Figures 18 to 20) and transmission electron microscopy (TEM) ( Figure 21).
  • ELP-K facilitates uptake of micelles in dendritic cells
  • BMDCs were exposed to the micelle formulations, each containing 8% FITC-labeled ELP (FITC-ELP).
  • FITC-ELP FITC-labeled ELP
  • 17 18 ELP/ELP-K (9:1) micelles were also included in this study.
  • the coiled coil sample contained TMR-labeled E-OVA323 (15% labeled). The use of different fluorescent dyes for E-OVA323 and ELP enabled us to study the antigen uptake independently from the micelle uptake.
  • Free E-OVA323-TMR peptide was included as a control for micelle-independent uptake.
  • BMDCs were incubated for 4 hours with the various samples and analyzed with flow cytometry, showing that micelle uptake is concentration dependent (Figure 23).
  • E-OVA323 was not internalized by the BMDCs, while conjugation to ELP-K containing micelles via coiled coil formation ensured efficient antigen uptake.
  • Most of the OVA (i.e. , TMR signal) positive cells were also ELP (i.e., FITC signal) positive: 93%, 80% and 59% for 270 nM, 90 nM and 30 nM OVA323, respectively.
  • mice uptake in BMDCs was confirmed with confocal microscopy ( Figure 13). No cell uptake was observed for ELP micelles or covalent micelles, which is not fully in line with the (relatively low number of) ELP positive cells measured in these samples with flow cytometry. The micelles may not have been internalized in these ELP positive cells, but merely adhered to the outside of a cell membrane. Cells were thoroughly washed before confocal microscopy imaging and noninternalized micelles and were therefore not observed. In contrast, micelles containing ELP-K (i.e., ELP/ELP-K, coiled coil and hybrid) were internalized efficiently inside cells. Thus, plain ELP micelles do not effectively enter the BMDCs at this concentration.
  • ELP-K i.e., ELP/ELP-K, coiled coil and hybrid
  • DC maturation is the first step to effectively stimulate T helper cells. Therefore, expression of the costimulatory molecule CD86, a marker for DC maturation, was quantified. ELP-K containing micelles induced more CD86 expression as compared to micelles without this polypeptide ( Figure 27). This shows that peptide K may stimulate the immune response by inducing both increased maturation and more efficient uptake of antigens. Peptide K is known to interact with membranes and potentially induces membrane disruption, 25 which could in turn result in enhanced BMDC maturation. 36
  • CD4 + T-cells were isolated from OT-II transgenic mice, which exclusively contain T-cells with OVA323-specific T-cell receptors. These T-cells were labeled with carboxyfluorescein diacetate N-succinimidyl ester (CFSE) and cocultured with BMDCs previously exposed to the micelle formulations. Finally, subsequent T-cells proliferation was quantified with flow cytometry.
  • CFSE carboxyfluorescein diacetate N-succinimidyl ester
  • the lower antigen loading per micelle may also be favorable.
  • the covalent micelles had a similar effect on T-cell proliferation as free OVA323.
  • the positive influence of increased uptake by the micelles may have compensated for the negative influence of antigen processing issues related to covalently attaching ELP to OVA323.
  • the coiled coil and hybrid groups did have significantly stronger effects on T-cell proliferation compared to their free peptide counterparts E-OVA323 and OVA323, respectively. These groups also outperformed the covalent group, suggesting the presence of peptide K provides an additional advantage over plain ELP micelles. Taken together, these results indicate the presence of ELP-K stimulates division of T-cells. This is likely caused by the increased uptake of ELP-K-containing formulations as well as the higher level of DC maturation.
  • ELP-E and ELP-K were successfully expressed and purified. Both peptide E and peptide K had a distinct effect on the inverse transition behavior of ELP.
  • ELP-E formed micelles similar to ELP, while ELP-K assembled into large aggregates. The CMT of ELP-E was higher compared to ELP, while the CAT of ELP-K was lower. Above the CMT, ELP-E had a higher particle concentration than ELP.
  • ELP-K was incorporated into micelles by co-assembly with ELP. Aggregation of these ELP/ELP-K mixed micelles could be prevented in the presence of an equimolar amount of peptide E.
  • the model antigen OVA323 was expressed as a fusion protein (ELP-OVA323) and the selfassembly behavior was comparable to unmodified ELP.
  • Covalent micelles were compared to coiled coil micelles and hybrid micelles. All micelle formulations had a comparable diameter and zeta potential.
  • ELP-K stimulated the uptake in DCs, as confirmed by flow cytometry and confocal microscopy studies.
  • coiled coil-associated OVA323 was colocalized with the micelles, which increases its immunogenicity. 12-14 Moreover, the colocalization of antigen and micelle confirms that coiled coil complexes remain intact at the ELP micelles surface in the presence of DCs.
  • ELP-K also induced increased DC maturation, in agreement with a previous study using peptide-K modified polymersomes.
  • BMDCs treated with micelles containing ELP-K induced the highest level of CD4 + T-cell proliferation.
  • a similar effect of peptide K on CD4 + T-cell proliferation was observed for liposomes with coiled coil-associated OVA323.
  • conjugation of OVA323 to ELP micelles negatively affected T-cell expansion.
  • peptide-K containing micelles enhance BMDC uptake and maturation, as well as subsequent CD4 + T-cell proliferation.
  • Keijzer, C. et al. PLGA, PLGA-TMC and TMC-TPP nanoparticles differentially modulate the outcome of nasal vaccination by inducing tolerance or enhancing humoral immunity.
  • Example 2 ELP micelle platform for birch pollen allergy immunotherapy
  • alum colloidal aluminum salts
  • SCIT subcutaneous allergen immunotherapy
  • ELP temperature-sensitive elastin-like polypeptide
  • the fusion protein ELP-Bet v 1 was expressed and purified by a combination of immunoaffinity chromatography and inverse transition cycling.
  • DLS dynamic light scattering
  • AFM atomic force microscopy
  • TEM transmission electron microscopy
  • the mixed ELP/ELP-Bet v 1 micelles showed to be hypoallergenic (10-fold), as determined by measuring mediator release from rat basophilic leukemia (RBL) cells, expressing the human IgE receptor and sensitized with human birch pollen specific IgE.
  • An increased protective antibody response with an earlier onset for ELP/ELP-Bet v 1 compared to alum- adsorbed Bet v 1 was observed in an in vivo study using naive mice.
  • ELP/ELP-Bet v 1 micelles did not induce a Th2 skewing immune response as evidenced by the induction of background to low levels of IL-4, IL-5 and IL-13 cytokines.
  • the hypoallergenic character and strong humoral immune response combined with the absence of a Th2 skewing T- cell response make ELP-based nanoparticles a promising candidate to replace alum in SCIT applications
  • Bet v 1 -displaying ELP micelles with the fusion protein ELP-Bet v 1 and 9 equivalents of plain ELP were prepared (Table 5) and by subsequently heating the resulting mixture
  • These “ELP/ELP- Bet v 1” micelles were characterized using static light scattering (SLS), dynamic light scattering (DLS), atomic force microscopy (AFM) and transmission electron microscopy (TEM).
  • SLS static light scattering
  • DLS dynamic light scattering
  • AFM atomic force microscopy
  • TEM transmission electron microscopy
  • CD circular dichroism
  • IgE binding assay the hypoallergenic character was studied by performing a rat basophil leukemia (RBL) assay.
  • ELP/ELP-Bet v 1 was compared to alum-adsorbed Bet v l in a mouse immunogenicity study.
  • Ultrapure water was obtained using a Milli-Q® system.
  • mice Six to eight weeks old female BALB/c mice were purchased from ENVIGO (The Netherlands). The animals were housed under specific, pathogen free conditions at the animal facility of the Academic Medical Center. All experiments were approved by the Animal Ethics Committee of the AMC.
  • the plasmid coding for ELP-Bet v 1 was constructed from the pET52b-ELP plasmid described in Example 1 .
  • pET52-ELP-Bet v 1 was constructed by cloning the Bet v 1 gene (BaseClear, Leiden, the Netherlands) into the Acc651 and Notl sites.
  • Bet v 1 (Bet v 1.0101) was purchased from the Department of Molecular Biology of the University of Salzburg, where it had been expressed in E. coli and purified according to previously established purification protocols.
  • ELP, ELP-Bet v 1 and ELP-K were expressed as described for ELP in Example 1 . Purification of ELP and ELP-K
  • ELP and ELP-K were purified as described in Example 1.
  • the lysate was first purified using immunoaffinity chromatography at 4 °C.
  • a Bet v 1 specific monoclonal antibody, 5H8H9 43 was coupled to cyanogen bromide-activated Sepharose 4B (GE Healthcare) according to manufacturer’s instructions.
  • the lysate was loaded on the column that had been equilibrated with 10 mM PB.
  • the column was washed with 5 column volumes of PB and eluted with 100 mM glycine pH 2.5 (Merck).
  • the fractions were immediately neutralized with 1 M Tris pH 8.8.
  • the elution fractions containing ELP-Bet v 1 were combined, concentrated and rebuffered to 10 mM PB.
  • the protein was further purified by one cycle of inverse transition cycling as described above for the purification of ELP.
  • the final ELP-Bet v 1 solution was dialyzed against 10 mM PB pH 7.8.
  • Protein analysis by SDS-PAGE was carried out as described for ELP in Example 1 .
  • the gels were stained with Coomassie Brilliant Blue-R250 (Bio-Rad).
  • ELP-Bet v 1 The mass of ELP-Bet v 1 was determined as described for ELP in Example 1 .
  • DLS and SLS measurements were done as described for ELP in Example 1 .
  • the measurements of the dilution into Tyrode’s buffer were done by first heating a cuvette containing 495 pL of Tyrode’s buffer (containing 9.5 g/L Tyrode's salts, 0.1% (w/v) BSA and 0.5 g/L NaHCOs) at 37 °C for 5 minutes inside the DLS device. Then 5 pL of cold 100 pM ELP or ELP/ELP-Bet v 1 was injected into the buffer and the measurement was started immediately.
  • the zeta potential of the samples was determined as described for ELP in Example 1.
  • IgE binding of ELP-Bet v 1 and ELP/ELP-Bet v 1 was determined by ImmunoCap IgE inhibition assay in triplicate. The samples were diluted in 10 mM PB, 280 mM sucrose, pH 7.4 to 540 nM Bet v 1 concentration. A pool of 36 birch pollen allergic patient sera was diluted to 12 kU/mL IgE and added 1 :1 (v/v) to the samples followed by incubation at room temperature for 1 hour. Uncomplexed IgE was measured on a Phadia 250 with rBet v 1 caps (t215), following the manufacturer’s instructions. Inhibition values were calculated using the uninhibited signal (PBS + serum) and maximally inhibited signal (PBS) as references. Bet v 1 and ELP were used as controls.
  • the fluorogenic substrate 4-methyl umbelliferyl-N-acetyl-beta-D-glucosaminide (Sigma-Aldrich) was used and measured at an excitation and emission wavelength of 360 nm and 465 nm, respectively.
  • the data were corrected for spontaneous release (untreated cells) and normalized to the maximal enzyme release caused by cell lysis (10% Triton X-100, Sigma-Aldrich).
  • the ELP-Bet v 1 group was replaced by a phosphate buffered sucrose alum group as negative control. Serum immunoglobulin levels were measured in serum samples taken via puncture of the vena saphena at days -1 , 6, 13 and 20.
  • mice received 100 pg/mL birch pollen extract in PBS intranasally under 3% (v/v) isoflurane anesthesia to further boost antibody production.
  • blood and lung draining lymph nodes were collected to analyze Bet v 1 specific lgG1 , lgG2a, and IgE levels, and the production of IL- 4, IL-5, IL-13, IL-10, IL-17A and IFN-y, respectively.
  • Bet v 1 specific IgE, lgG1 and lgG2a antibodies in serum were analyzed as described previously. 44 Briefly, NUNC Maxisorp plates were coated overnight with 5 pg Bet v 1 . The next day, the plates were blocked with FCS, followed by incubation with the serum samples. After washing, bound immunoglobulins were detected with horse radish peroxidase conjugated specific antibodies against mouse IgE, IgG 1 (Opteia, BD, San Diego, CA, USA) and lgG2a (eBioscience), according to the manufacturer’s instructions. Serum samples of all groups were diluted 10-fold for IgE detection.
  • Serum samples for lgG1 and lgG2a detection were diluted 100-fold except for the ELP/ELP-Bet v 1 and alum-adsorbed Bet v 1 groups which were diluted between 100- and 10000-fold depending on the measured time point.
  • Lung draining lymph node cell suspensions were plated in a 96 well round bottom plate at a density of 2 ⁇ 10 5 cells per well and were re-stimulated for 4 days with Bet v 1 .
  • Expression levels of cytokines IL-4, IL-5, IL-9, IL-13, IFN-y and I L-17A were determined in the supernatant by ELISA (eBioscience).
  • ELP-Bet v 1 was expressed in Escherichia Coli (E. Coli). After induction the cells produced a protein of approximately 60 kDa, which is illustrated by SDS PAGE ( Figure 33) by the extra band in the lane of the sample after induction (+) with respect to the sample before induction (-). The gel further shows the purification process of this protein, which consists of immunoaffinity chromatography and inverse transition cycling (ITC). The lysate containing ELP-Bet v 1 was loaded on a Bet v 1 specific monoclonal antibody column. Most proteins in the lysate did not bind to the column (FT) and were washed out (W1-3).
  • ELP-Bet v 1 was eluted at pH 2.5 (E1-4) and the elution fractions were immediately neutralized using Tris buffer pH 8.8. The combined elution fractions still contained some impurities.
  • PB phosphate buffer
  • E1-4 concentration of the resulting solution
  • NaCI was added (3 M) to precipitate the protein (P1), which was collected by centrifugation at room temperature. The supernatant did not contain any substantial amount of protein (S1).
  • the pellet was resuspended in cold 10 mM PB and centrifuged again at 4 °C. All protein impurities and some of the ELP-Bet v 1 remained in the pellet (P2).
  • the P2 lane shows a strong ELP-Bet v 1 band.
  • the actual amount of target protein lost in this step is estimated by comparing the ELP-Bet v 1 band in E1-4 with the same band in the supernatant of the cold centrifuging step (S2).
  • the bands have similar intensity and therefore, no substantial amount of ELP-Bet v 1 was lost in P2.
  • S2 contains pure ELP-Bet v 1 protein. The yield was 7 mg per liter culture, which is a factor 4 to 8 lower than the yields of ELP-based polypeptides reported in Example 1.
  • Conjugation of Bet v 1 to ELP increased the critical micelle temperature (CMT) from 22 °C to 28 °C ( Figure 36).
  • ELP/ELP-Bet v 1 had a CMT of approximately 24 °C.
  • the inverse transition temperature of ELP/ELP-Bet v 1 is in between those of ELP and ELP-Bet v 1 , even though the major component in this mixture is ELP. This indicates that conjugated Bet v 1 suppresses the ELP self-assembly, even of the ELP unimers.
  • ELP/ELP-Bet v 1 micelles At body temperature (37 °C), both ELP-Bet v 1 and the ELP/ELP-Bet v 1 mixture assembled into micelles with a size slightly larger size and polydispersity index as compared to ELP (Table 6).
  • ELP/ELP-Bet v 1 micelles had a near-neutral zetapotential as expected the formulation consists of 90% neutral ELP.
  • the ELP-Bet v 1 micelles however had a negative zetapotential of -12.0 mV, which is consistent with the net charge of ELP-Bet v 1 of -5.
  • the ELP/ELP-Bet v 1 micelles were visualized with both TEM ( Figure 38) and AFM ( Figure 39). Both techniques show the micelles are spherical in shape. The observed diameter by TEM imaging could not be measured accurately because it changed depending on how the sample was focused.
  • the height of the micelles as measured with AFM was similar to ELP micelles (15 vs 17 nm. The difference between AFM-determined height and DLS-determined hydrodynamic diameter is expected, as hydrodynamic diameters are usually larger than imaged diameters/heights by either electron microscopy or AFM. In addition DLS is strongly biased towards particles with the largest diameter. 25
  • hypotonic PB 10 mM, pH 7.6
  • the buffer was supplemented with 280 mM sucrose and the hydrodynamic diameters were measured. All micelle formulations appeared to be somewhat smaller in this isotonic buffer (Table 7) compared to the hypotonic 10 mM PB (Table 6).
  • the Pdl values are similar for both buffers. Possibly the presence of sucrose further drives the hydrophobic collapse of the hydrophobic ELP block, making the micelle more compact.
  • ELP particles induce strong humoral but weak T-cell responses in naive mice
  • mice injected with alum-adsorbed Bet v 1 were all similar to the buffer group in the first study, while these were significantly higher up from day 13 in the second study.
  • lgG2a levels induced by ELP/ELP-Bet v 1 were significantly higher than the levels induced by alum- adsorbed Bet v 1 (18.6-fold), while these levels were similar at the endpoint.
  • alum induces weak antigen specific humoral immune responses when administered via the subcutaneous route, while the humoral immune response induced by ELP micelles is stronger, with an earlier onset.
  • the results of both studies were different, despite the same set-up. Similar differences between the results of two such immunogenicity studies with the same design were observed by the Ferreira group. 3233 Additionally, the IgG responses to alum in that study were also detected only after all immunizations and challenges with birch pollen had been administered.
  • alum-adsorbed Bet v 1 showed significant expression of Th2 cytokines IL-4, IL-5 and IL-13.
  • alum-adsorbed Bet v 1 showed expression of regulatory T-cell (Treg) related IL-10 but not Th1 cytokine IFN-y and Th17- related cytokine IL-17A.
  • the Treg-associated IL-10 levels in the ELP/ELP-Bet v 1 group were similar to the alum- adsorbed Bet v 1 group, while Th2 cytokines IL-5 and IL-13 were significantly lower (2.6-fold and 1.8-fold, respectively) for ELP/ELP-Bet v 1 compared to alum-adsorbed Bet v l ELP-Bet v 1 induced a similar cytokine profile as ELP/ELP-Bet v 1 ( Figure 47).
  • alum in SCIT is able to induce a regulatory skewed immune response after repeated administrations, it is also known as a driver of Th2 immune responses.
  • Our murine in vivo data confirmed that alum is a typical Th2 adjuvant.
  • the ELP/ELP-Bet v 1 micelles showed a weak, Th2-skewed immune response, indicated by variable and low IL-4, IL-5 and IL-13 expression.
  • other pro-inflammatory cytokines such as IFN-y and IL-17A were not induced, suggestive of an overall weak T-cell response.
  • IFN-y is associated with lgG2a induction but in the alum-adsorbed Bet v 1 and ELP/ELP-Bet v 1 groups IFN-y was not detected, despite measuring significant lgG2a levels.
  • IFN-y producing T-cells necessary for lgG2a isotype switching were short-lived and therefore not detectable at the study endpoint.
  • the overall weak pro-inflammatory T-cell response could be related to the non-immunogenic character of ELP that was used in our design and because Bet v 1 requires pollen derived factors to stimulate DCs and induce T-cell polarization.
  • IL-10 produced by regulatory T- and B-cells is able to suppress the ongoing, Th2 associated allergic inflammation during SCIT.
  • 41 Besides a generally weak pro-inflammatory immune response the ELP/ELP-Bet v 1 micelles also induced Treg associated IL-10. These IL-10 levels were 1.4-fold lower than those induced by alum-adsorbed Bet v 1. This could be linked to the much weaker pro-inflammatory response of ELP/ELP-Bet v 1 , because Th2 cells can also produce IL-10.
  • allergen-specific regulatory T-cells producing IL-10 have been shown to be crucial to suppress Th2 associated allergic inflammation.
  • ELP/ELP-Bet v 1 micelles were investigated as a possible candidate to replace alum in SCIT.
  • Spherically shaped ELP/ELP-Bet v 1 micelles were produced, the ⁇ 50 nm size of which are suitable as an antigen delivery vehicle and as non-Th2 adjuvant.
  • characterization of these particles showed that the CMT is 24 °C, which is below body temperature of both mice and humans ensuring the presence of micelles inside the body.
  • This in vivo micellization is further supported by the CMC, which is a 465-fold lower than the concentration used for the immunogenicity study.
  • the data shows that rapid micelle formation occurs when a cold concentrated solution of the polypeptides is diluted into Tyrode’s buffer at 37 °C.
  • Bet v 1 was correctly folded and accessible on the corona of ELP/ELP-Bet v 1 micelles, as evidenced by the CAP inhibition assay. Despite being fully recognized by IgE antibodies, their IgE cross-linking capacity was reduced, as measured by the higher antigen concentrations required to get the same level of mediator release. These results are considered ideal for a SCIT candidate, since correctly folded of Bet v 1 seems more likely to induce blocking antibodies that can recognize natural Bet v 1 after exposure to birch pollen, while the hypoallergenic character of ELP/ELP-Bet v 1 can reduce adverse effects.
  • the ELP/ELP-Bet v 1 micelles induced earlier and stronger lgG1 and lgG2a antibody levels compared to alum adsorbed Bet v 1. Although alum-based SCIT is able to induce a regulatory skewed immune response after repeated administrations, it is also known as a driver of Th2 immune responses. 14 The murine in vivo data confirmed alum as a typical Th2 adjuvant. In contrast, the ELP/ELP-Bet v 1 micelles lacked a Th2 skewing effect, indicated by the low IL-5 and IL-13 expression.
  • T regulatory cytokine IL-10, Th1 cytokine IFN-y and IL-17A were also low and, in case of IFN-y and IL-17A, did not differ from the control groups, suggestive of an overall weak T helper cell response.
  • the ELPs not only facilitate the manufacturing of this vaccine but also induce specific immune responses that may improve the efficacy of allergy specific immunotherapy.
  • the strong humoral response might reduce the treatment frequency and duration whereas the weak Th2 skewing effect and hypoallergenic character of ELP based nanoparticles contribute to the safety of SCIT.

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Abstract

The present invention provides compositions for modulating an immune response in a subject. The compositions include micelles formed from elastin-like peptides (ELPs) that may be bound to immunomodulators. The invention also provides uses of such micelles as adjuvants. Also provided are pharmaceutical formulations including the compositions for use as medicaments, particularly for use in treating cancers, allergy and infectious disease.

Description

Immunotherapeutic compositions and adjuvants
The present invention provides compositions for modulating an immune response in a subject. The compositions include micelles formed from elastin-like peptides (ELPs) that may be bound to immunomodulators. The invention also provides uses of such micelles as adjuvants. Also provided are pharmaceutical formulations including the compositions for use as medicaments, particularly for use in treating cancers, allergy and infectious disease.
Background
Vaccination is an effective method of preventing infectious diseases and decreasing associated mortality. Traditional vaccination strategies are based on live attenuated or inactivated pathogens. These conventional vaccines are linked to various adverse effects. In the search for safer vaccines, research is becoming increasingly focused on subunit vaccines, containing antigenic components of pathogens and omitting any unnecessary and harmful elements. Subunit vaccines are considered safer than traditional vaccines, but at the cost of lower efficacy.
Since subunit vaccines do not generate the same level of immunity, they require an adjuvant. The most commonly used adjuvant is alum, which refers to micron-sized particles based on aluminium salts. However, injections with alum can cause local adverse effects and are also associated with some systemic side effects. Moreover, alum is unsuitable for inducing immunity against intracellular targets such as tuberculosis, legionella and malaria, because it primarily triggers a strong humoral response.
For over a century, subcutaneous allergen immunotherapy (SCIT) using allergen extracts has been used as a disease modifying allergy treatment. Despite its proven efficacy, the allergy market is still dominated by anti-symptomatic drugs. This can be explained by low therapy adherence due to both the 3-5 year duration of SCIT and the frequent occurrence of allergic side effects.
Allergen immunotherapy (AIT) and SCIT aim to induce lgG1 and lgG4 (in humans) or lgG1 and lgG2a (in mice), which compete with IgE to bind allergens, to prevent IgE-mediated effector mechanisms. These so-called blocking antibodies are produced by regulatory B-cells. The induction of regulatory T-cells is also considered a beneficial effect of AIT. Both types of regulatory cells produce interleukin 10 (IL-10). Besides these regulatory responses, a switch from a Th2- to a Th1-biased immune response is generally favourable for AIT. This switch is measured by the induction of interferon y (IFN-y). An allergen specific IgE response, as well as the cytokines IL-4, IL-5 and IL-13 are related to adverse effects. SCIT generally contains aluminium-based adjuvants (alum) such as aluminium hydroxide or aluminium phosphate. Even though alum has been regarded as a safe adjuvant in vaccines for infectious diseases and SCIT for a long time, there is growing concern about chronic alum exposure during SCIT, especially in the paediatric setting. Some reports have linked alum overexposure to systemic adverse events including renal failure and various neurodegenerative disease such as Alzheimer’s. Therefore, there is a growing need to replace alum with a different adjuvant in SCIT.
During SCIT, alum is known for inducing a mixed Th1/Th2/Treg immune response. SCIT aims to counteract Th2-skewed immune responses, and this is achieved by prolonged exposure to alum- adsorbed allergens. At the start of SCIT, the Th2 stimulation by alum results in a transient increase in IgE which may contribute to adverse events. Compared to alum-like microparticles, small particles (<600 nm) induce lower Th2 responses while causing increased DC maturation. Additionally nanoparticles (<200 nm) are also transported to the lymph nodes and taken up by lymph node resident DCs. Therefore, attention has focused on various types of nanoparticles (NPs) to replace micron-sized alum in allergy vaccine delivery systems.
Good adjuvants increase the uptake of the antigen into antigen presenting cells (APCs), such as dendritic cells (DCs) and also promote the subsequent step of DC maturation, which involves the development of additional dendrites and expressing major histocompatibility complex II (MHCII) and costimulatory molecules such as CD80 and CD86.8 These co-stimulatory molecules are presented on the DC surface and can interact with CD28 on CD4+ T-cells, thereby stimulating T- cells to expand and differentiate. Differentiation of T-cells has a major impact on the induced immune response. While differentiation into T helper 1 (Th1) cells allows the induction of type I cytokines like interferon y (IFN-y) and tumour necrosis factor a (TNF-a), leading to potentiation of the cellular immunity, differentiation into Th2 cells primarily leads to induction of interleukin 4 (IL- 4), IL-5 and IL-13, which strengthen the humoral arm of the immune response. Adjuvants can influence the differentiation of the immune response towards Th1 or Th2, depending on the physicochemical properties of the adjuvant nanoparticles, such as size, shape, surface charge, rigidity and particle composition.
There is a need for an improved immunoregulatory compositions that may be used as vaccines, such as subunit vaccines, or for immunotherapy. There is also a need for improved adjuvants for use in vaccines and immunotherapy.
Brief summary of the disclosure
Elastin-like polypeptides (ELPs) are composed of repeats of the pentapeptide VPGXG in which guest residue X can be any amino acid. ELP-based micelles comprise amphiphilic ELPs of which the hydrophobic block contains a different guest residue from the hydrophilic block. These polypeptide diblock copolymers self-assemble into spherical micelles above the critical micelle temperature (CMT). ELP assembly is induced by hydrophobic collapse of the hydrophobic ELP block, forming the micelle core with the hydrophilic blocks pointing outwards in solution. ELP- based micelles are a promising platform for drug delivery due to their biocompatibility, facile design regarding physicochemical properties1321-23 and simple modification with cargo. However, the adjuvant properties of ELP micelles have not been determined previously.
The invention is based on the surprising finding that ELP based micelles comprising an immunomodulator can act as adjuvants which show improved properties in comparison to alum (particularly due to their ability to induce a less T helper 2 (Th2)- and more Th1 -skewed immune response). In the context of vaccines for use in cancer immunotherapy or vaccination against infectious disease the ELPs provide an adjuvant that has an improved cellular mediated response and reduced humoral response. In the context of allergic diseases, the ELP based micelles have also been found to reduce IgE and increase IgG, demonstrating that ELP based micelles are prime candidates for use in allergy immunotherapy, particularly as an alternative adjuvant to alum for subcutaneous allergy immunotherapy.
In a first aspect of the invention, there is provided a composition comprising a micelle, wherein the micelle comprises one or more elastin-like polypeptides (ELP) and one or more immunomodulators. The composition can be used to modulate an immune response in a subject.
In certain embodiments, the one or more ELPs are amphiphilic. For example, the ELPs comprise at least one hydrophobic block and at least one hydrophilic block. In certain embodiments, the one or more ELPs comprise amphiphilic copolymers. In certain embodiments, the one or more ELPs comprise amphiphilic diblock copolymers. The use of amphiphilic ELPs provides a composition wherein the ELPs are configured to self-assemble into the micelles.
In certain embodiments, at least one of the one or more ELPs comprise the sequence (VPGXhbG)n(VPGXhPG)n (SEQ ID NO: 1) or (VPGXhPG)n(VPGXhbG)n (SEQ ID NO: 2) wherein XhP is any hydrophilic amino acid residue and Xhb is any hydrophobic amino acid residue. In certain embodiments, Xhp is serine. In certain embodiments, Xhb is isoleucine.
In certain embodiments, at least one of the one or more ELPs comprise the sequence MX1(VPGXhbG)48(VPGXhPG)48X2 (SEQ ID NO: 3), wherein X1 and X2 are each independently selected from any amino acid. In certain embodiments, at least one of the one or more ELPs comprise the sequence MG(VPGXhbG)n(VPGXhPG)nY (SEQ ID NO: 4). In certain embodiments, at least one of the one or more ELPs comprise the sequence (VPGIG)n(VPGSG)n (SEQ ID. NO: 5). In certain embodiments, at least one of the one or more ELPs comprise the sequence (VPGIG)48 (VPGSG)4s (SEQ ID NO: 6). In certain embodiments, at least one of the one or more ELPs comprise the sequence of MG(VPGIG)48(VPGSG)4sY (SEQ ID. NO: 7). In certain embodiments, the ELP comprises the sequence MG(VPGIG)4s(VPGSG)48 (SEQ ID NO: 32).
The ELPs may facilitate induction of a Th1 -skewed immune response in a subject in response to an immunomodulator. In addition, the ELPs may facilitate switching from a Th2- to a Th1-biased immune response to an immunomodulator.
In certain embodiments, the immunomodulator is covalently bound to the ELP. In certain embodiments, the immunomodulator is covalently bound to the ELP to form a ELP- immunomodulator fusion protein. In certain embodiments, the immunomodulator is covalently bound to the ELP by chemical conjugation.
In certain embodiments, the immunomodulator is covalently bound to the ELP so that the immunomodulator is located on the external surface of the micelle. In certain embodiments, the immunomodulator is covalently bound to a hydrophilic block of an ELP. In certain embodiments, the immunomodulator is covalently bound to the C-terminal of an ELP comprising a C-terminal hydrophilic block.
In certain embodiments, the ELP-immunomodulator fusion protein comprises the sequence “(VPGXhbG)n(VPGXhpG)n-immunomodulator” or “immunomodulator-(VPGXhPG)n(VPGXhbG)n”. In certain embodiments, the ELP-immunomodulator fusion protein comprises the sequence “(VPGIG)48(VPGSG)48-immunomodulator”. In certain embodiments, the ELP-immunomodulator fusion protein comprises the sequence “MG(VPGIG)48(VPGSG)48-immunomodulator” or “immunomodulator-MG(VPGIG)48(VPGSG)48”.
Thus in one embodiment, there is provided a composition comprising a micelle comprising one or more elastin-like polypeptides (ELP), wherein the immunomodulator is covalently bound to an ELP to form an ELP-immunomodulator fusion protein.
Binding of an immunomodulator to a hydrophilic block of an amphiphilic ELP provides a micelle that has an immunomodulator presented on the corona (external surface) of the micelle allowing for recognition and binding of the immunomodulator by immune cells or immune system molecules. The use of covalent attachment may also provide a more stably bound immunomodulator. In certain embodiments, the immunomodulator is non-covalently bound to the ELP. In certain embodiments, the immunomodulator is non-covalently bound to the ELP so that the immunomodulator is located on the external surface of the micelle. In certain embodiments, the immunomodulator is non-covalently bound to a hydrophilic block of an ELP. In certain embodiments, the immunomodulator is non-covalently bound to the C-terminal of an ELP comprising a C-terminal hydrophilic block.
In certain embodiments, the immunomodulator is non-covalently bound to the ELP via an intermediary binding molecule. The use of non-covalent bonding of an immunomodulator may allow for tuneable release of an immunomodulator in response to stimuli. For example, in response to pH, temperature, or enzymatic processing.
In certain embodiments, the immunomodulator comprises at least one epitope selected from the group consisting of: an allergenic epitope, a viral epitope, a bacterial epitope, a parasitic epitope, a disease-associated epitope, and a tumour-associated epitope.
In certain embodiments, the immunomodulator comprises an allergenic epitope.
In certain embodiments, the immunomodulator comprises an allergenic epitope from, or derived from, a Type I allergen.
In certain embodiments, the immunomodulator comprises an allergenic epitope from, or derived from, Bet v 1. In certain embodiments, the immunomodulator covalently bound to the ELP comprises the sequence:
GVFNYETETTSVIPAARLFKAFILDGDNLFPKVAPQAISSVENIEGNGGPGTIKKISFPEGFPFKY VKDRVDEVDHTNFKYNYSVIEGGPIGDTLEKISNEIKIVATPDGGSILKISNKYHTKGDHEVKAEQ VKASKEMGETLLRAVESYLLAHSDAYN (SEQ ID NO: 8).
In this context, the ELP-immunomodulator may comprise the sequence MG(VPGIG)48(VPGSG)48- Bet v 1 .
In certain embodiments, at least one of the ELPs is covalently bound to a first coiled coil forming peptide. In certain embodiments, the first coiled coil peptide is an intermediary binding molecule (e.g. peptide E). In certain embodiments, the first coiled coil peptide is an immunomodulator (e.g. peptide K). In certain embodiments, the first coiled coil peptide is an intermediary binding molecule and an immunomodulator (e.g., peptide K). In certain embodiments, wherein the first coiled coil peptide is an intermediary binding molecule, the immunomodulator is covalently bound to a cognate coiled coil forming peptide. For example, the immunomodulator may comprise a further coiled coil forming peptide configured to form a coiled coil (complex) with the first coiled coil forming peptide that is covalently bound to the ELP.
The coiled coil complex is stable, ensuring colocalization of the immunomodulator and micelle, but may dissociate at low pH, thus enabling endosomal escape.
In certain embodiments, the first coiled coil forming peptide comprises peptide K and/or peptide E.
In certain embodiments, the first coiled coil forming peptide comprises peptide K and the cognate coiled coil forming peptide comprises peptide E.
In certain embodiments, the first coiled coil forming peptide comprises peptide E and the cognate coiled coil forming peptide comprises peptide K.
Thus in one embodiment, there is provided a composition comprising a micelle, wherein the micelle comprises: a) one or more elastin-like polypeptides (ELP) covalently bound to a first coiled coil forming peptide; and b) one or more immunomodulators covalently bound to a cognate coiled coil forming peptide, wherein the immunomodulator is non-covalently bound to the ELP via the coiled coil forming peptides.
Thus in one embodiment, there is provided a composition comprising a micelle, wherein the micelle comprises a peptide K covalently bound to elastin-like polypeptide (ELP) and an immunomodulator covalently bound to a further ELP.
In certain embodiments, the first coiled coil forming peptide is covalently bound to the ELP so that the first coiled coil forming peptide is located on the external surface of the micelle. In certain embodiments, the first coiled coil forming peptide is covalently bound to a hydrophilic block of an ELP. In certain embodiments, the first coiled coil forming peptide is covalently bound to the C- terminal of an ELP comprising a C-terminal hydrophilic block.
In certain embodiments, the ELP covalently bound to peptide K comprises the sequence “MG(VPGIG)48(VPGSG)48YWSGGG(KIAALKE)n” (SEQ ID NO: 21) wherein n is an integer between 2 and 5. In certain embodiments, the ELP covalently bound to peptide K comprises the sequence “MG(VPGIG)48(VPGSG)48YWSGGG(KIAALKE)4” (SEQ ID NO: 9). In certain embodiments, the ELP covalently bound to peptide K comprises the sequence “MG(VPGIG)48(VPGSG)48YWSGGG(EIAALEK)n” (SEQ ID NO: 22) wherein n is an integer between 2 and 5. In certain embodiments, the ELP covalently bound to peptide E comprises the sequence “MG(VPGIG)48(VPGSG)48YWSGGG(EIAALEK)4” (SEQ ID NO: 10).
The presence of coiled coil domains may increase the composition’s ability to induce an immune response due to the tendency of coiled coil domains to interact with cellular membranes.
In certain embodiments wherein the first coiled coil peptide is an immunomodulator, the first coiled coil forming peptide may be peptide K. Peptide K may interact with and/or destabilize membranes, such as cell membranes of antigen presenting cells. Peptide K may interact with membranes, burying the hydrophobic face of the helix in the bilayer while the lysine side chains “snorkel” towards the polar/nonpolar interface.
In certain embodiments, the micelle comprises an average hydrodynamic diameter of from about 30 to about 70 nm.
In certain embodiments, the micelle comprises an average polydispersity index of from about 0.001 to about 0.25.
In certain embodiments, the micelle comprises an average zeta potential of from about -15 mV to about 15 mV.
In certain embodiments, the micelles in the composition are monodispersed.
In certain embodiments, the micelles are spherical.
Adjuvants, such as the micelles described herein, can influence the differentiation of the immune response towards Th1 or Th2, depending on the physicochemical properties of the adjuvant, such as nanoparticle size, shape, surface charge, and rigidity. For example, nanoscale spherical particles are best suited to induce Th 1 -biased responses.
In another aspect of the invention, there is provided a pharmaceutical formulation comprising a composition as described herein, further comprising a pharmaceutically acceptable carrier, excipient, diluent and/or adjuvant. In another aspect of the invention, there is provided use of a micelle comprising one or more ELPs as described herein for delivery of an immunomodulator as described herein.
In another aspect of the invention, there is provided use of a micelle comprising one or more ELPs as described herein for forming of an immunogenic vaccine.
In another aspect of the invention, there is provided use of a micelle comprising one or more ELPs as described herein as an adjuvant.
In another aspect of the invention, there is provided a pharmaceutical formulation as described herein for use as a medicament.
In another aspect of the invention, there is provided a pharmaceutical formulation as described for use in preventing and/or treating an allergic disease. In certain embodiments, pharmaceutical formulation is for use in preventing an allergic disease.
In another aspect of the invention, there is provided a method of preventing and/or treating an allergic disease, the method comprising administering to a subject a therapeutically effective amount of a pharmaceutical formulation described herein
In certain embodiments, the immunomodulator is an allergenic epitope as described herein. In certain embodiments, the pharmaceutical formulation is for use in allergy immunotherapy. In certain embodiments, the allergy immunotherapy is subcutaneous immunotherapy.
In certain embodiments, the immunomodulator is an epitope from or derived Bet v 1 as described herein.
In another aspect of the invention, there is provided a pharmaceutical formulation as described herein for use in preventing and/or treating cancer.
In another aspect of the invention, there is provided a method of preventing and/or treating cancer, the method comprising administering to a subject a therapeutically effective amount of a pharmaceutical formulation as described herein.
In certain embodiments, the immunomodulator is a tumour-associated epitope.
In certain embodiments the pharmaceutical formulation comprises a micelle comprising: a) one or more elastin-like polypeptides (ELP) covalently bound to a first coiled coil forming peptide; and b) an immunomodulator covalently bound to a cognate coiled coil forming peptide, wherein the immunomodulator is non-covalently bound to the ELP via the coiled coil forming peptides.
In certain embodiment, the pharmaceutical formulation comprises a micelle, wherein the micelle comprises a peptide K covalently bound to elastin-like polypeptide (ELP) and an immunomodulator covalently bound to a further ELP.
In another aspect of the invention, there is provided a pharmaceutical formulation as described herein for use in preventing and/or treating an infectious disease.
In another aspect of the invention, there is provided a method of preventing and/or treating an infectious disease, the method comprising administering to a subject a therapeutically effective amount of a pharmaceutical formulation as described herein.
In certain embodiments, the pharmaceutical formulation is a vaccine. In certain embodiments, the pharmaceutical formulation is for use in a method of vaccination of a subject.
In certain embodiments, the immunomodulator is a pathogen associated epitope. In certain embodiments, the immunomodulator is selected from the group of a viral epitope, a bacterial epitope, or a parasitic epitope.
In certain embodiments the pharmaceutical formulation comprises a micelle comprising: a) one or more elastin-like polypeptides (ELP) covalently bound to a first coiled coil forming peptide; and b) one or more immunomodulators covalently bound to a cognate coiled coil forming peptide, wherein the immunomodulator is non-covalently bound to the ELP via the coiled coil forming peptides.
In certain embodiments, the pharmaceutical formulation comprises a micelle, wherein the micelle comprises a peptide K covalently bound to elastin-like polypeptide (ELP) and an immunomodulator covalently bound to a further ELP. In another aspect of the invention, there is provided a method for inducing an immune response specific for an immunomodulator in a subject, the method comprising administering to a subject a therapeutically effective amount of a pharmaceutical formulation as described herein.
Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps.
Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.
Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.
Various aspects of the invention are described in further detail below.
Brief description of the Figures
Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:
Figure 1 shows micellar formulations displaying OVA323 used in this study. All formulations contain ELP and the covalent and hybrid micelles also contain ELP-OVA323, whereas the coiled coil micelles contain peptide E-OVA323 and ELP-K. ELP to OVA323 epitope ratio and ELP to peptide K ratio are 10:1. Cartoon is not to scale;
Figure 2 shows LC-MS spectrum of peptide E. Chromatogram is shown in the top and middle panels. Observed mass (bottom): 1271.84; calculated mass ([M+2H+]2+): 1272.97;
Figure 3 shows LC-MS spectrum of peptide K. Chromatogram is shown in the top and middle panels. Observed mass (bottom): 1281.77; calculated mass ([M+2H+]2+): 1283.07;
Figure 4 shows LC-MS spectrum of E-OVA323. Chromatogram is shown in the top and middle panels. Observed mass (bottom): 1432.89 and 1074.89; calculated mass ([M+3H+]3+and [M+4H+]4+): 1434.28 and 1075.96; Figure 5 shows LC-MS spectrum of E-OVA323-TMR. Chromatogram is shown in the top and middle panels. Observed mass (bottom): 1647.32 and 1235.46; calculated mass ([M+3H+]3+ and [M+4H+]4+): 1648.18 and 1236.39;
Figure 6 shows RP-HPLC analysis of ELP-E (top), ELP-K (middle) and ELP-OVA323 (bottom). Gradient of 0 to 90% acetonitrile in water containing 0.1% TFA. Traces were normalized to match the peak height of ELP;
Figure 7 shows expression, purification and identification of ELP-E (top), ELP-K (middle) and ELP-OVA323 (bottom). Left) Samples were analyzed on 10% SDS polyacrylamide gels with Coomassie Blue (ELP-E and ELP-K) or copper chloride (ELP-OVA323) staining. ELP-E, ELP-K and ELP-OVA323 are clearly visible at 43 kDa, 43 kDa and 40 kDa, respectively. In each gel, the black arrow marks the location of the purified polypeptide. Lys = lysate; C1-5 refer to samples taken after 1-5 cycles of ITC. Right) Mass spectra matched the theoretical masses of ELP-E, ELP-K and ELP-OVA323 without the N-terminal methionine. Expected masses were 43104 Da (ELP-E), 43100 Da (ELP-K) and 41456 Da (ELP-OVA323);
Figure 8 shows inverse transition behaviour of ELP-E and ELP-K compared to ELP. [polypeptide] = 10 pM; SLS measured in 10 mM PB pH 7.8 as a function of temperature;
Figure 9 shows CMC determination of ELP-E (top left) and ELP-OVA323 (bottom left) and CAC determination of ELP-K (top right). Measured in 10 mM PB pH 7.8 at 37 °C. Count rate was measured by SLS as a function of [ELP-E], [ELP-K] or [ELP-OVA323], Count rates were normalized to the count rate of the buffer. Gray data represent samples that did not contain particles according to the autocorrelation functions (see Figure 10); black data represent the samples for which the autocorrelation functions had a sigmoidal shape. The CMC of ELP-E was determined at 0.11 pM and the CAC of ELP-K as 56 nM by calculating the intercept of the trend lines;
Figure 10 shows autocorrelation functions of DLS measurements for CMC/CAC determinations of ELP-E (top), ELP-K (middle) and ELP-OVA323 (bottom). Each graph is an average of three measurements. Solid lines represent samples for which particles were detected; dotted lines represent samples for which particles could not be detected; dashed lines represent samples for which no particles were detected for the first measurement, but particles were detected for the last measurement; Figure 11 shows size distributions of ELP/ELP-E micelles in the absence (top) and presence (bottom) of equimolar amounts of peptide K. [polypeptide] = 20 pM; E/K ratio = 1 ; T = 37 °C; measured in 10 mM PB pH 7.8. Z-average values are included in Table 3;
Figure 12 shows size distributions of ELP/ELP-K micelles in the absence (top) and presence (bottom) of equimolar amounts of peptide E. [polypeptide] = 20 pM; E/K ratio = 1 ; T = 37 °C; measured in 10 mM PB pH 7.8. Z-average values are included in Table 3;
Figure 13 shows circular dichroism (CD) analysis of coiled coil formation on ELP micelle surface, [polypeptide] = 25 pM in 10 mM HEPES buffer pH 7.8; E/K ratio = 1 ; T = 37 °C;
Figure 14 shows colocalization of FITC-ELP and E-OVA323-TMR of coiled coil micelles. BMDCs were incubated for 4 hours with fluorescently labeled coiled coil micelles with 90 nM OVA323 (900 nM polypeptide). The BMDCs were subsequently washed thoroughly with medium to remove excess particles and imaged on a confocal microscope. The overlay of the bright field image, and the blue (cell nuclei), green (ELP), and red (E-OVA323) channels is shown (left); scale bar = 25 pM. The yellow color indicates colocalized ELP and E-OVA323. 94.4% of the E-OVA323 signal (red) is colocalized with the ELP signal (green). Along the white dashed line, the intensities of the ELP and E-OVA323 signals are correlated, which also shows the colocalization of ELP and E-OVA323 (right);
Figure 15 shows TEM imaging of ELP micelles displaying coiled coils. Left) ELP/ELP-E (9:1); right) ELP/ELP-K (9:1). Bottom) with an equimolar amount of the complementary peptide; top) without an equimolar amount of the complementary peptide, [polypeptide] = 20 pM in water; E/K ratio = 1 ; T = 37 °C; stained with 1 % uranyl acetate;
Figure 16 shows inverse transition behavior of ELP-OVA323 compared to ELP. [polypeptide] = 10 pM; DLS measured in 10 mM PB pH 7.8 as a function of temperature. The CMT was determined as the temperature value above which the count rate is stable: ~28 °C for both polypeptides;
Figure 17 shows AFM images of covalent (a, b), coiled coil (c, d) and hybrid (e, f) micelles, [polypeptide] = 2 pM in water, deposited on a silicon oxide surface and dried at 37 °C; height trace mode; imaged using medium (a, c, e) and high (b, d, f) magnifications. The medium magnifications were used to analyze the average height of the imaged micelles (g): 23 nm (covalent), 15 nm (coiled coil) and 11 nm (hybrid). Micelles are indicated with green arrows and clustered micelles are indicated with white arrows. Each sample was also imaged using error trace mode (Figure 17), at low magnification (Figure 18) and on a mica surface (Figure 19); Figure 18 shows AFM images of covalent (top), coiled coil (middle) and hybrid (bottom) micelles, [polypeptide] = 2 pM in water, deposited on a silicon oxide surface and dried at 37 °C; error trace mode; imaged using medium (left) and high (right) magnifications;
Figure 19 shows AFM images of covalent (top), coiled coil (middle) and hybrid (bottom) micelles, [polypeptide] = 2 pM in water, deposited on a mica surface and dried at 37 °C; height trace mode; imaged using medium (left) and high (right) magnifications;
Figure 20 shows AFM images of covalent (top), coiled coil (middle) and hybrid (bottom) micelles, [polypeptide] = 2 pM in water, deposited on a silicon oxide surface and dried at 37 °C; height trace (left) and error trace (right) mode; imaged using low magnifications;
Figure 21 shows TEM imaging of covalent (top left), coiled coil (top right) and hybrid (bottom left and right) micelles, [polypeptide] = 20 pM in water; E/K ratio = 1 ; T = 37 °C; stained with 1% uranyl acetate;
Figure 22 shows coiled coil association efficiency on the ELP micelle surface. Left) [polypeptide] = 20 pM; [E-OVA-TMR] = 2 pM; separation of unbound E-OVA323 from micelle in the presence (coiled coil) or absence (ELP/E-OVA323-TMR) of ELP-Kwas performed at 37 °C. The fluorescent signals of the resulting samples were normalized to the value of the coiled coil sample. Right) E- OVA323-TMR concentrations in the relevant range are linearly associated with the fluorescent signal;
Figure 23 shows BMDC uptake of FITC-labelled formulations with and without ELP-K. BMDCs were incubated for 4 hours with fluorescently labelled micelles or as a negative control with medium only. OVA323 concentrations were (from left to right) 270, 90, 30, 10, 3.3 and 1.1 nM. The BMDCs were subsequently analyzed with flow cytometry;
Figure 24 shows BMDC uptake of FITC-labelled formulations with and without ELP-K. [OVA323]: 270 nM (top left), 90 nM (top right), 30 nM (bottom left) and 10 nM (bottom right). BMDCs were incubated for 4 hours with fluorescently labelled micelles or with medium only as a negative control. The BMDCs were subsequently analysed with flow cytometry. Significant differences between BMDC uptake percentages of samples with ELP-K and samples without ELP-K were determined using a one-way ANOVA with a Tukey’s multiple comparison test. * = p < 0.05, ** = p < 0.01 , *** = p < 0.001 , **** = p < 0.0001 ;
Figure 25 shows BMDC uptake of free E-OVA323-TMR and coiled coil micelles containing both E-OVA323-TMR and FITC-ELP. BMDCs were incubated for 4 hours with peptide, micelle or with medium only as a negative control. [OVA323] (from left to right) = 270, 90 and 30 nM. The BMDCs were subsequently analysed with flow cytometry;
Figure 26 shows ELP-K-dependent uptake of ELP micelles into BMDCs. BMDCs were incubated for 4 hours with fluorescently labelled peptide or micelles containing 90 nM OVA323 (900 nM polypeptide). The BMDCs were subsequently washed thoroughly with medium to remove excess particles and imaged on a confocal microscope. Various images were taken from each sample: (a) bright field image, (b) blue channel (cell nuclei), (c) green channel (ELP), (d) red channel (E- OVA323), (e) overlay of all fluorescent channels, (f) complete overlay. The arrows in the complete overlay images indicate where the ELP was taken up into the cells. Scale bar = 25 pM;
Figure 27 shows maturation of BMDCs following exposure to micelles. BMDCs were incubated for 4 hours with micelles or with E-OVA323 peptide or medium only as negative controls. OVA323 concentrations were (from left to right) 270, 90, 30, 10, 3.3 and 1.1 nM and the plain ELP micelles were added in the same polypeptide concentration range as the other samples. The BMDCs were subsequently analysed with flow cytometry;
Figure 28 shows proliferation of OT-II cells in vitro. BMDCs were incubated for 4 hours with micelles, peptide or with medium only. OVA323 concentrations were (from left to right) 270, 90, 30, 10, 3.3 and 1.1 nM. The BMDCs were subsequently exposed for 3 days to OT-II cells containing fluorescent dye. The decrease of fluorescent signal in the OT-II population was then analysed with flow cytometry. As negative controls, BMDCs were pulsed with medium, plain ELP micelles or ELP/ELP-K micelles (containing 10% ELP-K). ELP micelles mixed with free OVA323 peptide (“ELP/OVA323”) was included as a positive control. The polypeptide concentrations of ELP and ELP-K and ELP/OVA323 was 2.7 pM, matching the highest polypeptide concentration of the covalent, coiled coil and hybrid groups;
Figure 29 shows proliferation of OT-II cells in vitro. [OVA323]: 270 nM (top), 90 nM (middle) and 30 nM (bottom). BMDCs were incubated for 4 hours with micelles, peptide or with medium only. The BMDCs were subsequently exposed for 3 days to OT-II cells containing fluorescent dye. The decrease of fluorescent signal in the OT-II population was then analysed with flow cytometry;
Figure 30 shows hydrodynamic diameters of ELP micelles with coiled coil-forming peptides, [polypeptide] = 10 pM; T = 37 °C; measured in 10 mM PB pH 7.8;
Figure 31 shows hydrodynamic diameter of ELP and ELP-OVA323 micelles, [polypeptide] = 10 pM; T = 37 °C; measured in 10 mM PB pH 7.8; Figure 32 shows hydrodynamic diameter of covalent, coiled coil and hybrid micelles, [polypeptide] = 10 pM; T = 37 °C; measured in 10 mM PB pH 7.8;
Figure 33 shows expression, purification and identification of ELP-Bet v 1 . The samples were analysed on 10% SDS polyacrylamide gels with Coomassie Blue staining. ELP-Bet v 1 is clearly visible at around 60 kDa. In both gels, the black arrow marks the location of the purified protein, top) Expression and purification by immune-affinity chromatography. Lanes marked with - and + are before and after adding IPTG; Lys is lysate; FT is flow through; W1-3 refer to wash fractions; E1-4 refer to elution fractions, bottom left) Further purification by ITC. P is pellet; S is supernatant; hot spin is the centrifugation step at 22 °C; cold spin is the centrifugation step at 4 °C. bottom right);
Figure 34 shows RP-HPLC analysis of ELP-Bet v 1. Gradient of 0 to 90% acetonitrile in water containing 0.1 % TFA. Trace was normalized to match the peak height of ELP;
Figure 35 shows endotoxin level determination using mTLR4 HEK reporter cell assay. ELP contained 0.38 EU LPS/mg and ELP/ELP-Bet v 1 contained 0.11 EU LPS/mg
Figure 36 shows Inverse transition behaviour of ELP-Bet v 1 and ELP/ELP-Bet v 1 compared to ELP. [polypeptide] = 10 pM; SLS measured in 10 mM PB pH 7.8 as a function of temperature. The CMT was determined as the temperature value above which the count rate is stable: ~22 °C (ELP), 24 °C (ELP/ELP-Bet v 1) and ~28 °C (ELP-Bet v 1);
Figure 37 shows CMC determination of ELP-Bet v 1 (left) and 9:1 ELP/ELP-Bet v 1 (right). Measured in 10 mM PB pH 7.8 at 37 °C. Count rate was measured by SLS as a function of [ELP- Bet v 1] or [ELP/ELP-Bet v 1], Count rates were normalized to the count rate of the buffer. Gray data points represent samples that did not contain particles according to the autocorrelation functions; black data points represent the samples for which the autocorrelation functions had a sigmoidal shape. The CMC was determined to be 1.1 pM (ELP-Bet v 1) and 0.22 pM (ELP/ELP- Bet v 1) respectively by calculating the intercept of the trend lines;
Figure 38 shows TEM image of ELP/ELP-Bet v 1 particles. Grid was prepared with 37 °C 20 pM polypeptide solution in water and stained with 1% uranyl acetate;
Figure 39 shows AFM images of ELP/ELP-Bet v 1 micelles, [polypeptide] = 2 pM in water, deposited on a silicon oxide surface and dried at 37 °C; height trace mode; imaged using low (top left), medium (top right) and high (bottom left) magnifications. Micelles are indicated with green arrows and clustered micelles are indicated with white arrows. The height of each micelle in the medium magnified image were used for size analysis (bottom right);
Figure 40 shows rapid micellization of ELP and ELP/ELP-Bet v 1 upon dilution of cold sample into 37 °C TB. [polypeptide] = 1 pM; diluted from 100 pM; T = 37 °C; measured in 10 mM PB pH 7.8. or TB containing 9.5 g/L Tyrode's salts, 0.1 % (w/v) BSA and 0.5 g/L sodium NaHCCh;
Figure 41 shows IgE binding capacities of Bet v 1 -displaying micelles. The samples were diluted in 10 mM PB, 280 mM sucrose pH 7.4 to various concentrations and incubated with a diluted serum pool of 36 birch pollen allergic patients. Then, unbound IgE was captured by immobilized Bet v 1 and subsequently measured on a Phadia 250 device;
Figure 42 shows Circular dichroism (CD) analysis of ELP-Bet v 1. Each sample was measured at 25 °C (solid lines) and 37 °C (dashed lines);
Figure 43 shows mediator release of patients 1-10. Rat basophil cells transfected with human IgE receptor were sensitized with human sera from birch pollen allergic patients. The cells were then stimulated with the respective antigen (Bet v l , plain ELP micelles mixed with Bet v l “ELP/Bet v 1”, ELP/ELP-Bet v 1 and ELP-Bet v 1) in concentrations ranging from 80 pg/mL to 0.024 fg/mL. The release of mediator p-hexosaminidase was measured using the fluorogenic substrate 4-methyl umbelliferyl-N-acetyl-beta-D-glucosaminide. The level of mediator release in the absence of antigen is shown with a dashed line;
Figure 44 shows hypoallergenicity of ELP/ELP-Bet v 1 and ELP-Bet v 1. Rat basophil cells transfected with human IgE receptor were sensitized with human sera from birch pollen allergic patients. The cells were then stimulated with the respective antigen (Bet v 1 , plain ELP micelles mixed with Bet v 1 “ELP/Bet v 1”, ELP/ELP-Bet v 1 and ELP-Bet v 1) in concentrations ranging from 80 pg/mL to 0.024 fg/mL. The release of mediator p-hexosaminidase was measured using the fluorogenic substrate, 4-methyl umbelliferyl-N-acetyl-beta-D-glucosaminide. The data shown in Figure 43 were used to determine for each patient the antigen concentration required to induce half the maximum level of mediator release. Significant differences between these concentrations among each group were determined using a one-way ANOVA with a Tukey’s post-hoc analysis test. * = p < 0.05, ** = p < 0.01 , *** = p < 0.001 , **** = p < 0.0001 ;
Figure 45 shows immunization protocol. Mice were immunized with 36 pg Bet v 1on day 0 by subcutaneous injection followed by booster immunizations on days 7 and 14 (red arrows). Blood samples were taken for serum immunoglobulin analyses on days -1 , 6, 13 and 20. The mice received intranasal BPE challenges to further boost immunoglobulin production on days 28, 29 and 30. At day 31 the mice were sacrificed;
Figure 46 shows Serum immunoglobulin levels in immunized mice of pilot study. Bet v 1 specific lgG1 (a) and lgG2a (b) at different time points, c) Bet v 1 specific IgE levels at day 31. The dotted lines represent average endpoint lgG1 , lgG2a and IgE levels of a buffer control group from other immunogenicity experiments to indicate the background signal;
Figure 47 shows Cytokine expression in re-stimulated lymph node cultures from pilot study. Expression of IL-4 (a), IL-5 (b), IL-10 (c), IL-13 (d), IFN-y (e) and IL-17 (f) in lung draining lymph nodes, stimulated ex vivo with Bet v 1 . * p<0.05, ** p<0.01
Figure 48 shows Serum immunoglobulin levels in immunized mice. Bet v 1 specific lgG1 (a) and lgG2a (b) at different time points, c) Bet v 1 specific IgE levels at day 31 ;
Figure 49 shows Cytokine expression in re-stimulated lymph node cultures. Expression of IL-4 (a), IL-5 (b), IL-10 (c), IL-13 (d), IFN-y (e) and IL-17 (f) in lung draining lymph nodes, stimulated ex vivo with Bet v 1 . * p<0.05, ** p<0.01 ;
The patent, scientific and technical literature referred to herein establish knowledge that was available to those skilled in the art at the time of filing. The entire disclosures of the issued patents, published and pending patent applications, and other publications that are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference. In the case of any inconsistencies, the present disclosure will prevail.
Various aspects of the invention are described in further detail below.
Detailed Description
Elastin-like Peptides
The compositions provided herein include micelles that include elastin-like peptides. Elastin-like- polypeptides (ELPs) are a genetically engineered polypeptide with unique phase behavior (see for e.g. S. R. MacEwan, et al., Biopolymers 94(1) (2010) 60-77) which promotes recombinant expression, protein purification and self-assembly of nanostructures (see for e.g. A. Chilkoti, et al., Advanced Drug Delivery Reviews 54 (2002) 1093-1111). ELPs have potential advantages over chemically synthesized polymers. First, because they are biosynthesized from a genetically encoded template, ELPs can be made with precise molecular weight. Chemical synthesis of long linear polymers does not typically produce an exact length, but instead a range of lengths. Consequently, fractions containing both small and large polymers yield mixed pharmacokinetics and biodistribution. Second, ELP biosynthesis produces very complex amino acid sequences with nearly perfect reproducibility. This enables very precise selection of the location of attachment of additional moieties, such as an immunomodulator. Thus an additional moiety can be selectively placed on the corona of an ELP micelle, buried in the core of an ELP micelle, or dispersed equally throughout the polymer. Third, ELP can self-assemble into multivalent nanoparticles. Fourth, because ELP are designed from native amino acid sequences found extensively in the human body they are biodegradable, biocompatible, and tolerated by the immune system. Fifth, ELPs undergo an inverse phase transition temperature, Tt, above which they phase separate into structures such as micelles.
ELPs are artificial polypeptides composed of repeated pentapeptide sequences, (VPGXG)n (SEQ ID. NO: 11) derived from human tropoelastin, where X is a “guest residue” which is any amino acid. This peptide motif displays rapid and reversible de-mixing from aqueous solutions above a transition temperature, Tt. Below Tt, ELPs adopt a highly water soluble random coil conformation; however, above Tt, they separate from solution, coalescing into a second aqueous phase. The Tt of ELPs can be tuned by choosing the guest residue and ELP chain length as well as fusion peptides at the design level (see for e.g. MacEwan S R, et al., Biopolymers 94(1): 60-77). The ELP phase is both biocompatible and highly specific for ELPs or ELP fusion proteins, even in complex biological mixtures. Genetically engineered ELPs are monodispersed, biodegradable, and non-toxic. Throughout this description, ELPs are identified by the single letter amino acid code of the guest residue followed by the number of repeat units, n. N may be any number, for example n may be from 1 to 100.
For example, I48S48 represents a diblock copolymer of ELP comprising 48 isoleucine (I) pentamers (VPGIG)48 (SEQ ID. NO: 12) at the amino terminus and 48 serine (S) pentamers (VPGSG)48, (SEQ ID. NO: 13) at the carboxy terminus. A “diblock” refers to an ELP with two blocks or structural units of repeated polypeptide sequence. For example, the diblock (VPGIG)4S (VPGSG)48 (SEQ ID. NO: 6) comprises 48 repeated units of a polypeptide having the sequence VPGIG (SEQ ID. NO: 14) and 48 repeated units of a polypeptide having the sequence VPGSG (SEQ ID NO: 15).
In some examples, the ELPs include polymeric or oligomeric repeats of the pentapeptide VPGXG (SEQ ID NO: 16), where the guest residue X is any amino acid. X may be a naturally occurring or non-naturally occurring amino acid.
The guest residue X may be a non-classical (non-genetically encoded) amino acid. Examples of non-classical amino acids include: D-isomers of the common amino acids, 2, 4-diaminobutyric acid, a-amino isobutyric acid, A-aminobutyric acid, Abu, 2-amino butyric acid, y-Abu, c-Ahx, 6- amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t- butylalanine, phenylglycine, cyclohexylalanine, p-alanine, fluoro-amino acids, designer amino acids such as p-methyl amino acids, C a-methyl amino acids, N a-methyl amino acids, and amino acid analogs in general.
Selection of X is independent in each ELP structural unit (e.g., for each structural unit defined herein having a guest residue X). For example, X may be independently selected for each structural unit as an amino acid having a positively charged side chain, an amino acid having a negatively charged side chain, or an amino acid having a neutral side chain, including in some examples, a hydrophobic side chain.
In some examples, the structural units, or in some cases polymeric or oligomeric repeats, of the ELP sequences may further include (e.g. at the N- or C- terminus of each structural unit) one or more amino acid residues that do not eliminate the overall effect of the molecule, that is, in imparting certain improvements to the immunomodulator component as described. In some examples, such one or more amino acids also do not eliminate or substantially affect the phase transition properties of the ELP component (relative to the deletion of such one or more amino acids). For example, the ELP may have a sequence MX1(VPGXhbG)48(VPGXhpG)4sX2 (SEQ ID NO: 3), wherein X1 and X2 are each any amino acid and wherein Xhp is any hydrophilic amino acid residue and Xhb is any hydrophobic amino acid residue. In some examples, Xhp is serine. In some examples, Xhb is isoleucine. For example, the ELP diblock polymer may have the sequence MG(VPGXhbG)n(VPGXhpG)nY (SEQ ID NO: 4). In some examples, the methionine residue at the N-terminal of the ELP may not be included. For example, when the ELP is synthetically synthesised.
The Ttof the ELP component can be modified by varying ELP chain length, as the Tt generally increases with decreasing MW. For polypeptides having a molecular weight >100,000, the hydrophobicity scale developed by Urry et al. (WO9632406, which is hereby incorporated by reference in its entirety) is preferred for predicting the approximate Ttof a specific ELP sequence. However, in some examples, ELP component length can be kept relatively small, while maintaining a target Tt, by incorporating a larger fraction of hydrophobic guest residues (e.g., amino acid residues having hydrophobic side chains) in the ELP sequence.
While the Tt of the ELP, and therefore of the ELP coupled to an immunomodulator or coiled coil peptide as described herein, is affected by the identity and hydrophobicity of the guest residue, X, additional properties of the molecule may also be affected. Such properties include, but are not limited to solubility, bioavailability, persistence, and half-life of the molecule.
In some examples, the ELP is a diblock copolymer having a first ELP structural unit (i.e. a first block) that includes a guest residue (X) that is a hydrophobic amino acid residue and a second ELP structural unit (i.e. second block) that includes a guest residue (X) that is a hydrophilic amino acid residue. Thus, in some examples, the ELP component includes a first hydrophobic unit and second hydrophilic unit thus providing an amphiphilic ELP molecule.
"Hydrophobic amino acid" refers to an amino acid having a side chain that is uncharged at physiological pH and that is repelled by aqueous solution. Examples of genetically encoded hydrophobic amino acids are glycine, alanine, isoleucine, leucine, valine proline, phenylalanine, methionine, and tryptophan. An example of a non-genetically-encoded hydrophobic amino acid is t-BuA. Hydrophobic amino acids include amino acids having aromatic or nonpolar side chains.
Hydrophilic amino acids include amino acids having acidic, basic or polar side chains. "Hydrophilic amino acid" refers to an amino acid having a side chain that is attracted by aqueous solution. Examples of genetically encoded hydrophilic amino acids are, serine, arginine, lysine, glutamine, aspartic acid, glutamic acid, asparagine, and histidine. Examples of non-genetically- encoded hydrophilic amino acids are Cit and hCys. For example the ELP may comprise the sequence (VPGXhbG)n(VPGXhPG)n (SEQ ID NO: 1) or (VPGXhPG)n(VPGXhbG)n (SEQ ID NO: 2) wherein Xhp is any hydrophilic amino acid residue and Xhb is any hydrophobic amino acid residue. In some examples, Xhp is serine. In some examples, Xhb is isoleucine.
Amphiphilic ELPs self-assemble into spherical micelles above the critical micelle temperature (CMT) and/or at a critical micelle concentration (CMC). ELP assembly is induced by hydrophobic collapse of the hydrophobic ELP block, forming the micelle core with the hydrophilic blocks pointing outwards in solution. In some examples, the ELP includes a sequence of (VPGIG)n(VPGSG)n (SEQ ID NO: 5). In some examples, the ELP has a sequence of MG(VPGIG)48(VPGSG)48Y (SEQ ID NO:7).
Some of the ELPs that make up the micelle may be covalently or non-covalently bound to additional moieties as described herein. Binding of immunomodulators and other specific additional moieties is described below in more detail. In some examples, additional moieties, such as an immunomodulator, coiled coil forming peptide as described herein or any other additional moieties, may be bound so that they are configured to be located on the external surface of the micelle. In some examples, additional moieties (other than immunomodulators or intermediary linking molecules) may be configured to be located inside the micelle, for example, in the core of the micelle. For example, in the case of diblock ELPs described herein having a hydrophobic and hydrophilic block, the additional moieties may be bound to the hydrophilic block. For example, for (VPGXhbG)n(VPGXhpG)n (SEQ ID NO: 1 ) the additional moiety may be bound to the C-terminal of the ELP. For (VPGXhPG)n(VPGXhbG)n (SEQ ID NO: 2) the additional moiety may be bound to the N-terminal of the ELP.
In some examples wherein the additional moiety is proteinaceous, the ELP may be covalently bound to the additional moiety by recombinant fusion methods, for example, by providing an ELP additional moiety fusion protein. A recombinantly-produced ELP fusion protein may include the ELP and the additional moiety associated with one another by genetic fusion. For example, the fusion protein may be generated by translation of a polynucleotide encoding the therapeutic component cloned in-frame with the ELP (or vice versa). As such, also provided herein are nucleic acids encoding ELP fusion proteins described herein as well as the amino acid sequences encoding ELP fusion proteins described herein.
The ELP and the additional moieties can be fused using a linker peptide of various lengths to provide greater physical separation and allow more spatial mobility between the fused portions, and thus maximize the accessibility of the additional moieties, for instance, for binding to its cognate receptor. The linker peptide may consist of amino acids that are flexible or rigid. For example, a flexible linker may include amino acids having relatively small side chains, and which may be hydrophilic. Without limitation, the flexible linker may contain a stretch of glycine and/or serine residues. More rigid linkers may contain, for example, more sterically hindering amino acid side chains, such as (without limitation) tyrosine or histidine. The linker may be less than about 50, 40, 30, 20, 10, or 5 amino acid residues. The linker can be covalently linked to and between an ELP and an additional moiety, for example, via recombinant fusion.
The linker or peptide spacer may be protease-cleavable or non-cleavable. By way of example, cleavable peptide spacers include, without limitation, a peptide sequence recognized by proteases (in vitro or in vivo) of varying type, such as Tev, thrombin, factor Xa, plasmin (blood proteases), metalloproteases, cathepsins (e.g., GFLG, etc.), and proteases found in other corporeal compartments. In some examples, employing cleavable linkers, the fusion protein may be inactive, less active, or less potent as a fusion, which is then activated upon cleavage of the spacer in vivo. Alternatively, a non-cleavable spacer may be employed. The non-cleavable spacer may be of any suitable type known in the art.
In other examples, the ELP may be covalently bound to an additional moiety via chemical conjugation. The conjugates can be made by chemically coupling an ELP to an additional moiety by any number of methods well known in the art (See e.g. Nilsson et al., 2005, Ann Rev Biophys Bio Structure 34: 91-118). In some examples, the chemical conjugate can be formed by covalently linking the additional moiety to the ELP, directly or through a short or long linker moiety, through one or more functional groups on the additional moiety, e. g., amine, carboxyl, phenyl, thiol or hydroxyl groups, to form a covalent conjugate. Various conventional linkers can be used, e. g., diisocyanates, diisothiocyanates, carbodiimides, bis (hydroxysuccinimide) esters, maleimide- hydroxysuccinimide esters, glutaraldehyde and the like. Therefore, in some examples there is provided a micelle that includes ELP covalently bound to an additional moiety. In some examples, the additional moiety may be conjugated via click chemistry. “Click chemistry” refers to a chemical approach to conjugation introduced by Sharpless in 2001 and describes chemistry tailored to generate substances quickly and reliably by joining units together. See, e.g., Kolb, Finn and Sharpless Angewandte Chemie International Edition 2001 40, 2004-2021; Evans, Australian Journal of Chemistry 2007 60, 384-395). Exemplary coupling reactions (some of which may be classified as “click chemistry”) include, but are not limited to, formation of esters, thioesters, amides (e.g., such as peptide coupling) from activated acids or acyl halides; nucleophilic displacement reactions (e.g., such as nucleophilic displacement of a halide or ring opening of strained ring systems); azide-alkyne Huisgen cycloaddition; thiol-yne addition; imine formation; Michael additions (e.g., maleimide addition reactions); and Diels-Alder reactions (e.g., tetrazine [4+2] cycloaddition). Examples of click chemistry reactions and click-chemistry handles can be found in, e.g., Kolb, H. C.; Finn, M. G. and Sharpless, K. B. Angew. Chem. Int. Ed. 2001 , 40, 2004-2021. Kolb, H. C. and Sharless, K. B. Drug Disc. Today, 2003, 8, 112-1137; Rostovtsev, V. V.; Green L. G.; Fokin, V. V. and Shrapless, K. B. Angew. Chem. Int. Ed. 2002, 41 , 2596-2599; Tomoe, C. W.; Christensen, C. and Meldal, M. J. Org. Chem. 2002, 67, 3057-3064. Wang, Q. et al. J. Am. Chem. Soc. 2003, 125, 3192-3193; Lee, L. V. et al. J. Am. Chem. Soc. 2003 125, 9588- 9589; Lewis, W. G. et al. Angew. Chem. Int. Ed. 2002, 41 , 1053-41057; Manetsch, R. et al., J. Am. Chem. Soc. 2004, 126, 12809-12818; Mocharla, V. P. et al. Angew. Chem., Int. Ed. 2005, 44, 116-120.
In some examples, the micelle may include ELPs non-covalently bound to an additional moiety. For example, the ELP may be directly or indirectly (i.e. via an intermediary binding molecule) bound to an additional moiety via hydrogen bonding, ionic bonding, or Van der Waals forces. For example, the ELP may be bound to an immunomodulator as described herein via a intermediatory moiety such as a coiled coil forming peptide as described herein. The intermediatory moiety may be covalently bound to the ELP as described herein and mediate non-covalent binding to an additional moiety such as an immunomodulator that includes a cognate binding partner for the intermediatory moiety as described herein.
In some cases, in addition to moieties such as immunomodulators, the ELPs may also be associated with a detectable label that allows for the visual detection of in vivo uptake of the ELPs. Suitable labels include, for example, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, a detectable label sold under ALEXA-FLUOR®, stilbene, Lucifer Yellow, CASCADE BLUE®, and TEXAS RED®. Other suitable optical dyes are described in Haugland, Richard P. (1996) MOLECULAR PROBES™ Handbook (which is a Guide to Fluorescent Probes and Labeling Technologies).
The ELPs described (including any additional moieties or not) may be crosslinked. Crosslinked ELPs may be formed by producing a dimer of an amphiphilic ELP as described herein. Dimeric amphiphilic ELPs include two ELP molecules joined via a linker between the terminal of the hydrophobic block of each ELP. Crosslinked ELPs may provide an altered CMT and/or CMC. Methods of crosslinking may include chemically crosslinking ELPs. Any suitable agent that is capable of binding two amine groups (i.e. amine to amine crosslinkers may be used). For example, the ELPs may be crosslinked by reaction with agents such as bis(sulfosuccinimidyl)suberate (BS3), disuccinimidyl suberate (DSS), or N-hydroxysuccinimide esters (NHS esters). In some examples, the ELP, such as an amphiphilic ELPs described herein may include one or more cysteine residues, for example at the hydrophobic terminus. This may allow for binding of two ELP molecules together via a disulphide bond (disulphide bridge). Alternatively, crosslinked ELPs may be produced by recombinantly expressing dimeric amphiphilic ELPs. Such dimeric ELPs may include a protein linker expressed between each hydrophobic terminal. It will be understood that in the case that the ELP has a hydrophilic block at the N-terminal of the ELP, the ELPs in a dimer may be linked via the N-terminals of the ELPs.
The ELPs described herein (including any additional moieties or not) may include modifications. Such modifications include acetylation, carboxylation, glycosylation, phosphorylation, lipidation and/or acylation. Numerous methods of modifying proteins are known in the art. For example, modifications may be added to ELPs in vivo by host cell post-translational modification systems or may be added in vitro by chemical or enzymatic based methods.
The ELPs described herein (e.g., including any additional moieties or not) may be configured to self-assemble into micelles. In some examples, the micelles may include a mixture of ELPs as described herein. For example, the micelles described herein may include ELPs (without any additional moieties bound) and one or more ELPs each with one or more independently selected additional moiety attached.
ELPs and other recombinant proteins described herein can be prepared by expressing polynucleotides encoding the polypeptide sequences described herein in an appropriate host cell, i.e., a prokaryotic or eukaryotic host cell This can be accomplished by methods of recombinant DNA technology known to those skilled in the art. It is known to those skilled in the art that modifications can be made to any peptide to provide it with altered properties. Polypeptides described herein can be modified to include unnatural amino acids. Thus, the peptides may comprise D-amino acids, a combination of D- and L-amino acids, and various “designer” amino acids (e.g., p-methyl amino acids, C-a-methyl amino acids, and N-a-methyl amino acids, etc.) to convey special properties to peptides. Additionally, by assigning specific amino acids at specific coupling steps, peptides with a-helices, turns, p sheets, a-turns, and cyclic peptides can be generated.
The ELPs can be expressed and purified from a suitable host cell system. Suitable host cells include prokaryotic and eukaryotic cells, which include, but are not limited to bacterial cells, yeast cells, insect cells, animal cells, mammalian cells, murine cells, rat cells, sheep cells, simian cells and human cells. Examples of bacterial cells include Escherichia coli, Salmonella enterica and Streptococcus gordonii. In one embodiment, the host cell is E. coli. The cells can be purchased from a commercial vendor such as the American Type Culture Collection (ATCC, Rockville Md., USA) or cultured from an isolate using methods known in the art. Examples of suitable eukaryotic cells include, but are not limited to 293T HEK cells, as well as the hamster cell line BHK-21 ; the murine cell lines designated NIH3T3, NSO, C127, the simian cell lines COS, Vero; and the human cell lines HeLa, PER.C6 (commercially available from Crucell) U-937 and Hep G2. A non-limiting example of insect cells include Spodoptera frugiperda. Examples of yeast useful for expression include, but are not limited to Saccharomyces, Schizosaccharomyces, Hansenula, Candida, Torulopsis, Yarrowia, or Pichia. See e.g., U.S. Pat. Nos. 4,812,405; 4,818,700; 4,929,555; 5,736,383; 5,955,349; 5,888,768 and 6,258,559.
The phase transition behaviour of the ELPs allows for easy purification. For example, the ELPs may be purified from host cells using methods known to those skilled in the art. These techniques involve the crude fractionation of the cellular milieu to polypeptide and non-polypeptide fractions. Having separated the polypeptide from other proteins, the polypeptide of interest may be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suited to the preparation of a pure peptide or polypeptide are filtration, ion-exchange chromatography, exclusion chromatography, polyacrylamide gel electrophoresis, affinity chromatography, or isoelectric focusing. A particularly efficient method of purifying peptides is fast protein liquid chromatography or even HPLC. In the case of ELP compositions protein purification may also be aided by the thermal transition properties of the ELP domain as described in U.S. Pat. No. 6,852,834.
Generally, “purified” will refer to a protein or peptide composition that has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity. Where the term “substantially purified” is used, this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the proteins in the composition.
Various methods for quantifying the degree of purification of the protein or peptide will be known to those of skill in the art. These include, for example, determining the specific activity of an active fraction, or assessing the amount of polypeptides within a fraction by SDS/PAGE analysis.
Various techniques suitable for use in protein purification will be well known to those of skill in the art. These include, for example, precipitation with ammonium sulfate, PEG, antibodies and the like or by heat denaturation, followed by centrifugation; chromatography steps such as ion exchange, gel filtration, reverse phase, hydroxylapatite and affinity chromatography; isoelectric focusing; gel electrophoresis; and combinations of such and other techniques. As is generally known in the art, it is believed that the order of conducting the various purification steps may be changed, or that certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified protein or peptide.
Immunomodulators
The micelles provided herein include at least one immunomodulator. Immunomodulator refers to a substance capable of altering (e.g., inhibiting, decreasing, increasing, enhancing or stimulating) the working of any component of the innate, humoral and/or cellular immune system of a mammal.
The immunomodulator may include at least one epitope. An epitope, also known as antigenic determinant, is the part of an antigen that is recognized by the immune system, for example by antibodies, B cells, or T cells. The epitope is the molecular region of an antigen which is bound by the antibody, B cell or T cell. Although epitopes are usually non-self-proteins, sequences derived from the host that can be recognized (as in the case of autoimmune diseases) are also epitopes.
“Epitope" includes any determinant capable of specific binding to an immunoglobulin or otherwise interacting with an immune system cell or molecule. Epitopes generally consist of chemically active surface groupings of molecules such as amino acids or carbohydrate or sugar side chains and can have specific three-dimensional structural characteristics, as well as specific charge characteristics. An epitope may be "linear" or "conformational."
The epitope may be of any chemical nature, including without limitation, peptides, carbohydrates, lipids, glycopeptides and glycolipids. The epitope may be at least substantially the same as a naturally occurring epitope. It may be identical to a naturally occurring epitope, or a modified form of a naturally occurring epitope. The term "linear epitope" refers to an epitope with all of the points of interaction between the protein and the interacting molecule (such as an antibody) occur linearly along the primary amino acid sequence of the protein (continuous). The term "conformational epitope" refers to an epitope in which discontinuous amino acids that come together in three-dimensional conformation. In a conformational epitope, the points of interaction occur across amino acid residues on the protein that are separated from one another. Generally, binding molecules (such as antibodies) for a particular target antigen will preferentially recognize an epitope on the target antigen in a complex mixture of proteins and/or macromolecules. Regions of a given polypeptide that include an epitope can be identified using any number of epitope mapping techniques, well known in the art. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, New Jersey. For example, linear epitopes may be determined by concurrently synthesizing large numbers of peptides on solid supports, the peptides corresponding to portions of the protein molecule, and reacting the peptides with antibodies while the peptides are still attached to the supports. Such techniques are known in the art and described in, e.g., U.S. Patent No. 4,708,871 ; Geysen et al., (1984) Proc. Natl. Acad. Sci. USA 8:3998-4002; Geysen et al., (1985) Proc. Natl. Acad. Sci. USA 82:78-182; Geysen et al., (1986) Mol. Immunol. 23:709-715. Similarly, conformational epitopes are readily identified by determining spatial conformation of amino acids such as by, e.g., hydrogen/deuterium exchange, x-ray crystallography and two-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols, supra. Antigenic regions of proteins can also be identified using standard antigenicity and hydropathy plots, such as those calculated using, e.g., the Omiga version 1.0 software program available from the Oxford Molecular Group. This computer program employs the Hopp/Woods method, Hopp et al., (1981) Proc. Natl. Acad. Sci USA 78:3824-3828; for determining antigenicity profiles, and the Kyte- Doolittle technique, Kyte et al., (1982) J. Mol. Biol. 157:105-132; for hydropathy plots.
In some examples, the epitope may be referred to by the source that it has been derived from/from which it originates. For example, the epitope may be selected from one or more of: an allergenic epitope, a viral epitope, a bacterial epitope, parasitic epitope, disease associated epitope, and a tumour associated epitope.
In this context, an “allergenic epitope” refers to any antigenic determinant/component part of (or derived from) an allergen (the antigen) that is capable of specific binding to an immunoglobulin or otherwise interacting with an immune system molecule or cell (e.g. T cell or B cell). In other words, an allergenic epitope may be a linear or conformational epitope that is a (small) part of the allergen. The epitope may be present within a longer sequence (for example, the a larger part, or all of the allergen may be present). Similarly, a “viral epitope” refers to any antigenic determinant/component part of (or derived from) a virus (the antigen) that is capable of specific binding to an immunoglobulin or otherwise interacting with an immune system molecule or cell (e.g. T cell or B cell). In other words, a viral epitope may be a linear or conformational epitope that is a (small) part of a viral protein for example. The epitope may be present within a longer sequence (for example, the a larger part, or all of a viral protein may be present).
Similarly, a “bacterial epitope” refers to any antigenic determinant/component part of (or derived from) bacteria (the antigen) that is capable of specific binding to an immunoglobulin or otherwise interacting with an immune system molecule or cell (e.g. T cell or B cell). In other words, a bacterial epitope may be a linear or conformational epitope that is a (small) part of a bacterial protein for example. The epitope may be present within a longer sequence (for example, a larger part, or all of a bacterial protein may be present).
Equivalent definitions for “parasitic epitope”, “disease-associated epitope”, and “tumour- associated epitope” would be clear to a person of skill in the art. In this context, a “tumour- associated epitope” can be an epitope from a tumour associated antigen (TAA).
The epitope may be an epitope derived from a naturally occurring antigen or epitope. A derived epitope may differ from a naturally occurring epitope by 1 , 2, 3, 4, or more amino acid residues, as compared to the native epitope. These modifications as compared to a naturally occurring epitope may independently be selected from: a substitution of an amino acid as compared to the original sequence; a deletion of an amino acid residue as compared to the original sequence; and introduction of an additional amino acid residue as compared to the original sequence.
The epitope may be recognised and bound by a B cell, immunoglobulin or antibody, for example. Epitopes that are bound by B cells are typically at least five amino acids, more often at least six amino acids, still more often at least seven or eight amino acids in length, that may be continuous (“linear”) or discontinuous (“conformational”) (the latter being formed by the folding of a protein to bring non-contiguous parts of the primary amino acid sequence into physical proximity). B-cells may also bind to carbohydrate epitopes.
The epitope may be recognised and bound by a T cell (via a T cell receptor for example). Epitopes that are recognised by T cell receptors are typically presented to the T cell receptor in the context of class I or class II MHC molecules. The class I epitopes are usually 8 to 15, more often 9-11 amino acids in length. The class II epitopes are usually 5-24 (a 24 mer is the longest peptide which can fit in the Class II groove), more often 8-24 amino acids. Carbohydrate epitopes (as small as a single sugar unit (e.g., Tn)) may also be recognised by T cells. They are preferably no larger than five sugars. Many such epitopes are known.
Allergenic epitopes
In some examples, the epitope is derived from or part of an allergenic antigen. In other words, the immunomodulator may comprise an allergenic epitope. In some examples, the immunomodulator may comprise part of an allergen (which includes an allergenic epitope). In other examples, the immunomodulator may be an allergen (which includes an allergenic epitope).
Allergenic epitopes refer to an epitope derived from an antigen that causes an allergic reaction (i.e. and allergen). The term “allergen” encompasses "allergen extracts" and "allergenic epitopes”. Examples of allergens include but are not limited to pollens; house dust and dust mites; animal allergens; mold and fungus; insect bodies and insect venom; feathers; food; and drugs (e.g., penicillin).
In some examples, the allergen from which the epitope is derived may be a Type I allergen. In some examples, the allergen may be a type II allergen.
Type I allergens are allergens that illicit a Type I allergic reaction. Type I allergic reaction (hypersensitivity) is also known as an immediate reaction and involves immunoglobulin E (IgE) mediated release of antibodies against the soluble antigen. This results in mast cell degranulation and release of histamine and other inflammatory mediators.
Pollen Epitopes
Pollen allergens include for example, grass pollen, tree pollen (e.g. birch, alder, hazel, olive, cypress, cedar, plane tree) and weed pollen (e.g. ragweed, mugwort, pellitory, Russian thistle, plantain, goosefoot).
In some examples, the allergen is birch pollen allergen. In some examples, the allergenic epitope is derived from or is a Bet v protein, for example, Bet v 1 , Bet v 2, Bet v 3, Bet v 4, Bet v 5, Bet v 6, or Bet v 7. In some examples, the allergenic epitope includes a Bet v 1 epitope, is derived from or is Bet v 1 .
Other exemplary pollen allergens that the epitope may be (derived) from include Amb a 1 family of pectate lyases (e.g., UniProt accession numbers P27759, P27760 , P27761 , P27762 from Ambrosia artemisiifolia (short ragweed)); the defensin-like Art v 1 family (e.g., from mugwort and feverfew, e.g., UniProt ace. no. Q84ZX5 from Artemisia vulgaris (mugwort)); the Ole e 1-like allergens, Pla 1 1 from plantain, and Che a 1 from goosefoot, and the nonspecific lipid transfer proteins Parj 1 and Parj 2 from pellitory (Gadermaier G, et al. Curr Allergy Asthma Rep. 4(5):391- 400, 2004).
Dust mite allergens that the epitope may be (derived) from include, for example, dust mite protein Der f 1‘, Der f 2, Der p 1 , Der p 2, Der p 5, Der p 7, dust mite protein Der p 10, Der p 11 , Der p 20, Der p 21 , Der p 23, Bio t 5, Bio 1 10, Bio 1 21 , from Dermatophagoides pteronyssinus, Dermatophagoides farinae, and Blomia tropicalis, respectively.
Animal allergens may occur in dander, feathers, hair, saliva, and excretions (e.g., urine) from animals such as cat, dog, camel, chinchilla, cow, deer, gerbil, goat, guinea pig, hamster, hog, horse, mohair, monkey, mouse, rabbit, sheep. For example, domesticated animals such as cats (Fells domesticus) and dogs (Canis lupus familiaris) are common sources of allergy. Fel d 1 (UniProt acc. no. P30438 (chain 1); UniProt acc. no. P30440 (chain 2)), Fel d 3, and Fel d 4 are major cat allergens. Can f 1 (UniProt acc. no. 018873) and Can f 2 (UniProt acc. no. 018874) are major dog allergens. Rodents such as mice (e.g., Mus musculus), rats (e.g., Rattus norvegicus), and rabbits (e.g. European rabbit [Oryctolagus cuniculus]) are common sources of allergy. Identified allergens include, e.g., Mus m 1 , Rat n 1 , and Ory c 1 , in these species, respectively.
Fungi (e.g., fungal spores or fragments (e.g., hyphal fragments)) are significant sources of allergy. Alternaria (e.g., Alternaria alternata (Altemaria rot fungus)), Cladosporium (e.g., Cladosporium herbarum, Cladosporium cladosporioides), Aspergillus (e.g., Aspergillus fumigatus, Aspergillus niger), Fusarium, Penicillium are exemplary allergenic fungi of interest. In some examples, an allergen the epitope is derived from comprises a protein found in or produced by Alternaria, Cladosporium, Aspergillus, Fusarium, Penicillium, or other fungus. For example, an allergen the epitope is derived from can comprise an Alt a, Asp a, Asp n, Cla or Pen protein.
Insects and insect venoms are notable sources of allergens. Cockroach allergens are significant causes of allergy in many areas of the world. Cockroach species include, for example, Blattella germanica (German cockroach) and Periplaneta americana (American cockroach), and Blatta orientalis (Oriental cockroach) Cockroach allergens include, for example, Bia g 1 , Bia g 2, Bia g 5, Bia g 5, Bia g 6, Bia g 7, and Bia g 8 (from B. germanica) and Per a 1 , Per a 3, Per a 6, Per a 7, Per a 9, and Per a 10 (from P. Americana). In some examples, an allergen comprises an epitope derived from a Bia g, Per a, or Bia o allergen. Ant, moths, fleas, flies (e.g., house fly, horse fly, mayfly), and mosquitos are also sources of allergens. In some examples, an allergen the epitope may be derived from is a cockroach, ant, moth, flea, fly, or mosquito protein.
Insect venoms, (e.g., from insects of the order Plymenoptera, e.g., bees, hornets, or wasps) that are potential causes of severe allergic reactions include venoms from European Hornet (Vespa crabr ), Honey Bee (Apis mellifera), Hornet (Dolichovespula spp.), Paper Wasp (Polistes spp.), Yellow Jacket (Vespula spp.), White (Bald)-faced Hornet (Dolichovespula maculata), Yellow Hornet (Dolichovespula arena ria). In some examples, an allergen the epitope may be derived from is a venom (or extract or component thereof) of a bee, wasp, or hornet. For example, an allergen the epitope may be derived from can comprise an Api, Doi, or Ves protein.
Food allergens that the epitope may be (derived) from include, but are not limited to proteins in legumes such as peanuts, peas, lentils and beans, as well as the legume-related plant lupin, tree nuts such as almond, cashew, walnut, Brazil nut, filbert/hazelnut, pecan, pistachio, beechnut, butternut, chestnut, chinquapin nut, coconut, ginkgo nut, lychee nut, macadamia nut, nangai nut and pine nut, egg, fish, shellfish such as crab, crawfish, lobster, shrimp and prawns, molluscs such as clams, oysters, mussels and scallops, milk, soy, wheat, gluten, corn, meat such as beef, pork, mutton and chicken, gelatine, sulphite, seeds such as sesame, sunflower and poppy seeds, and spices such as coriander, garlic and mustard, fruits, vegetables such as celery, and rice.
Methods of obtaining allergens are well known in the art. For example, pollens can be collected from the respective plants, which may be cultivated or in the wild. Fungal extracts can be produced from pure culture mycelial mats or allergens can be isolated from culture medium. Rusts and smuts can be obtained from natural growths. Epithelial extracts can be produced from the hide, hair, or feathers containing the natural dander, or from separated dander. Insect and mite extracts can be produced from the whole body of the insects or mite, respectively. In the case of insect venoms, venom or venom-containing organs can be isolated or a whole-body extract can be used. House dust can he made from various dusts ordinarily found in the home (e.g., upholstery dust, mattress dust, or general dust accumulating on surfaces). Other dusts (e.g., grain dust, wood dust, cotton dust) can be collected from the appropriate location. Food extracts can be prepared from the edible portions of the respective foods, e.g., freshly obtained foods.
Methods suitable for allergen processing, e.g., production of allergen extracts, purification of allergen molecules, etc., are well known in the art. Very briefly, source allergen material (e.g., pollen, insect, dander) can be subjected initially to pulverization, drying, defatting (by extraction using organic solvent), or other steps as appropriate for the particular allergen. Centrifugation can be used, e.g., to separate solid or particulate matter. Resulting material can be incubated in an aqueous medium (e.g., water or suitable buffered solution, e.g., ammonium bicarbonate, phosphate buffered saline, etc.) for a suitable period of time to at least partly solubilize proteins. Crude extract can be processed using, e.g., dialysis, filtration, fractionation, chromatography, etc.
Extracts of allergens specifically processed for safe use in human immunotherapy are available commercially. For example, GREER Laboratories Tnc. Allergy and Immunotherapy division publishes a brochure entitled “Human Allergy Products and Sendees” available on-line at the company website currently at: http://www.greerlabs.com/files/catalogs/HumanAllergyCatalog.pdf. GREER also publishes a brochure entitled “Source Materials Products and Sendees” available online at the company website currently at -192017200098 06Jan 2017: http://www.greerlabs.com/files/catalogs/SourceMateiialsCatalog.pdf, which details available allergens that can be used as raw materials for production of allergen extracts or more highly purified allergen protein preparations. Both publications are incorporated herein by reference.
Viral epitopes
In some examples, the epitope is derived from or part of a viral antigen. In other words, the immunomodulator may comprise a viral epitope. In some examples, the immunomodulator may comprise part of a viral protein (which includes a viral epitope). In other examples, the immunomodulator may be a viral protein (which includes a viral epitope). In some examples, the epitope is derived from or part of a viral antigen, such as an epitope derived from an infectious virus.
Examples of infectious virus include: Retroviridae (e.g., human immunodeficiency viruses, such as HIV-1 (also referred to as HTLV-III, LAV or HTLV- lll/LAV, or HLV-III; and other isolates, such as HIV-LP; Picornaviridae (e.g., polio viruses, hepatitis A virus; enteroviruses, human coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g., strains that cause gastroenteritis); Togaviridae (e.g., equine encephalitis viruses, rubella viruses); Flaviridae (e.g., dengue viruses, encephalitis viruses, yellow fever viruses); Coronaviridae (e.g., coronaviruses); Rhabdoviridae (e.g., vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g., ebola viruses); Paramyxoviridae (e.g., parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenza viruses); Bungaviridae (e.g., Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses); Arena viridae (hemorrhagic fever viruses); Reoviridae (e.g., reoviruses, orbiviurses and rotaviruses); Birnaviridae', Hepadnaviridae (Hepatitis B virus); Parvoviridae (parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae (most adeno viruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), hecpes viruses'); Poxviridae (variola viruses, vaccinia viruses, pox viruses); and Iridoviridae (e.g., African swine fever virus); and unclassified viruses (e.g., the etiological agents of Spongiform encephalopathies, the agent of delta hepatitis (thought to be a defective satellite of hepatitis B virus), the agents of non-A, non-B hepatitis (class 1 = internally transmitted; class 2 = parenterally transmitted (i.e. , Hepatitis C); Norwalk and related viruses, and astro viruses).
Bacterial epitopes In some examples, the epitope is derived from or part of a bacterial antigen. In other words, the immunomodulator may comprise a bacterial epitope. In some examples, the immunomodulator may comprise part of a bacterial protein (which includes a bacterial epitope). In other examples, the immunomodulator may be a bacterial protein (which includes a bacterial epitope). In some examples, the epitope is derived from or part of a bacterial antigen, such as an epitope derived from an infectious bacteria.
Examples of infectious bacteria include: Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia, Mycobaderia sps (e.g. M. tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcus pneumoniae, pathogenic Campylobacter spp., Enterococcus spp., Haemophilus influenzae, Bacillus antracis, Corynebaderium diphtheriae, Corynebacterium spp., Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides spp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue, Leptospira, and Actinomyces israelii.
Parasitic epitopes
In some examples, the epitope is derived from or part of a parasitic antigen. In other words, the immunomodulator may comprise a parasitic epitope. In some examples, the immunomodulator may comprise part of a parasitic protein (which includes a parasitic epitope). In other examples, the immunomodulator may be a parasitic protein (which includes a parasitic epitope). In some examples, the epitope is derived from or part of a parasitic antigen, such as a protein from Hookworm (Necator americanus), Scabies mite (Sarcoptes scabiei var. hominis), Roundworm (Ascaris lumbricoides), Flatworm blood fluke (Schistosoma mansoni, S. haematobium, S. japonicum), Tapeworm (Taenia solium), Pinworm (Enterobius vermicularis), Toxoplasma gondii, Giardia lamblia or Entamoeba histolytica.
Disease-associated epitope
In some examples, the epitope is a disease-associated epitope.
An epitope may be indirectly associated with a disease if the epitope is of an antigen which is specifically produced or overproduced by infected cells of the subject, or which is specifically produced or overproduced by other cells of the subject in specific, but non-immunological, response to the disease, e.g., an angiogenic factor which is overexpressed by nearby cells as a result of regulatory substances secreted by a tumour. The term “disease-associated epitope” also includes any non-naturally occurring epitope which is sufficiently similar to an epitope naturally associated with the disease in question so that antibodies or T cells which recognize the natural disease epitope also recognize the similar nonnatural epitope.
Tumour-associated epitopes
An epitope may be said to be directly associated with a particular tumour if it is an intracellular, surface or secreted antigen that is present on/in said tumour. It need not be present on all cells of the tumour type in question, or on all cells of a particular tumour, or throughout the entire life of the tumour. It need not be specific to the tumour in question. An epitope may be said to be “tumour-associated” in general if it is so associated with any tumour (cancer, neoplasm).
The tumour-associated epitope may be a tumor-associated antigen (TAA), or derived from a TAA. In some examples, the epitope is derived from or part of a TAA. In other words, the immunomodulator may comprise a TAA epitope. In some examples, the immunomodulator may comprise part of a TAA (which includes a tumour-associated epitope). In other examples, the immunomodulator may be a TAA (which includes a tumour-associated epitope). Several TAAs are known.
Tumours may be of mesenchymal or epithelial origin. Cancers include cancers of the colon, rectum, cervix, breast, lung, stomach, uterus, skin, mouth, tung, lips, larynx, kidney, bladder, prostate, brain, and blood cells.
In the case of a “tumour-specific” epitope, the epitope is more frequently associated with that tumour that with other tumours, or with normal cells. Preferably, there should be a statistically significant (p=0.05) difference between its frequency of occurrence in association with the tumour in question, and its frequency of occurrence in association with (a) normal cells of the type from which the tumour is derived, and (b) at least one other type of tumour. An epitope may be said to be “tumour-specific” in general if it is associated more frequently with tumours (of any or all types) than with normal cells. It need not be associated with all tumours.
The term “tumour specific epitope” also includes any non-naturally occurring epitope which is sufficiently similar to a naturally occurring epitope specific to the tumour in question (or as appropriate, specific to tumours in general) so that antibodies or T cells stimulated by the similar epitope will be essentially as specific as cytotoxic T lymphocytes stimulated by the natural epitope.
In general, tumour-versus-normal specificity is more important than tumour-versus-tumour specificity as (depending on the route of administration and the particular normal tissue affected) higher specificity generally leads to fewer adverse effects. Tumour-versus-tumour specificity is more important in diagnostic as opposed to therapeutic uses.
The term “specific” is not intended to connote absolute specificity, merely a clinically useful difference in probability of occurrence in association with a pathogen or tumour rather than in a matched normal subject.
Tumour-associated epitopes include, but are not limited to, peptide epitopes such as those of mutant p53, the point mutated Ras oncogene gene product, her 2/neu, c/erb2, and the MLIC1 core protein, and carbohydrate epitopes such as sialyl Tn (STn), TF, Tn, CA 125, sialyl Lex, sialyl Lea and P97.
Carbohydrate epitopes are also of interest. For example, any of three types of tumour-associated carbohydrate epitopes which are highly expressed in common human cancers may be presented. These particularly include the lacto series type 1 and type 2 chains, cancer associated ganglio chains, and neutral glycosphingolipids. Examples of the lacto series Type 1 and Type 2 chains are as follows: Lewis a, dimeric Lewis a, Lewis b, Lewis b/Lewis a, Lewis x, Lewis, y, Lewis a/Lewis x. dimeric Lewis x, Lewis y/Lewis x, trifucosyl Lewis y, trifucosyl Lewis b, sialosyl Lewis x, sialosyl Lewis y, sialosyl dimeric Lewis x, Tn, sialosyl Tn, sialosyl TF, TF. Examples of cancer- associated ganglio chains are as follows: GM3. GD3, GM2, GM4, GD2, GM1 , GD-1a, GD-1b. Neutral sphingolipids include globotriose, globotetraose, globopentaose, isoglobotriose, isoglobotetraose, mucotriose, mucotetraose, lactotriose, lactotetraose, neolactotetraose, gangliotriose, gangliotetraose, galabiose, and 9-O-acetyl-GD3. Numerous antigens of clinical significance bear carbohydrate determinants. One group of such antigens comprises the tumour- associated mucins (Roussel, et al., Biochimie 70, 1471 , 1988).
Generally, mucins are glycoproteins found in saliva, gastric juices, etc., that form viscous solutions and act as lubricants or protectants on external and internal surfaces of the body. Mucins are typically of high molecular weight (often >1 ,000,000 Dalton) and extensively glycosylated. The glycan chains of mucins are O-linked (to serine or threonine residues) and may amount to more than 80% of the molecular mass of the glycoprotein. Mucins are produced by ductal epithelial cells and by tumours of the same origin, and may be secreted, or cell-bound as integral membrane proteins (Burchell, et al., Cancer Res., 47, 5476, 1987; Jerome, et al., Cancer Res., 51 , 2908, 1991).
Other immunomodulators
In some examples, the immunomodulator may be a peptide K molecule as described herein. Peptide K is a coiled coil forming peptide as described herein, but as shown by the examples provided herein peptide K may act as an immunomodulator. Peptide K binds parallel to membranes and due to cationic and amphiphilic properties peptide K may have cell penetrating peptide (CPP) activity. In addition, coiled coil domains of peptide K may interact with scavenger receptors from cells such as dendritic cells. Both membrane binding and CPP characteristics potentially increase the uptake of coiled coils and conjugates thereof into cells. Thus, peptide K may act to alter immune response in a subject as well as acting as an intermediary binding molecule as described herein.
Attachment of Immunomodulators to ELP
The micelles provided herein include ELP and an immunomodulator. As described above, additional moieties, such as immunomodulators may be attached to an ELP using a variety of methods.
In some examples, the immunomodulator is attached to the ELP so that the immunomodulator is displayed on the outside of a micelle (i.e. the corona of the micelle). This allows for the immunomodulator and any epitopes thereof to be accessible to binding molecules of the immune system and thus lead to alterations in immune response.
In some examples, the immunomodulator is bound to a hydrophilic block of an amphiphilic ELP. In examples, where the hydrophilic block is located at the N-terminal of an ELP, the immunomodulator may be bound to the N-terminal of the ELP. In some examples, where the hydrophilic block is located at the C-terminal of an ELP, the immunomodulator may be bound to the C-terminal of the ELP. For example, for the ELP I48S48 the immunomodulator may be bound to the C-terminal carboxylic group of the ELP.
The orientation of the immunomodulator may be selected so that once attached, epitopes of the immunomodulator are accessible to immune system binding molecules. When the immunomodulator is a peptide or polypeptide the immunomodulator may be attached via the N- terminal, C-terminal or via sidechains of intrachain amino acids of the peptide or polypeptide.
In some examples, an immunomodulator may be attached to the ELP molecule covalently. In some examples, an immunomodulator as described herein may be bound or attached to an ELP by recombinantly expressing the ELP and immunomodulator as a fusion protein. Alternatively, the immunomodulator may be covalently attached an ELP via chemical methods as described herein.
As such, provided herein are ELP-immunomodulator fusion proteins. In addition, provided herein are nucleic acids encoding such fusion proteins. For example, the immunomodulator may be Bet v 1 and the ELP may be I48S48. In such an example, the Bet v 1 may be bound to the C-terminal ELP via the N-terminal amide group or C-terminal carboxylic group of the Bet v 1 peptide. For example, the ELP fusion protein may have a sequence of:
ELP-Bet v 1
MG(VPGIG)48(VPGSG)48-
GVFNYETETTSVIPAARLFKAFILDGDNLFPKVAPQAISSVENIEGNGGPGTIKKISFPEGFPFKY VKDRVDEVDHTNFKYNYSVIEGGPIGDTLEKISNEIKIVATPDGGSILKISNKYHTKGDHEVKAEQ VKASKEMGETLLRAVESYLLAHSDAYN (SEQ ID NO: 31).
In some examples, the immunomodulator is non-covalently bound to the ELP molecule. Non- covalent binding strategies are well known such as specific ligand- and protein-binding methods such as biotin-avidin interaction, antigen-antibody interaction, Ni-NTA-hexahistidine interaction, aptamer-protein binding, and specific-sequence DNA and DNA-binding protein interactions. Non- covalent attachment may allow for controllable reversible binding of an immunomodulator to the ELP.
In some examples, the immunomodulator is bound via an intermediary binding molecule that may be covalently bound to the ELP molecule and a cognate of the intermediary binding molecule.
The terms “cognate binding partner” and “cognate” refer to a second molecule, which specifically interacts with the intermediary binding molecule. In some examples, the “intermediary binding molecule” and “cognate binding partner” comprise a binding pair. In some examples, multiple intermediary binding molecules have the same cognate binding partner, and in some examples, multiple cognate binding partners bind the same intermediary binding molecule.
For example, the ELP may be bound to a peptide that is configured to form a coiled coil motif with its cognate partner, the cognate partner being bound to the immunomodulator, thus providing a system that allows for the immunomodulator to be non-covalently attached to the ELP (i.e. via the intermediary binding molecule).
A coiled coil motif or coiled coil refers to a peptide/protein sequence usually with a contiguous pattern of hydrophobic residues spaced 3 and 4 residues apart, which assembles (folds) to form a multimeric bundle of helices. The sequences, structures, and interactions of coiled-coils have been studied extensively and well documented in for example, Mason J.M., Arndt K.M. Coiled coil domains: Stability, specificity, and biological implications. Chembiochem. 2004;5:170-176. doi: 10.1002/cbic.200300781 ; Lupas A.N., Gruber M. The structure of a-helical coiled coils. Adv. Protein Chem. 2005;70:37-38; Woolfson D.N., Bartlett G.J., Bruning M., Thomson A.R. New currency for old rope: From coiled-coil assemblies to a-helical barrels. Curr. Opin. Struct. Biol. 2012;22:432-441. doi: 10.1016/j.sbi.2012.03.002; Apostolovic B., Danial M., Klok H.-A. Coiled coils: Attractive protein folding motifs for the fabrication of self-assembled, responsive and bioactive materials. Chem. Soc. Rev. 2010;39:3541-3575. doi: 10.1039/b914339b; and rigoryan G., Keating A.E. Structural specificity in coiled-coil interactions. Curr. Opin. Struct. Biol. 2008;18:477-483. doi: 10.1016/j.sbi.2008.04.008 all of which are expressly incorporated herein by reference.
The most commonly observed type of coiled coil is left-handed; here each helix has a periodicity of seven (a heptad repeat), with anywhere from two (in designed coiled coils) to 200 of these repeats in a protein. The helices are amphipathic, being encoded by a relatively straightforward sequence repeat of hydrophobic (H) and polar (P)residues, (HPPHPPP)n. This repeat is usually denoted (a-b-c-d-e-f-g)n in one helix, and (a’-b’-c’-d’-e’-f’-g’)n in the other. A helical diagram of a typical dimeric coiled coil shows that hydrophobic amino acid residues are placed in the positions of a and d, while polar residues are in the positions of e and g. The hydrophobic-polar patterns of amino acid residues at the interface between coiled-coils directly influence the interaction between the helices. The number of helices of a coiled coil (e.g., dimeric, trimeric, tetrameric or multimeric) is decided by packing in the hydrophobic core at the positions of a and d.
As such, the ELP may be bound to a first coiled coil forming peptide, and the immunomodulator may be bound to a second coiled coil forming peptide that is configured to form a coiled coil (complex) with the first coiled coil forming peptide. In some examples, a micelle may include ELPs each having a different first coiled coil forming peptide allowing for multiple immunomodulators, each being bound to a respective cognate partner for each first coiled coil forming peptide.
In some examples, the first coiled coil forming peptide may be multivalent (i.e. may be able to form a trimer, tetramer, or pentamer etc.) and configured to form a coiled coil with multiple cognate partners, thus allowing binding of multiple immunomodulators to a single ELP including the first coiled coil forming peptide.
Coiled coil forming peptides can form a homomeric coiled coil (i.e., wherein the first coiled coil forming peptide and any further coiled coil forming peptides are the same) or a heteromeric coiled coil (i.e. wherein the first coiled coil forming peptide and any further coiled coil forming peptides are different).
The coiled coil formed by the first and any further coiled coil forming peptides may be parallel or anti-parallel. For example, in a two-stranded parallel coiled-coils, a knobs insert into a’-d’-g’- d’ holes, but in antiparallel coiled-coils, the hole consists of residues at e’-a’-d’-a’ positions (prime indicates positions on the opposing a-helix).
In some examples, the first coiled coil peptide (i.e. , covalently bound to an ELP) may be a peptide comprising the sequence (KIAALKE)n (SEQ ID NO: 17) referred to herein as peptide K. The cognate partner of peptide K may be a peptide comprising the sequence (EIAALEK)n (SEQ ID NO: 18) referred to herein as peptide E.
In some examples, peptide E has the sequence “Ac-YG(EIAALEK)3-NH2” (SEQ ID NO: 19). In some examples, peptide K has the sequence “Ac-(KIAALKE)3GW-NH2” (SEQ ID NO: 20).
In some examples, an ELP covalently bound to peptide K has the sequence: “MG(VPGIG)48(VPGSG)48YWSGGG(KIAALKE)4” (SEQ ID NO: 9).
In some examples, an ELP covalently bound to peptide E has the sequence: “MG(VPGIG)48(VPGSG)48YWSGGG(EIAALEK)4” (SEQ ID NO: 10).
In some examples, the ELP comprises peptide E and the immunomodulator comprises peptide K. In other examples, the ELP comprises peptide K and the immunomodulator comprises peptide E.
Other examples of coiled coil peptide systems that may be used include GCN4 pLI, Leucine zipper peptide, JR2KC/JR2E, KV/KI/EV/EI, SVLP (SEQ ID NO: 23), SAGE (SEQ ID NO: 24), SAPN (SEQ ID NO: 25),CCE/CCK, and Helix A and Helix B. Further examples of coiled coil peptide systems include those described in Lapenta, Fabio, et al. "Coiled coil protein origami: from modular design principles towards biotechnological applications." Chemical Society Reviews 47.10 (2018): 3530-3542.
Coiled coil peptides may have a number of advantages such as being responsive to external stimuli, allowing controllable or tuneable release of moieties linked via such coiled coil peptides. For example, binding of the coiled coil peptide to its cognate peptide may be modulated by pH, temperature, and/or enzymatic processing.
Micelles
The compositions described herein include micelles that comprise ELPs as described herein. The micelles may include a mixture of ELPs described herein. For example, the micelles may include ELPs (i.e. not bound to an immunomodulator or intermediary binding molecule as described herein), one or more ELPs covalently bound to an immunomodulator, one or more ELPs bound non-covalently bound to an immunomodulator, and/or one or more ELPs covalently bound to a coiled coil peptide (e.g. wherein the immunomodulator does not comprise a cognate of the coiled coil forming peptide).
Amphiphilic ELPs are capable of self-assembling into micelles when heated to the CMT of the ELPs or mixture thereof. As mentioned above, the CMT of ELPs or mixtures thereof may be determined by known methods.
The ELPs described herein may be configured to form micelles at a desired temperature. For example, the ELPs may be designed via selection of the number of repeat structural units and/or the guest residue in each unit to have a desired CMT. The formation of micelles may also be altered by the amounts of different ELPs included in a micelle (i.e. , by the mixture of ELPs used). The CMT may be understood as the temperature that the ELPs self-assemble into micelles. The CMT of ELPs may be determined by any methods known in the art, for example by dynamic light scattering methods.
In some examples the CMT of ELPs described herein or mixture thereof may be at least 15°C. In some examples the CMT of ELPs described herein or mixture thereof may be at least 16°C. In some examples the CMT of ELPs described herein or mixture thereof may be at least 17°C. In some examples the CMT of ELPs described herein or mixture thereof may be from 15°C to 40°C. In some examples, the CMT of the ELPs described herein or mixture thereof may be from 16°C to 37°C. In some examples, the CMT of the ELPs described herein or mixture thereof may be from 16°C to 27°C. In some examples, the CMT of the ELPs described herein, or mixture thereof may be about 16°C to 26°C. In some examples, the CMT of the ELPs described herein or mixture thereof may be about 16°C, 17°C, 18°C, 19°C, 20°C, 21 °C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 21°C, 32°C, 33°C, 34°C, 35°C, 36°C, or 37°C.
In particular, the ELPs may be designed or selected so that micelles are formed and stabilised in a subject (e.g., a human). Thus, the CMT may be less than 37°C, less than 36°C, less than 35°C, less than 34°C, less than 32°C, less than 31 °C, less than 30°C, less than 29°C, less than 28°C, less than 27°C, less than 26°C, or less than 25°C. The ELPs described herein may be designed to have a CMT that allows for micelle formation in a subject (e.g., a human).
The ELPs described herein may also be designed or selected to have a CMC that is below a predetermined threshold. For example, a CMC below a known immunogenicity concentration or below a concentration that is toxic to a subject. CMC may be determined by known methods, such as static light scattering methods. In some examples, the ELPs or mixtures thereof may have a CMC of at least 0.004 pM. In some examples, the ELPs or mixture thereof may have a CMC from 0.05 to 1.2 pM. In some examples, the ELPs or mixture thereof may have a CMC from 0.1 to 1.1 pM. In some examples, the ELPs or mixture thereof may have a CMC less than 110 pM.
The micelles formed by the ELPs or mixture of ELPs described herein may have an average hydrodynamic diameter in the nanoscale. That is to say that the micelles may have an average hydrodynamic diameter of less than 1000 nm. For example, the micelles may have an average hydrodynamic diameter of less than 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 55, 50, 45, 40, 35, or 30 nm. In some examples, the micelles have average hydrodynamic diameter of from 10 nm to 1000 nm. In some examples, the micelles have average hydrodynamic diameter of from 10 nm to 100 nm. In some examples, the micelles have average hydrodynamic diameter of from 10 nm to 70 nm. In some examples, the micelles have average hydrodynamic diameter of from 30 nm to 70 nm. In some examples, the micelles have average hydrodynamic diameter of from 40 nm to 70 nm. In some examples, the micelles have average hydrodynamic diameter of from 50 nm to 60 nm.
The micelles formed by the ELPs or mixture of ELPs described herein may have an average polydispersity index from 0.001 to 0.5. Polydispersity index refers to the ratio of “weight average molecular weight” to “number average molecular weight” for a particular polymer. The polydispersity index represents the distribution of individual molecular weights within the polymer sample. In some examples, the ELPs may have an average polydispersity index from 0.001 to 0.2. In some examples, the ELPs may have an average polydispersity index less than 0.25. In particular, the micelles may be monodispersed. Monodispersed refers to a composition including micelles that all have substantially same molecular weight and are not aggregated. The micelles may be any shape. For example, the micelles may be substantially spherical.
The micelles formed by the ELPs or mixture of ELPs described herein may have an average zeta potential from -15 mV to 15 mV. In some examples, the ELPs may have an average zeta potential from -15 mV to -2 mV.
"Zeta potential" refers to measured electrical potential of a colloidal particle in aqueous environment, measured with an instrument such as a Zetasizer 3000 using Laser Doppler microelectrophoresis under the conditions specified. The zeta potential describes the potential at the boundary between bulk solution and the region of hydrodynamic shear or diffuse layer. The term is synonymous with "electrokinetic potential" because it is the potential of the particles which acts outwardly and is responsible for the particle's electrokinetic behaviour. The concentration of a specific ELP described herein in a micelle may be from 1 to 100% of the total amount of ELP in a micelle. For example, a micelle comprising 10% of an ELP bound to an immunomodulator consists of at least 10% of ELPs bound to an immunomodulator and 90% or less ELPs not bound to immunomodulators (i.e. ELPs not bound to the immunomodulator, any other immunomodulator or any other additional moiety (such as an intermediary binding molecule).
In some examples, the amount of an ELP bound to an immunomodulator that is present in a micelle is from 5 % to 100 % of the total amount of ELP in the micelle. In some examples, the amount is from 20 % to 100 % of the total amount of ELP in the micelle. In some examples, the amount is from 5 % to 50 % of the total amount of ELP in the micelle. In some examples, the amount is from 5 % to 20 % of the total amount of ELP in the micelle.
In one example there is provided a micelle that includes 10% ELP bound to an immunomodulator and 90% of other ELPs not bound to the same or any immunomodulator. For example, a micelle may have 10% of ELP covalently bound to an immunomodulator and 90% of ELP comprising (VPGXhbG)n(VPGXhpG)n (SEQ ID NO: 1) or (VPGXhpG)n(VPGXhbG)n (SEQ ID NO: 2) not bound to an immunomodulator. In some examples, the ELP bound to the immunomodulator comprises ((VPGXhbG)n(VPGXhPG)n-immunomodulator) or (immunomodulator-(VPGXhPG)n(VPGXhbG)n). In some examples, the ELP covalently bound to the immunomodulator and the ELP not bound to the immunomodulator is the ELP I48S48.
In one example there is provided a micelle that includes 20% ELP bound to an immunomodulator and 80% of other ELPs not bound to an immunomodulator, for example, 20% of ELP covalently bound to an immunomodulator and 80% of an ELP comprising (VPGXhbG)n(VPGXhPG)n (SEQ ID NO: 1) or (VPGXhPG)n(VPGXhbG)n (SEQ ID NO: 2) not bound to an immunomodulator. In one example there is provided a micelle that includes 30% ELP bound to an immunomodulator and 70% of other ELPs not bound to an immunomodulator, for example, 30% of ELP covalently bound to an immunomodulator and 70% of an ELP comprising (VPGXhbG)n(VPGXhPG)n (SEQ ID NO:1) or (VPGXhpG)n(VPGXhbG)n (SEQ ID NO: 2) not bound to an immunomodulator. In one example there is provided a micelle that includes 40% ELP bound to an immunomodulator and 60% of other ELPs not bound to an immunomodulator, for example, 40% of ELP covalently bound to an immunomodulator and 60% of an ELP comprising (VPGXhbG)n(VPGXhpG)n (SEQ ID NO: 1) or (VPGXhpG)n(VPGXhbG)n (SEQ ID NO: 2) not bound to an immunomodulator. In one example there is provided a micelle that includes 50% ELP bound to an immunomodulator and 50% of other ELPs not bound to an immunomodulator, for example, 50% of ELP covalently bound to an immunomodulator and 50% of an ELP comprising (VPGXhbG)n(VPGXhpG)n (SEQ ID NO: 1) or (VPGXhpG)n(VPGXhbG)n (SEQ ID NO: 2) not bound to an immunomodulator. In one example there is provided a micelle that includes 60% ELP bound to an immunomodulator and 40% of other ELPs not bound to an immunomodulator, for example, 60% of ELP covalently bound to an immunomodulator and 40% of an ELP comprising (VPGXhbG)n(VPGXhpG)n (SEQ ID NO: 1) or (VPGXhpG)n(VPGXhbG)n (SEQ ID NO: 2) not bound to an immunomodulator. In one example there is provided a micelle that includes 70% ELP bound to an immunomodulator and 30% of other ELPs not bound to an immunomodulator, for example, 70% of ELP covalently bound to an immunomodulator and 30% of an ELP comprising (VPGXhbG)n(VPGXhpG)n (SEQ ID NO: 1) or (VPGXhpG)n(VPGXhbG)n (SEQ ID NO: 2) not bound to an immunomodulator. In one example there is provided a micelle that includes 80% ELP bound to an immunomodulator and 20% of other ELPs not bound to an immunomodulator, for example, 80% of ELP covalently bound to an immunomodulator and 20% of an ELP comprising (VPGXhbG)n(VPGXhpG)n (SEQ ID NO: 1) or (VPGXhpG)n(VPGXhbG)n (SEQ ID NO: 2) not bound to an immunomodulator. In one example there is provided a micelle that includes 90% ELP bound to an immunomodulator and 10% of other ELPs not bound to an immunomodulator, for example, 90% of ELP covalently bound to an immunomodulator and 10% of an ELP comprising (VPGXhbG)n(VPGXhpG)n (SEQ ID NO: 1) or (VPGXhpG)n(VPGXhbG)n (SEQ ID NO: 2) not bound to an immunomodulator. In one example there is provided a micelle that includes 100% ELP bound to an immunomodulator, for example, 100% of an ELP comprising (VPGXhbG)n(VPGXhpG)n (SEQ ID NO: 1) or (VPGXhpG)n(VPGXhbG)n (SEQ ID NO: 2) covalently bound to an immunomodulator. In some examples, the ELP bound to the immunomodulator comprises “(VPGXhbG)n(VPGXhpG)n-immunomodulator” or “immunomodulator-(VPGXhpG)n(VPGXhbG)n”. In some examples, the ELP covalently bound to the immunomodulator and the ELP not bound to the immunomodulator is the ELP I48S48. For example the micelle may comprise or consist of the ELPs “(VPGIG)4s(VPGSG)48- immunomodulator” and the ELP I48S48. This may be referred to herein as a coiled coil micelle (i.e. a micelle that includes an ELP species covalently bound to an immunomodulator).
In some examples, the ELP is covalently bound to an intermediary binding molecule, which allows for non-covalent binding of an immunomodulator with a cognate partner. Accordingly, in one example there is provided a micelle that includes 5% ELP covalently bound to an intermediary binding molecule and 95% of ELPs not bound to an immunomodulator or intermediary binding molecule, for example, 5% of ELP covalently bound to an immunomodulator and 95% of an ELP comprising (VPGXhbG)n(VPGXhpG)n (SEQ ID NO: 1) or (VPGXhpG)n(VPGXhbG)n (SEQ ID NO: 2) not bound to an immunomodulator or intermediary binding molecule. In one example there is provided a micelle that includes 10% ELP bound to an intermediary binding molecule and 90% of ELPs not bound to an immunomodulator or intermediary binding molecule, for example, 10% of ELP covalently bound to an immunomodulator and 90% of an ELP comprising (VPGXhbG)n(VPGXhpG)n (SEQ ID NO: 1) or (VPGXhpG)n(VPGXhbG)n (SEQ ID NO: 2) not bound to an immunomodulator or intermediary binding molecule. In one example there is provided a micelle that includes 20% ELP bound to an intermediary binding molecule and 80% of ELPs not bound to an immunomodulator or intermediary binding molecule, for example, 20% of ELP covalently bound to an immunomodulator and 80% of an ELP comprising (VPGXhbG)n(VPGXhpG)n (SEQ ID NO: 1) or (VPGXhpG)n(VPGXhbG)n (SEQ ID NO: 2) not bound to an immunomodulator or intermediary binding molecule. In one example there is provided a micelle that includes 30% ELP bound to an intermediary binding molecule and 70% of other ELPs not bound to an immunomodulator or intermediary binding molecule, for example, 30% of ELP covalently bound to an immunomodulator and 70% of an ELP comprising (VPGXhbG)n(VPGXhpG)n (SEQ ID NO: 1) or (VPGXhpG)n(VPGXhbG)n (SEQ ID NO: 2) not bound to an immunomodulator or intermediary binding molecule. In one example there is provided a micelle that includes 40% ELP bound to an intermediary binding molecule and 60% of other ELPs not bound to an immunomodulator or intermediary binding molecule, for example, 40% of ELP covalently bound to an immunomodulator and 60% of an ELP comprising (VPGXhbG)n(VPGXhpG)n (SEQ ID NO: 1) or (VPGXhpG)n(VPGXhbG)n (SEQ ID NO: 2) not bound to an immunomodulator or intermediary binding molecule. In one example there is provided a micelle that includes 50% ELP bound to an intermediary binding molecule and 50% of other ELPs not bound to an immunomodulator or intermediary binding molecule, for example, 50% of ELP covalently bound to an immunomodulator and 50% of an ELP comprising (VPGXhbG)n(VPGXhpG)n (SEQ ID NO: 1) or (VPGXhpG)n(VPGXhbG)n (SEQ ID NO: 2) not bound to an immunomodulator or intermediary binding molecule. In one example there is provided a micelle that includes 60% ELP bound to an intermediary binding molecule and 40% of other ELPs not bound to an immunomodulator or intermediary binding molecule, for example, 60% of ELP covalently bound to an immunomodulator and 40% of an ELP comprising (VPGXhbG)n(VPGXhpG)n (SEQ ID NO: 1) or (VPGXhpG)n(VPGXhbG)n (SEQ ID NO: 2) not bound to an immunomodulator or intermediary binding molecule. In one example there is provided a micelle that includes 70% ELP bound to an intermediary binding molecule and 30% of other ELPs not bound to an immunomodulator or intermediary binding molecule, for example, 70% of ELP covalently bound to an immunomodulator and 30% of an ELP comprising (VPGXhbG)n(VPGXhpG)n (SEQ ID NO: 1) or (VPGXhpG)n(VPGXhbG)n (SEQ ID NO: 2) not bound to an immunomodulator or intermediary binding molecule. In one example there is provided a micelle that includes 80% ELP bound to an intermediary binding molecule and 20% of other ELPs not bound to an immunomodulator or intermediary binding molecule, for example, 80% of ELP covalently bound to an immunomodulator and 20% of an ELP comprising (VPGXhbG)n(VPGXhpG)n (SEQ ID NO: 1) or (VPGXhpG)n(VPGXhbG)n (SEQ ID NO: 2) not bound to an immunomodulator or intermediary binding molecule. In one example there is provided a micelle that includes 90% ELP bound to an intermediary binding molecule and 10% of other ELPs not bound to an immunomodulator or intermediary binding molecule, for example, 90% of ELP covalently bound to an immunomodulator and 10% of an ELP comprising (VPGXhbG)n(VPGXhpG)n (SEQ ID NO: 1) or (VPGXhpG)n(VPGXhbG)n (SEQ ID NO: 2) not bound to an immunomodulator or intermediary binding molecule. In one example there is provided a micelle that includes 100% ELP bound to an intermediary binding molecule, for example, 100% of ELP covalently bound to an intermediary binding molecule such as 100% of an ELP comprising (VPGXhbG)n(VPGXhpG)n (SEQ ID NO: 1) or (VPGXhpG)n(VPGXhbG)n (SEQ ID NO: 2) covalently bound to an intermediary binding molecule. In some examples, the ELP bound to the intermediary binding molecule comprises “(VPGXhbG)n(VPGXhpG)n-intermediary binding molecule” or “intermediary binding molecule- (VPGXhpG)n(VPGXhbG)n”. In some examples, the ELP covalently bound to the intermediary binding molecule and the ELP not bound to the intermediary binding molecule is the ELP I48S48. For example, the micelle may comprise or consist of the ELPs “MG(VPGIG)48(VPGSG)48YWSGGG(KIAALKE)4” (SEQ ID NO: 9) and the ELP l48S48. For example, the micelle may comprise or consist of the ELPs “MG(VPGIG)48(VPGSG)48YWSGGG(EIAALEK)4” (SEQ ID NO: 10) and the ELP l48S48. This may be referred to herein as a coiled coil micelle (i.e. , a micelle that includes an ELP species covalently bound to an intermediary binding molecule to allow for non-covalent binding of an immunomodulator that comprises a cognate of intermediary binding molecule).
In one example there is provided a micelle that includes 10% ELP bound to a first immunomodulator, 10% ELP bound to a second immunomodulator and/or an intermediary binding molecule, and 80% of ELPs not bound to an immunomodulator or intermediary binding molecule, for example, 10% of ELP covalently bound to a first immunomodulator, 10% of ELP covalently bound to peptide K and 80% of an ELP comprising (VPGXhbG)n(VPGXhpG)n (SEQ ID NO: 1) or (VPGXhpG)n(VPGXhbG)n (SEQ ID NO: 2) not bound to an immunomodulator or intermediary binding molecule. In some examples, the ELP covalently bound to the first immunomodulator, the ELP covalently bound to the second immunomodulator ad/or intermediary binding molecule and the ELP not bound to the intermediary binding molecule is the ELP I48S48. In some examples, the micelle may comprise or consist of the ELPs: “(VPGIG)4s(VPGSG)48- immunomodulator”; “MG(VPGIG)48(VPGSG)48YWSGGG(KIAALKE)4” (SEQ ID NO: 9); and the ELP I48S48-
Alternatively, the concentration of each ELP in a micelle be described by the ratio of each ELP in the micelle. For example, a micelle may have a ratio of ELP not bound to an immunomodulator or intermediary binding molecule described herein to ELP bound to an immunomodulator or intermediary binding molecule as described herein of 9:1 . For example, the composition includes 9 parts of an ELP comprising (VPGXhbG)n(VPGXhpG)n (SEQ ID NO: 1) or (VPGXhpG)n(VPGXhbG)n (SEQ ID NO: 2) not bound to an immunomodulator or intermediary binding molecule to 1 part ELP comprising “(VPGXhbG)n(VPGXhpG)n-immunomodulator”, “immunomodulator- (VPGXhpG)n(VPGXhbG)n”, “(VPGXhbG)n(VPGXhpG)n-intermediary binding” or “intermediary binding-(VPGXhpG)n(VPGXhbG)n”. In some examples the ELP covalently bound to an immunomodulator may be “(VPGIG)48(VPGSG)48-immunomodulator”. In some examples the ELP covalently bound to an intermediary binding molecule may be “(VPGIG)48(VPGSG)48-intermediary binding molecule”. In some examples the ELP not bound to an immunomodulator, or intermediary binding molecule may be the ELP I48S48.
In some examples, a micelle may have a ratio of ELP not bound to an immunomodulator or intermediary binding molecule described herein: ELP bound to an immunomodulator: and ELP bound to a second immunomodulator or intermediary binding molecule of 8: 1 : 1. For example, the composition includes 8 parts of an ELP comprising (VPGXhbG)n(VPGXhpG)n (SEQ ID NO: 1) or (VPGXhpG)n(VPGXhbG)n (SEQ ID NO: 2) not bound to an immunomodulator or intermediary binding molecule to 1 part ELP comprising “(VPGXhbG)n(VPGXhpG)n-immunomodulator”, or “immunomodulator-(VPGXhpG)n(VPGXhbG)n”, and 1 part ELP comprising “(VPGXhbG)n(VPGXhpG)n-intermediary binding” or “intermediary binding- (VPGXhpG)n(VPGXhbG)n”. In some examples the ELP covalently bound to an immunomodulator may be “(VPGIG)48(VPGSG)48-immunomodulator”. In some examples the ELP covalently bound to an intermediary binding molecule may be “(VPGIG)48(VPGSG)48-intermediary binding molecule”. In some examples the ELP not bound to an immunomodulator, or intermediary binding molecule may be the ELP I48S48. This may be referred to herein as a hybrid micelle (i.e. , a micelle that includes two species of ELPs covalently bound to different immunomodulators or one ELP species covalently bound to an immunomodulator and one ELP bound to an intermediary binding molecule such as peptide K [which may also be considered an immunomodulatory molecule]).
In order to form the micelles described herein, the amphiphilic ELPs described herein are mixed at an appropriate ratio in a buffer and heated to a temperature above the CMT. For example, the ELPs in buffer may be heated to a temperature of at least 16°C. In some examples, the ELPs in buffer are heated to a temperature of at least 20°C. In some examples, the ELPs in buffer are heated to a temperature of at least 30°C. the ELPs in buffer are heated to a temperature of at least 37°C.
The buffer may be any suitable buffer. In particular, the buffer may be physiologically acceptable buffer such as phosphate buffered saline or phosphate buffer. Other suitable buffers are well known in the art.
The micelles may be formed so that additional agents are encapsulated within the core of the micelle. Such agents may be hydrophobic and as such when the ELPs self-assemble, the agents are automatically incorporated into the core of the micelles. For example, additional agents that may be incorporated into the micelles may include hydrophobic drugs or hydrophobic immunomodulatory molecules that may for example be used in immunotherapy such as allergen immunotherapy such as anti-histamines, anti-lgE monoclonal antibodies such as omalizumab, or steroids. In some examples, the micelles may comprise vitamin D3 encapsulated within the core of the micelle.
In some examples, additional agents may be attached to the hydrophobic block of the ELP so that when the ELPs self-assemble the additional agents are incorporated into the core of the micelle.
In examples that include micelles with non-covalently bound immunomodulator, the buffer may include an immunomodulator bound to an intermediary binding molecule that is the cognate partner of an intermediary binding molecule bound to ELPs of the micelle. For example, if a micelle includes ELPs covalently bound to peptide E, an immunomodulator covalently bound to peptide K may be added to the buffer prior to heating and formation of micelles. Alternatively, if a micelle includes ELPs covalently bound to peptide K, an immunomodulator covalently bound to peptide E may be added to the buffer prior to heating and formation of micelles. The amount of immunomodulator bound to a cognate intermediary binding molecule may be equal to the amount of ELP bound to an intermediary binding molecule. For example, it may be an equimolar amount. For example, the immunomodulator bound to a cognate intermediary binding molecule may be added to the buffer at a ratio of 1 :1 to the amount of ELP bound to an intermediary binding molecule. For example, if a micelle includes one part of ELPs covalently bound to peptide K, the same amount of an immunomodulator covalently bound to peptide E may be added to the buffer and vice versa.
In some examples, a free cognate intermediary binding molecule may be added to the buffer. This may help reduce homodimerization of intermediary binding molecules bound to ELPs which may lead to aggregation of micelles. For example, when the micelle includes ELPs covalently bound to peptide K, free peptide E may be added to the buffer. This may help prevent peptide K molecules binding to other peptide K molecules thus preventing aggregation of micelles. The free cognate intermediary binding partner may be added at an equimolar amount to the amount of intermediary binding molecule bound to ELPs.
In some examples, when a micelle includes more than 20% ELPs bound to an intermediary binding molecule, such as peptide K, a free cognate intermediary binding molecule may be added to the buffer. In some examples, when a micelle includes more than 25% ELPs bound to an intermediary binding molecule, such as peptide K, a free cognate intermediary binding molecule may be added to the buffer. In some examples, when a micelle includes more than 30% ELPs bound to an intermediary binding molecule, such as peptide K, a free cognate intermediary binding molecule may be added to the buffer.
Uses
As shown by the examples provided herein, ELPs and micelles formed therefrom have a number of advantages over common adjuvants such as alum. “Adjuvant” refers to a substance that modulates the immunogenicity of an antigen.
Provided herein is the use of ELPs as described herein and micelles formed therefrom as adjuvants. In some examples, the ELPs as described herein, and micelles formed therefrom may be for use as adjuvants for vaccines. ELPs as described herein, and micelles formed therefrom may lead to a strong cellular immune response in comparison to known adjuvants such as alum. In some examples, the ELPs as described herein, and micelles formed therefrom provide a reduced Th2 (or a lower Th2 skewed) response in comparison to known adjuvants such as alum. As such, the use of the ELPs as described herein and micelles formed therefrom may be particular advantageous for vaccination against intracellular pathogens such viruses (e.g., CMV HIV), bacteria (e.g., Listeria, Mycobacteria, Salmonella (e.g., S. typhi) enteropathogenic Escherichia coli (EPEC), enterohaemorrhagic Escherichia coli (EHEC), Yersinia, Shigella, Chlamydia, Chlamydophila, Staphylococcus, Legionella), protozoa (e.g., Taxoplasma), fungi, and intracellular parasites (e.g., Plasmodium (e.g., P. vivax, P. falciparum, P. ovale, and P. malariae). Furthermore, the ELPs as described herein, and micelles formed therefrom may reduce humoral response against an immunogenic immunomodulator.
In addition, the increase in uptake of antigens (immunomodulators) bound to the ELPs as described herein and micelles formed therefrom may increase antigen presenting cell maturation and/or increase T cell expansion and differentiation. In particular, the ELPs as described herein, and micelles formed therefrom may increase differentiation of T cells to Th1 cells.
As such, it is shown that the ELPs described herein, and micelles formed therefrom may be used for delivery of an immunomodulator to a subject. In some examples, the ELPs described herein, and micelles formed therefrom may be used for delivery of an immunomodulator to specific cell types, for example delivery of an immunomodulator to antigen presenting cells such as dendritic cells.
The ELPs described herein, and micelles formed therefrom may be used as adjuvants for cancer immunotherapy. The ELPs described herein, and micelles formed therefrom may be used as adjuvants for allergen immunotherapy. It is shown that when an allergenic immunomodulator is used, the ELPs described herein, and micelles formed therefrom may decrease production of I g E in response to the allergen. In addition, they may increase IgG production and lead to earlier IgG production in comparison to known adjuvants such as alum. In addition, the use of the ELPs described herein and micelles formed therefrom may reduce the overall T cell response to the allergen in comparison to known adjuvants such as alum. Furthermore, the ELPs described herein, and micelles formed therefrom may reduce cellular mediated immune response to the allergen.
Given the simple methods of forming micelles that display immunomodulators on the outer surface of the micelle, the ELPs described herein, and micelles formed therefrom may be used for forming immunogenic vaccines.
Compositions
Provided herein are compositions for modulating an immune response, for example, modulating an immune response in a subject such as a human. The terms “modulating”, “altering” and “regulating” are used to interchangeably to refer to any change in cells of the immune system or the activity of such cells. Such control includes increasing or decreasing the number of various cell types, increasing or decreasing the activity of these cells, or stimulating or suppressing the immune system, which may be caused by other possible changes in the immune system.
The compositions provided herein may modulate certain parts of the immune response differently. For example, the compositions provided herein may increase cell-mediated immune response and/or reduce humoral immune response. In some examples, the compositions provided herein may increase cell-mediated immune response and/or increase humoral immune response. In some examples, the compositions provided herein may reduce cell-mediated immune response and/or reduce humoral immune response.
"Immune response” refers to a response by a cell of the immune system of a subject (for example of a human), such as a B cell, T cell (CD4 + or CD8 +), regulatory T cell, antigen-presenting cell, dendritic cell, monocyte, macrophage, NKT cell, NK cell, basophil, eosinophil, or neutrophil, to a stimulus. The response may be for a particular antigen (an “antigen specific response”), and may be a response by a CD4 T cell, CD8 T cell , or B cell via their antigen-specific receptor. In some embodiments, an immune response is a T cell response, such as a CD4+ response or a CD8+ response. Such responses by these cells can include, for example, cytotoxicity, proliferation, cytokine or chemokine production, trafficking, or phagocytosis, and can be dependent on the nature of the immune cell undergoing the response. Exemplary immune responses include humoral immune responses (e.g., production of antigenspecific antibodies) and cell-mediated immune responses (e.g., production of antigen-specific T cells). Assays for assessing an immune response are known in the art and may comprise in vivo assays, such as assays to measure antibody responses and delayed type hypersensitivity responses. For example, an assay to measure antibody responses primarily may measure B-cell function as well as B-cell/T-cell interactions. For the antibody response assay, antibody titers in the blood may be compared following an antigenic challenge. In vitro assays may comprise determining the ability of cells to divide, or to provide help for other cells to divide, or to release lymphokines and other factors, express markers of activation, and lyse target cells. Lymphocytes in mice and man can be compared in in vitro assays. In some examples, the lymphocytes from similar sources such as peripheral blood cells, splenocytes, or lymph node cells, are compared. It is possible, however, to compare lymphocytes from different sources as in the non-limiting example of peripheral blood cells in humans and splenocytes in mice. For an in vitro assay, cells may be purified (e.g., B-cells, T-cells, and macrophages) or left in their natural state (e.g., splenocytes or lymph node cells). The cells can be tested in vitro for their ability to proliferate using mitogens or specific antigens. The ability of cells to divide in the presence of specific antigens can be determined using a mixed lymphocyte reaction (MLR) assay. Supernatant from the cultured cells can be tested to quantitate the ability of the cells to secrete specific lymphokines. The cells can be removed from culture and tested for their ability to express activation antigens. This can be done by any method that is suitable as in the non-limiting example of using antibodies or ligands which bind to the activation antigen as well as probes that bind the RNA coding for the activation antigen.
The term "humoral immune response" refers to the stimulation of antibody production. Humoral immune response also refers to the accessory proteins and events that accompany antibody production, including T helper cell activation and cytokine production, affinity maturation, and memory cell generation.
The term "cell-mediated immune response" refers to the immunological defence provided by lymphocytes, such as the defence provided by sensitized T cell lymphocytes when they directly lyse cells expressing foreign antigens and secrete cytokines (e.g., IFN-gamma.), which can modulate macrophage and natural killer (NK) cell effector functions and augment T cell expansion and differentiation.
In some examples, the compositions of the invention may lead to an altered T cell response. For example, the compositions may alter T helper 1 cell (Th1) response. In some examples, the compositions provided herein may alter Th2 cell response. T helper (Th) cells provide helper functions to other cells of the immune system, especially the antigen-presenting cells (APCs) such as macrophages, dendritic cells, and B cells and are important for their activation and maturation. There are distinct subsets of CD4+ Th cells, including Th1 , Th2, Th17, and T regulatory cells, each activated by a specific set of cytokines and transcription factors and characterized by the cytokines they secrete and effector functions they perform.
Th1 cells derive from the alpha:beta lineage of T cells and recognize antigens presented by major histocompatibility complex (MHC) class I or II molecules. Th1 cells play important roles in the identification and eradication of intracellular pathogens such as viruses and bacteria, including Mycobacterium tuberculosis, Mycobacterium leprae, and Leishmania. These pathogens typically reside in phagocytic vesicles within cells such as macrophages and often evade intracellular killing by preventing lysosomal fusion. Th1 cells help to activate macrophages against these pathogens and overcome these microbial evasion strategies. In contrast, Th2 cells, which are the other major subset of CD4+ T cells, help to recognize extracellular pathogens such as helminths and parasites and activate B cell-mediated antibody responses. The two cytokines that play a critical role in Th1 differentiation are Interferon Gamma (IFNy) and IL-12. IL-12 secreted by APCs upon T cell engagement drives differentiation into Th1 effector cells through the activation of STAT4 transcription factor. STAT4 induces IFNy production, which is another driver towards Th1 differentiation by activating transcription factors STAT1 and Tbet. Tbet is considered the master regulator of Th1 differentiation since it induces more expression of IFNy, leading to a positive feedback loop to strengthen the Th1 response. The main effector functions of Th1 cells are in cell-mediated immunity and inflammation, including the activation of cytolytic and other effector functions of other immune cells such as macrophages, B cells, and CD8+ cytotoxic T lymphocytes (CTLs). One of the first steps in Th1-mediated activation of other immune cells is the interaction of CD40 Ligand (CD40L) on the surface of Th1 cells with CD40 expressed on the surface of macrophages, B cells, and dendritic cells. Effector Th1 cells also secrete copious amounts of IFNy that, besides further expanding the Th1 population, also activate cytolytic activities of macrophages through the induction of more than 200 target genes. This increased cytotoxic activity in macrophages is key to killing intracellular pathogens such as viruses, intracellular bacteria, and protozoa. A classic example of Th1-dependent immune response is during Mycobacteria infection. Mycobacteria escape lysosomal fusion within macrophages; however, peptides derived from these pathogens displayed by MHC Class II on the surface of infected macrophages lead to activation of Th1 responses, which turns on the cytolytic properties of macrophages.
In addition to clearing intracellular infection, Th1 responses play crucial roles in activating CD8+ cytotoxic T lymphocytes (CTLs) to target and destroy tumours, in addition to leading to increasing CTL survival and memory. CD40 also activates class switching in B cells to produce lgG2a antibodies.
Th2 cells mediate the activation and maintenance of the humoral, or antibody-mediated, immune response against extracellular parasites, bacteria, allergens, and toxins. Th2 cells mediate these functions by producing various cytokines such as IL-4, IL-5, IL-6, IL-9, and IL-13. These cytokines are responsible for a strong antibody production, eosinophil activation, and inhibition of several macrophage functions, thus providing phagocyte-independent protective responses. These cytokines also counteract the Th1 responses that allow for the Th2 responsiveness to IL-4. Functionally, Th2 cytokines have effects on many cell types in the body as the cytokine receptors are widely expressed on numerous cell types. Th2 cells stimulate and recruit specialized subsets of immune cells, such as eosinophils and basophils, to the site of infection or in response to allergens or toxins leading to tissue eosinophilia and mast cell hyperplasia. They induce mucus production, goblet cell metaplasia, and airway hyper-responsiveness. Th2 cells also control the regulation of B cell class-switching to IgE. Because of their influence on the production of antibodies and allergic responses, over activation of Th2 cells appears to be responsible for the exacerbation of allergies (Type-1 , immediate hypersensitivity reactions), autoimmune reactions such as chronic graft-versus host disease, progressive systemic sclerosis, and systemic lupus erythematosus. Additionally, Th2 cells are also known to be responsible for the development of asthma and other allergic inflammatory diseases. Interestingly, Th2 cells also produce the growth factor amphiregulin and IL-24 which have anti-tumor effects. The surrounding cytokine environment drives polarization of naive T cells into specific cell lineages, where the Th2 cell type is specifically triggered by interleukin (I L)-4 and IL-2.
Differentiated effector Th cells migrate to sites of inflammation in the periphery, where they reencounter their cognate antigen and secrete effector cytokines, thus driving an antigen-specific immune response. IL-5, IL-9, and IL-13 are secreted by Th2 cells only once they have reached inflamed tissue sites. The cytokine milieu in the surrounding inflammatory microenvironment drives different Th2 effector activities. As a result, there are several phenotypically and functionally distinct Th2 memory cell subsets that carry out different effector roles depending on the cytokine influence. For instance, Th2 cells that produce high levels of IL-5 have been shown to contribute to the pathogenesis of allergic asthma, while cells that produce IL-5, IL-17, and IFN- y, in addition to IL-4 and IL-13, have been identified as noncanonical memory Th2 cells that drive chronic allergic inflammatory diseases.
Memory Th2 cells can be divided into at least four distinct subpopulations based on the levels of expressed chemokine (C-X-C motif) receptor 3 (CXCR3) and CD62L. All four subpopulations characteristically produce large amounts of IL-4 and IL-13 in response to antigenic re-stimulation. However, only the CXCR3|OW CD62L|OW subpopulation produces IL-5 and is considered pathogenic, denoted as memory-type Tpath cells. IL-33 stimulation induces production of IL-5 and IL-13 in memory Th2 cells and type 2 innate lymphoid cells (ILC2), thereby driving a type 2 immunological response. Similar to IL-33, the combination of IL-2 and IL-25 also drives IL-5 production in Th2 memory cells and ILC. ILC are immune cells derived from common lymphoid progenitors and are grouped based on their cytokine and transcription factor profiles, where ILC2 function in part to regulate Th2 phenotype. Whereas IL-33 can induce a strong Th2 immunity and eosinophilic inflammation by amplifying the Th2 cytokine response in lung and intestine tissue, IL-33 has little effect on the Th2 effector subset and instead mediates inflammation via memorytype Tpath cells. Exposure to an allergen, for example, may initiate a Th2 response in the airway by stimulating epithelial and endothelial cells to produce IL-33, thereby driving IL-5 production by Th2 memory cells and exacerbating the eosinophilic inflammation.
Th2 cells producing IL-4, IL-5, and IL-13 are implicated in a number of inflammatory diseases, including rhinitis, rhino-conjunctivitis, asthma, chronic rhinosinusitis, atopic dermatitis, food allergy and eosinophilic gastrointestinal disorders such as ulcerative colitis. Th2 cytokines drive pathogenesis of human asthma and murine allergic airway inflammation models by promoting eosinophilic infiltration, increased mucus production through goblet cell metaplasia, and tissue fibrosis. Th2 cell-derived IL-4, IL-5, and IL-13 contribute to B cell proliferation and isotype class switching from immunoglobulin lgG1 to IgE, a key antibody involved in parasitic helminth infection and allergic diseases associated with Th2 cells. Th2 cells have also been shown to induce the alternate activation (M2) macrophage phenotype, which mediates resolution of the inflammatory stage and initiates tissue repair in wound healing processes. Effector Th2 cells mediate immune responses to parasitic helminth infections, venoms, certain bacterial infections, and promote wound healing by inducing alternate activation (M2) macrophage phenotype.
Methods of studying, identifying and/or detecting Th cells include but are not limited cytokine profiling, cytokine ELISAs, and flow cytometry.
Depending on the use of the compositions provided herein the effects on T cell response in a subject may differ. In some examples, the effects of the compositions provided herein may be a change in immune response, such as an increase or decrease, in comparison to a composition comprising alum. In some examples, the change may be in comparison to a subject that has not been administered a composition of the invention.
In some examples, the compositions may be for use as a vaccine. As such, in some examples, provided herein are immunogenic compositions. In some examples, the compositions are vaccine compositions. The terms "immunogenic composition" and "immunological composition" and "immunogenic or immunological composition" refer to compositions that elicit an immune response against an antigen or immunogen after administration into a subject. The terms "vaccine" and "vaccine composition" refers to compositions that induce a protective immune response against the antigen of interest, or which efficaciously protects against the antigen; for instance, after administration to the subject, elicits a protective immune response against the targeted antigen or immunogen.
For example, the compositions provided herein may be for use as a vaccine and increase immunogenicity of an antigen or immunogen of interest. In some examples, the compositions may lead to an increased uptake of immunogen. In some examples, the compositions provided herein may increase T-cell proliferation in comparison to free immunogen. In some examples, the compositions may increase dendritic cell maturation.
For example, the compositions provided herein may be for use in treating an allergic disease and the compositions may provide an increased humoral response and a weak Th2 skewing effect in comparison to a composition comprising alum. In some examples, the compositions may provide a decreased overall T helper cell response.
In some examples, the compositions provided herein may increase levels of IL10. In some examples, the compositions provided herein may increase in IFN-y. In some examples, the compositions provided herein may decrease Th2 response. For example, the compositions provided herein may decrease levels of IgE. In some examples, the compositions provided herein may increase levels of IgG, such as levels of lgG2a and/or lgG4 in a human subject (or IgG 1 in a murine subject). In some examples, the compositions provided herein may cause earlier production of IgG antibodies earlier (i.e., sooner after administration). In some examples, the compositions provided herein may reduce IgE crosslinking. In some examples, the compositions provided herein may decrease IL-4 levels. In some examples, the compositions provided herein may decrease IL-13 levels.
Inhibiting production of IgE antibodies, therefore, is a promising target to protect against allergies. This should be possible by attaining a desired Th cell response. Th1 cells secrete interferongamma and other cytokines which trigger B cells to produce IgG antibodies. In contrast, a critical cytokine produced by Th2 cells is IL-4, which drives B cells to produce IgE. In many experimental systems, the development of Th1 and Th2 responses is mutually exclusive since Th1 cells suppress the induction of Th2 cells and vice versa. Thus, antigens that trigger a strong Th1 response simultaneously suppress the development of Th2 responses and hence the production of IgE antibodies. The presence of high concentrations of IgG antibodies may prevent binding of allergens to mast cell bound IgE, thereby inhibiting the release of histamine. Thus in some examples, the compositions provided herein may reduce the release of histamines in a subject in response to an allergen.
In some examples, the compositions provided herein may include additional agents. For example, the compositions may include vitamins. In some examples, the compositions may include vitamin A and/or vitamin D3. In some examples, compositions may include vitamin A. In some examples, compositions may include vitamin D. In some examples, compositions may include vitamin D3.
In some examples wherein the micelles include ELPs bound to intermediary binding molecules, the compositions may also include cognate intermediary binding molecules. These may help to reduce aggregation of micelles. For example, when micelles include ELPs bound to peptide K, the compositions may also include free peptide E. In some examples, when micelles include at least 20% ELPs bound to peptide K, the compositions may also include free peptide E. In some examples, when micelles include at least 25% ELPs bound to peptide K, the compositions may also include free peptide E. In some examples, when micelles include at least 30% ELPs bound to peptide K, the compositions may also include free peptide E.
In some examples, the compositions provided herein may be provided as part of a pharmaceutical formulation. Advantageously, such formulations may be administered to a human subject in need thereof (as described elsewhere herein).
A pharmaceutical formulation and the compositions described herein may comprise a composition or micelle as described herein along with a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier.
Compositions and formulations may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, supplementary immune potentiating agents such as adjuvants and cytokines and optionally other therapeutic agents or compounds.
As used herein, "pharmaceutically acceptable" refers to a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the selected micelle without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical formulation in which it is contained.
Excipients are natural or synthetic substances formulated alongside an active ingredient (e.g. a micelle or composition as provided herein), included for the purpose of bulking-up the formulation or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating drug absorption or solubility. Excipients can also be useful in the manufacturing process, to aid in the handling of the active substance concerned such as by facilitating powder flowability or non-stick properties, in addition to aiding in vitro stability such as prevention of denaturation over the expected shelf life. Pharmaceutically acceptable excipients are well known in the art. A suitable excipient is therefore easily identifiable by one of ordinary skill in the art. By way of example, suitable pharmaceutically acceptable excipients include water, saline, aqueous dextrose, glycerol, ethanol, and the like.
Adjuvants are pharmacological and/or immunological agents that modify the effect of other agents in a formulation. Pharmaceutically acceptable adjuvants are well known in the art. A suitable adjuvant is therefore easily identifiable by one of ordinary skill in the art. Merely by way of example, a pharmaceutical formulation may comprise an adjuvant selected from the group consisting of: AS03; AddaS03; AS04; MF59; AddaVax; Poly l:C; R848; Cpg; virus-like particles; virosomes; MPL; and flagellin protein.
A suitable adjuvant may be an agonist of Toll-like receptors (TLRs). For example, a suitable adjuvant may be an agonist of a TLR selected from the group consisting of: TLR3; TLR4; TLR7; TLR8; and TLR9.
Polyinosic:polycytidylic acid (also referred to as Poly I :C) is suitable example of an adjuvant of this sort that is an agonist of TLR3. MLP is an example of an adjuvant that is an agonist of TLR4. R848 (an imidazoquinoline) is an example of an adjuvant that is an agonist of TLR7 or TLR8. CpG oligodeoxynucleotides (also referred to as CpG, or CpG ODNs) constitute an example of an adjuvant that is an agonist of TLR9.
Diluents are diluting agents. Pharmaceutically acceptable diluents are well known in the art. A suitable diluent is therefore easily identifiable by one of ordinary skill in the art.
Carriers are non-toxic to recipients at the dosages and concentrations employed and are compatible with other ingredients of the formulation. The term “carrier” denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. Pharmaceutically acceptable carriers are well known in the art. A suitable carrier is therefore easily identifiable by one of ordinary skill in the art.
Medical uses
The compositions and formulations provided herein may be for use as a medicament. Cancer
The compositions and formulations described herein may be for use in preventing and/or treating cancer.
The compositions provided herein may help to induce effective tumour-reactive T-cell responses to a tumour. When for use in treating cancer the immunomodulator may include a tumour- associated epitope. The compositions and formulations described herein may effectively help generate a population of immune cells, in particular of CD8+ effector T cells (also known as cytotoxic T lymphocytes (CTLs)). The immune cells induced by administration of the compositions or formulations described herein may be reactive to the epitope, or epitopes, presented on the micelles. These immune cells are then primed for the killing of cancer cells that present the same or similar epitopes. Such compositions and formulations for use in treating cancer may be referred to as “cancer vaccines” or “cancer immunotherapy vaccines”.
The medical uses and methods of treating cancer may include administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical formulation or composition as described herein.
The medical uses and methods of treatment described herein may be used in the treatment of a wide range of cancers. Tumours may be of mesenchymal or epithelial origin. Cancers include cancers of the colon, rectum, cervix, breast, lung, stomach, uterus, skin, mouth, tung, lips, larynx, kidney, bladder, prostate, brain, and blood cells. The medical uses and methods of treatment described herein may be used in the treatment of solid tumours.
Suitably, a cancer to be treated by a medical use or method of treatment described herein may be a solid tumour selected from but not limited to the group consisting of: pancreatic ductal adenocarcinoma; pancreatic cancer; breast cancer; melanoma; non-small cell lung cancer; small cell lung cancer; nasopharyngeal cancer; hepatocellular cancer; colorectal cancer; oesophageal cancer; gastric cancer; anal cancer; small intestine cancer; mesothelioma; kidney cancer; renal cell carcinoma; bladder cancer; prostate cancer; ovarian cancer; vulval cancer; cervical cancer; penile cancer; uveal melanoma; retinoblastoma; sarcoma; osteosarcoma; glioblastoma; adrenocortical carcinoma; neuroblastoma; Wilms tumour; endometrial cancer; and thyroid cancer.
In reference to cancer, the terms " treatment" and " treating" should be taken as encompassing therapy undertaken in order to prevent, slow down, or reduce an undesired physiological change or disorder, such as the growth, development or spread of cancer. Beneficial or desired results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized state of disease (which is to say, disease that is not worsening), delay or slowing of disease progression, de-staging the tumour (e.g., changing from borderline resectable to amendable for surgical resection), amelioration or palliation of the disease state, and remission (either partial or total).
Treatment may bring about prolonged survival as compared to expected survival if not receiving treatment. Alternatively, or additionally, treatment may provide a patient with an improved standard of life as compared to that which would be expected if not receiving treatment.
The medical uses and methods of treatment described herein may be of particular benefit in preventing the growth, progression or metastasis of tumours in subjects receiving treatment. In particular, the medical uses and methods of treatment described herein may be of particular benefit in preventing growth, or reducing size, of such tumours. It will be appreciated that these effects are able to beneficially reduce a subject’s tumour burden.
The medical use and methods of treating cancer provided herein may include administration of at least additional therapeutic agent. For example, the additional therapeutic agent may be an agent currently used for prevention or treatment of cancer (i.e. , an anti-cancer agent).
Examples of anti-cancer agents include, but are not limited to, antibodies, antibody fragments, conjugates, drugs, cytotoxic agents, proapoptotic agents, toxins, nucleases (including DNAses and RNAses), hormones, immunomodulators, chelators, boron compounds, photoactive agents or dyes, radioisotopes or radionuclides, oligonucleotides, interference RNA, peptides, anti- angiogenic agents, chemotherapeutic agents, cytokines, chemokines, prodrugs, enzymes, binding proteins or peptides or combinations thereof.
For example, chemotherapeutic drugs include vinca alkaloids, anthracyclines, epidophyllotoxins, taxanes, antimetabolites, tyrosine kinase inhibitors, alkylating agents, antibiotics, Cox-2 inhibitors, antimitotics, antiangiogenic and proapoptotic agents, doxorubicin, methotrexate, taxol, other camptothecins, and others from these and other classes of anticancer agents, and the like. Other cancer chemotherapeutic drugs include nitrogen mustards, alkyl sulfonates, nitrosoureas, triazenes, folic acid analogs, pyrimidine analogs, purine analogs, platinum coordination complexes, hormones, and the like. Suitable chemotherapeutic agents are described in REMINGTON'S PHARMACEUTICAL SCIENCES, 19th Ed. (Mack Publishing Co. 1995), and in GOODMAN AND GILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, 7th Ed. (MacMillan Publishing Co. 1985), as well as revised editions of these publications. Other suitable chemotherapeutic agents, such as experimental drugs, are known to those of skill in the art.
Exemplary drugs include, but are not limited to, 5-fluorouracil, afatinib, aplidin, azaribine, anastrozole, anthracyclines, axitinib, AVL-101 , AVL-291 , bendamustine, bleomycin, bortezomib, bosutinib, bryostatin-1 , busulfan, calicheamycin, camptothecin, carboplatin, 10- hydroxycamptothecin, carmustine, Celebrex, chlorambucil, cisplatin (CDDP), Cox-2 inhibitors, irinotecan (CPT-1 1), SN-38, carboplatin, cladribine, camptothecans, crizotinib, cyclophosphamide, cytarabine, dacarbazine, dasatinib, dinaciclib, docetaxel, dactinomycin, daunorubicin, doxorubicin, 2-pyrrolinodoxorubicine (2P-DOX), cyano- morpholino doxorubicin, doxorubicin glucuronide, epirubicin glucuronide, erlotinib, estramustine, epidophyllotoxin, erlotinib, entinostat, estrogen receptor binding agents, etoposide (VP 16), etoposide glucuronide, etoposide phosphate, exemestane, fingolimod, floxuridine (FLIdR), 3',5'-0-dioleoyl-FudR (FLIdR- dO), fludarabine, flutamide, farnesyl- protein transferase inhibitors, flavopiridol, fostamatinib, ganetespib, GDC-0834, GS-1101 , gefitinib, gemcitabine, hydroxyurea, ibrutinib, idarubicin, idelalisib, ifosfamide, imatinib, L- asparaginase, lapatinib, lenolidamide, leucovorin, LFM-A13, lomustine, mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, navelbine, neratinib, nilotinib, nitrosurea, olaparib, plicomycin, procarbazine, paclitaxel, PCI-32765, pentostatin, PSI-341 , raloxifene, semustine, sorafenib, streptozocin, Sil 11248, sunitinib, tamoxifen, temazolomide (an aqueous form of DTIC), transplatinum, thalidomide, thioguanine, thiotepa, teniposide, topotecan, uracil mustard, vatalanib, vinorelbine, vinblastine, vincristine, vinca alkaloids and ZD 1839.
Allergy
The compositions and formulations described herein may be for use in preventing and/or treating an allergic disease.
An “allergic disease” refers to a condition caused by hypersensitivity of the immune system to typically harmless substances in the environment. Allergic diseases include, but are not limited to, asthma, hypersensitivity lung diseases, rhinitis, rhino-conjunctivitis, rhinosinusitis, atopic eczema, contact dermatitis, allergic conjunctivitis (intermittent and persistent), vernal conjunctivitis (hay fever), atopic keratoconjunctivitis, giant papillary conjunctivitis, urticaria (hives), angioedema, hypersensitivity pneumonitis, eosinophilic bronchitis, vasculitis, hypersensitivity vasculitis, antineutrophil cytoplasmic antibody (ANCA) associated vasculitis, Wegner's granulomatosis, Churg Strauss vasculitis, microscopic polyangiitis, temporal arteritis, celiac disease, mastocytosis, and anaphylaxis.
“Allergy symptom” or “allergic reaction” refers to the body's response to an allergen. An allergic reaction can be localized to one area (skin that came into contact with allergen) or generalized. Allergic reactions may include, but are not limited to, rash, itching, hives, swelling, difficulty breathing, wheezing, angioedema, difficulty swallowing, nasal congestion, runny nose, shortness of breath, nausea, stomach cramps, abdominal pain, vomiting and/or low blood pressure. Subjects in need of treatment may identified or diagnosed as having an allergic disease by any suitable method known in the art. For example, diagnosis made through any one or more of clinical history, detection of specific IgE molecules in the blood stream (i.e., via blood tests), skin prick testing, patch test methods, challenge test methods or elimination methods (such as elimination diets).
The medical uses and methods of preventing and/or treating an allergic disease may include administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical formulation or composition as described herein.
In particular, compositions and formulations described herein that include an immunomodulator that includes an allergenic epitope may be used in medical uses and method for treating allergy. It will be apparent that in the majority of cases that the allergenic epitope is an epitope from or derived from an allergen that causes allergy symptoms or allergic reaction in the subject.
The compositions and formulations described herein that include an immunomodulator that includes an allergenic epitope may provide an increased level of IgG and/or decreased levels of IgE in a subject. The use of micelles as described herein may increase the amount of allergenic epitope required to produce a detectable IgE response. In addition, use of micelles as described herein may lead to a weaker pro-inflammatory response as well as a reduced Th2-skewed response to the allergenic epitope.
In some examples, the compositions and formulations described herein that include an immunomodulator that includes an allergenic epitope may be for use in methods of allergy immunotherapy (AIT). In particular, subcutaneous allergy immunotherapy (SCIT). Compositions and formulations for use in AIT and/or SCIT may be referred to as allergy vaccines.
In general AIT comprises administering an allergen to the patient in order to treat an allergy to that allergen of the patient, i.e., reducing current or future immune response, such as an allergenspecific IgE response and/or histamine release by mastocytes and/or granulocytes induced by the allergen, and/or manifestation of clinical symptoms of allergy. Immunotherapy is conventionally carried out by administering repeatedly a mono-dose or incremental doses of an allergen to a patient in need thereof, thereby resulting in an adaptive immune response of the patient who becomes desensitised to the allergen.
During AIT or SCIT, increasing doses of the allergen or allergenic epitope are administered, followed by a maintenance dose for several years, with the goal of inducing immunological changes leading to symptom amelioration while on therapy, as well as sustained desensitization off AIT or SCIT (immune tolerance). Typically, at the start of AIT or SCIT, subjects receive increasing doses of the allergen or allergenic epitope at weekly intervals over several weeks to months, under tightly monitored medical supervision. The gradual dose escalation enables tolerability to therapy and mitigates risk of severe hypersensitivity reactions related to allergen administration.
For example, the dosing regimen for AIT or SCIT includes a cluster dosing regimen. The cluster dosing regimen may include an up-titration regimen followed by a maintenance regimen. The up- titration regimen may include administering increasing doses of the antigen or antigenic epitope over a period of at least 4 weeks or from 1 to 24 weeks (e.g., over a period of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, or 24 weeks, e.g., from 4-10 weeks, from 4-8 weeks, from 6-12 weeks, from 6-10 weeks, from 6-8 weeks, from 8-12 weeks, or from 8-10 weeks). In some examples the up-titration regimen be over a period of more than 24 week. The maintenance regimen may include administering one or more maintenance doses of the allergen or allergenic epitope at the highest dose administered during the up-titration regimen. In some examples, the maintenance regimen includes administering maintenance doses every 1 to 4 weeks for at least 8 weeks (e.g., for at least s weeks, 10 weeks, 12 weeks, 14 weeks, 16 weeks, or longer). In some examples, the up-titration regimen may include up-titrating from a dose of 1 bioequivalent allergy unit (BAU) to a dose of at least about 4,000 BAU (e.g., over a period of 4, 5, 6, 7, 8, 9, 10, 11 , or weeks) and the maintenance regimen may include administering one or more maintenance doses at least about 4,000 BAU.
The terms “prevent,” “preventing,” or the like, as used with reference to an allergic reaction or allergic condition, refer to preventing development of allergy, an allergic reaction or an allergic condition. The term, as used herein, also includes reducing or abrogating allergen sensitization to prevent an allergic reaction.
“Response” of a subject to treatment indicates that the subject manifests a reduction in the clinical symptoms. Clinical symptoms may be assessed over the course of treatment, i.e. symptoms before treatment may be compared to symptoms during and after treatment. Alternatively, a reduction in symptoms may be determined by comparison to a baseline level established before treatment. Concerning allergy, this approach is particularly useful where, for example, immunotherapy is carried out in subjects not currently experiencing symptoms, as may be the case for seasonal grass pollen allergy sufferers, who may be treated before the pollen season. Symptoms may be assessed by standard methods, such as patient self-assessment or recording of the amount of medication required. The degree of a subject’s response to treatment may be assessed by measuring the degree of reduction of severity in symptoms. The medical use and methods of treating allergic disease provided herein may include administration of at least additional therapeutic agent.
In some examples, the additional therapeutic agent is selected from the group consisting of: a steroid, an antihistamine, a decongestant, and an anti-lgE agent. For example, the additional therapeutic agent is a steroid (e.g., a corticosteroid, such as an inhaled corticosteroid (ICS)). For example, the additional therapeutic agent is an antihistamine (e.g., loratadine, fexofenadine, cetirizine, diphenhydramine, promethazine, carbinoxamine, desloratadine, hydroxyzine, levocetirizine, triprolidine, brompheniramine, or chlorpheniramine). For example, the additional therapeutic agent is a decongestant (e.g., pseudoephedrine or phenylephrine). For example, the additional therapeutic agent is an anti-lgE agent (e.g., omalizumab).
Infectious Disease
The compositions and formulations described herein may be for use in preventing and/or treating an infectious disease.
The medical uses and methods of preventing and/or treating an infectious disease may include administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical formulation or composition as described herein.
“Infectious disease” refers to a disease which results from an infection. An infection is a condition caused by the invasion of an organism by a foreign agent (i.e., infectious agent). Infectious agents include, but are not limited to, bacteria, fungi, viruses, viroids, nematodes (e.g., parasites such as roundworms and pinworms), anthropods (e.g., mites, fleas, lice, ticks), and macroparasites (e.g., tapeworms). Common infectious diseases include bacterial and viral infections.
The compositions and formulations described herein when for use in methods of treating infectious disease may include an epitope or antigen from or derived from the pathogen causing the infectious disease.
The compositions and formulations described herein may be particularly useful in prevention or treatment of infectious diseases caused by intracellular pathogens. For example, viruses (e.g., CMV HIV), bacteria (e.g., Listeria, Mycobacteria, Salmonella (e.g., S. typhi) enteropathogenic Escherichia coli (EPEC), enterohaemorrhagic Escherichia coli (EHEC), Yersinia, Shigella, Chlamydia, Chlamydophila, Staphylococcus, Legionella), protozoa (e.g., Taxoplasma), fungi, and intracellular parasites (e.g., Plasmodium (e.g., P. vivax, P. falciparum, P. ovale, and P. malariae). The compositions and formulations described herein may reduce humoral response against an immunogenic immunomodulator including epitopes derived from such intracellular pathogens and increase cellular mediated response.
In some examples, the methods of preventing and/or treating infectious disease is a method of vaccination.
"Vaccination" refers to the administration of a composition or formulation as described herein intended to generate an immune response, for example to a disease-causing pathogen. For Vaccination can be administered before, during, and/or after exposure to a disease-causing pathogen, and in some examples, before, during, and/or shortly after exposure to the agent. In some examples, vaccination includes multiple administrations, appropriately spaced in time, of a composition or formulation as described herein.
"Treatment” in relation to infectious diseases refers to any administration of a composition or formulation as described herein that partially or completely alleviates, ameliorates, relieves, inhibits, delays onset of, reduces severity of and/or reduces incidence of one or more symptoms or features of an infectious disease or the predisposition toward the disease. Such treatment may be of a subject who does not exhibit signs of the relevant disease, and/or of a subject who exhibits only early signs of the disease. Alternatively or additionally, such treatment may be of a subject who exhibits one or more established signs of the relevant disease. As such the term "treating" in reference to infectious diseases refers to the vaccination of a subject. Prevention" refers to a delay of onset of an infectious disease. Prevention may be considered complete when onset of an infectious disease, disorder has been delayed for a predefined period of time.
As used herein, the terms “treat”, “treating” and "treatment" generally are taken to include an intervention performed with the intention of preventing the development or altering the pathology of a condition, disorder or symptom (e.g., an allergic disease, infectious disease, etc). Accordingly, "treatment" refers to both therapeutic treatment and prophylactic or preventative measures (such as vaccination), wherein the object is to prevent or slow down (lessen) the targeted condition, disorder or symptom. “Treatment” therefore encompasses a reduction, slowing or inhibition disease symptoms, for example at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% when compared to before treatment.
As used herein the term “subject” generally refers to an individual, e.g., a human, having or at risk of having a specified condition, disorder or symptom. The subject may be a patient i.e., a subject in need of treatment in accordance with the invention. The subject may have received treatment for the condition, disorder or symptom. Alternatively, the subject has not been treated prior to treatment in accordance with the present invention. The compositions and formulations described herein generally can be administered to the subject by any conventional route, including injection or by gradual infusion over time. The administration may, for example, by intramuscular, intravascular, intracavity, intracerebral, intralesional, rectal, subcutaneous, intradermal, epidural, intrathecal, percutaneous administration.
The compositions, formulations, methods of treatment, and medical uses described herein may provide micelles as described herein to a recipient via any suitable route of administration.
The compositions and formulations can be administered via any desired route of administration. Compositions and formulations, or medical uses, may make use of a route of administration selected from the group consisting of: intravenous (iv) administration; subcutaneous (sc) administration; intramuscular (im) administration; intradermal (id) administration; sublingual (si) administration; and intranasal administration.
The skilled person will be able to determine suitable forms of the compositions of the invention for use with the desired route of administration.
The compositions and formulations described herein are for administration in an effective amount. An “effective amount” is an amount that alone, or together with further doses, produces the desired (therapeutic or non-therapeutic) response. The effective amount to be used will depend, for example, upon the therapeutic (or non-therapeutic) objectives, the route of administration, and the condition of the patient/subject. For example, the suitable dosage of a compositions or formulations of the invention for a given patient/subject will be determined by the attending physician (or person administering the composition), taking into consideration various factors known to modify the action of the compositions or formulations of the invention for example severity and type of disease, body weight, sex, diet, time and route of administration, other medications and other relevant clinical factors. The dosages and schedules may be varied according to the particular condition, disorder or symptom the overall condition of the patient/subject. Effective dosages may be determined by either in vitro or in vivo methods.
Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. For example, Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 2d Ed., John Wiley and Sons, NY (1994); and Hale and Marham, The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991) provide those of skill in the art with a general dictionary of many of the terms used in the invention. Although any methods and materials similar or equivalent to those described herein find use in the practice of the present invention, the preferred methods and materials are described herein. Accordingly, the terms defined immediately below are more fully described by reference to the Specification as a whole. Also, as used herein, the singular terms "a", "an," and "the" include the plural reference unless the context clearly indicates otherwise. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context they are used by those of skill in the art.
Aspects of the invention are demonstrated by the following non-limiting examples.
EXAMPLES
Example 1 - conjugation of OVA323 to ELP micelles
In this Example the elastin-like polypeptide (ELP) diblock copolymer micelles were studied as an adjuvant for vaccine delivery. The micelles can be readily functionalized with peptide or protein antigens by standard recombinant techniques. The resulting micelles are decorated covalently with antigens at the periphery, similar to spike proteins on viruses. However, the requirement for intracellular processing of covalently conjugated antigens can negatively influence the desired immune response.28 In contrast, coiled coil-mediated conjugation is reversible and can release the potential protein. Moreover, the coiled coil complex is stable, ensuring colocalization, but dissociates at low pH, enabling endosomal escape.29 The aim of this example was therefore to study whether the immune response is effected by the method of antigen conjugation to the nanoparticle adjuvant. Covalent antigen attachment is compared to attachment via a heterodimeric coiled coil complex. The dissociation constant of the E3/K3 coiled coil complex is low (Kd = 73 nM),30 making this heterodimeric coiled coil complex suitable to bind antigens to adjuvant nanoparticles in a non-covalent yet stable fashion.
Coiled coil-mediated association of antigens to adjuvants is of interest for three reasons. The first is to study the relation between the strength of antigen attachment to the adjuvant and the induced immune response by examining the difference between covalent and non-covalent attachment strategies. Furthermore, each strategy has implications on internal processing and the resulting ability to present the OVA323 epitopes on MHCII. Lastly, the presence of coiled coil domains may increase the immune response due to its tendency to interact with cellular membranes.
The peptide antigen “OVA323” (amino acids 323-339 from ovalbumin) was chosen as a model antigen to study the immunogenicity of ELP adjuvants. Three differently functionalized micelles, called “covalent”, “coiled coil” and “hybrid” were designed (Figure 1), referring to the conjugation strategy of OVA323 to ELP. Since the coiled coil and covalent micelles differ both in the strength of attachment and in the presence of peptide K on the micelle corona, the hybrid micelles were designed to compare the strength of attachment with the coiled coil micelles and the presence of ELP-K with the covalent micelles.
The amount of antigen cargo per micelle can be controlled by changing the ratio of plain ELP to functionalized ELP, enabling optimization of the desired immune response. In this example, the covalent, coiled coil and hybrid formulations are compared to non-functionalized micelles (“ELP”) and fully functionalized micelles (“ELP-OVA323”).
In this example, the effect of functionalization with coiled coil peptides “E” and “K” on the ELP micelles is studied. The sequences of the polypeptides and peptides are listed in Table 1. Static light scattering (SLS) studies were used to determine the critical micelle temperature (CMT) and the critical micelle concentration (CMC), while the hydrodynamic diameter of the micelles as a function of composition was measured with dynamic light scattering (DLS). Micelle morphology was investigated using atomic force microscopy (AFM) and transmission electron microscopy (TEM). Subsequently, micelle uptake in BMDCs was studied using flow cytometry and confocal microscopy. Flow cytometry analysis of these BMDCs also included quantification of DC maturation and CD4+ T-cell stimulation.
Figure imgf000066_0001
Sequences are given in one letter amino acid abbreviations. Ac = Acetyl, NH2 = amide, TMR = tetramethyl rhodamine, attached to cysteine side chain.
Table 1 : Names and sequences of polypeptides and peptides Materials and Methods
Chemicals and reagents
Ultrapure water was obtained using a Milli-Q® system.
Plasmids
The plasmids coding for ELP-E, ELP-K and ELP-OVA323 were constructed from the pET52b- ELP plasmid described below. pET52-ELP-E, pET52-ELP-K, and pET52-ELP-OVA323 were constructed by cloning the respective DNA fragments (BaseClear, Leiden, the Netherlands) into the Acc651 and Notl sites.
A pET25 expression vector coding for ELP was provided by the MacKay laboratory.13 The Xbal- Acul (blunted by T4 DNA polymerase treatment) DNA fragment comprising the ELP sequence was recloned into Xbal-Smal digested pET52b (+) and maintained in E.coli XL10-Gold. In the resulting pET52b-ELP plasmid Xbal and BseRI sites are available for inserting DNA fragments upstream, and Acc65l, BamHI, BsrGI, Sall, Eagl, Notl, Sacl, and Avril sites are available for inserting DNA fragments downstream of the ELP coding sequence.
Expression and purification of ELP, ELP-E, ELP-K and ELP-OVA323
ELP, ELP-E, ELP-K and ELP-OVA323 were expressed and purified as described for ELP below.
ELP (MG(VPGIG)48(VPGSG)4SY (SEQ ID NO: 7) was expressed by transforming the plasmids into E. coli BL21(DE3) cells using the heat shock and calcium methods.35 The cell culture was grown in LB medium containing ampicillin (250 pg/mL) at 37 °C. A starter culture of 10 mL was added to 1 L of medium and the cells were cultured until an OD600 of ~0.5. The cultures were cooled to 18 °C and induced overnight with 0.05 mM IPTG. The cells were harvested, washed with 0.9% NaCI solution and the cell pellet was frozen at -80 °C. Cells were lysed in PB containing 1 mM pefablock (Roche Diagnostics), 1 mg/mL lysozyme (Thermo Scientific)), 2 mM MgCh, 25 u/mL benzonase (250 u/pL; Sigma-Aldrich) in a total volume of 10 mL. The mixture was incubated at 4 °C for approximately 45 minutes and sonicated with a 13 mm probe on ice at 25% amplitude for 5 minutes in 5 second intervals. The solids were removed from the lysate by centrifugation at 4 °C (37000 rpm, 228783 ref) for 30 minutes.
ELP was purified by inverse transition cycling (ITC). NaCI was added to the lysate to reach a concentration of 4 M. After incubation for 30 minutes at room temperature the mixture was centrifuged (10000 rpm, 17100 ref) for 30 minutes. The pellet was suspended in cold PB and incubated for at least 30 minutes at 4 °C. After centrifugation (10000 rpm, 17100 ref) for 30 minutes at 4 °C, the soluble ELP was collected by decanting the supernatant. 4 additional cycles of ITC were applied, using 3 M NaCI for the incubation at room temperature. The final supernatant was dialyzed against PB. The ELP concentration was determined using UV-Vis spectroscopy. ELP was frozen in liquid N2 and then stored at -20 °C.
FITC labelling of ELP
To a solution of ELP (276 mM) in 0.1 M NaHCCh pH 9 was added 8 equivalents of fluorescein isothiocyanate or tetramethylrhodamine isothiocyanate from a 5 mg/mL stock in DMSO. The mixture was light protected and incubated overnight at 4 °C. The reaction was quenched with 0.5 M NH4CI (25 equivalents with respect to the dye) for 2 hours in the dark at 4 °C. Excess dye was removed on a PD-10 desalting column (8.3 mL bed volume, GE Healthcare) according to the manufacturer’s instructions. The concentration was determined using the initial polypeptide concentration corrected for the dilution during the reaction and purification. The labeling factor was determined using UV-Vis spectroscopy. Labeled ELPs were frozen in liquid N2 and then stored at -20 °C.
Peptide synthesis and purification
Peptide E was synthesized on a Biotage Syro I fully automated parallel peptide synthesizer using standard Fmoc chemistry. Rink amide resin with a loading of 0.55 mmol/g (Sigma-Aldrich) was used as a support. Coupling reactions were performed with 0.5 M HCTU (Novabiochem) in DMF (Biosolve), 2 M DIPEA (Carl Roth) in a 1 :1 mixture of NMP (Biosolve) and DMF and 0.5 M of the Fmoc-protected amino acid (Novabiochem) in DMF. Deprotection steps were carried out with 40% piperidine (Biosolve) in DMF. All solutions contained 1 g/L LiCI (Sigma-Aldrich). Up from the 15th coupling step, amino acids were coupled to the peptide using double coupling steps. The N- terminus was acetylated with 0.5 M acetic anhydride (Biosolve) and 0.125 M DIPEA in NMP for 2 hours. The peptide was cleaved from the resin using a mixture of 2.5% triisopropylsilane (Sigma- Aldrich), 2.5% water and 95% TFA (Biosolve) and subsequently precipitated in cold diethyl ether (Honeywell). The precipitate was collected by centrifugation, dissolved in water and lyophilized.
Peptide K, E-OVA323 and E-OVA323-GC were synthesized on a CEM Liberty Blue automated peptide synthesizer using similar methods as described above.
The purity and identity of each peptide was confirmed with LC-MS (Figures 2 to 4).
Fluorescent labelling of E-OVA323-GC
1.85 mg E-OVA323-GC (0.42 pmol) was dissolved in 2.07 mL PBS containing 10 mM EDTA and 1 mM TCEP. 1 mg TMR maleimide (21 pmol) in another 2.07 mL buffer was added and the mixture was incubated at room temperature in the dark for 2 hours. Excess dye was removed using a centrifugal spin column (2 kDa MWCO, Sartorius Vivaspin) and peptide E-OVA323-TMR was purified by reverse phase HPLC. Successful labeling of the peptide was confirmed with LC- MS (Figure 5).
Preparative RP-HPLC
Peptides were purified using preparative reverse phase HPLC on a Shimadzu system consisting of two LC-8A pumps and an SPD-10AVP LIV-VIS detector and equipped with a Kinetex Evo C18 column. The following gradients of acetonitrile (Biosolve) in water, both containing 0.1 % TFA, were used: 10% to 35% followed by a plateau at 35% for peptide E, 10% to 80% for peptide K and 20% to 80% for E-OVA323, E-OVA323-GC and E-OVA323-TMR. The purity of the fractions was confirmed by LC-MS. Pure fractions were combined and lyophilized to obtain the peptide as a powder.
LC-MS
LC-MS was measured on a Thermo Scientific TSQ access MAX device for mass detection combined with an Ultimate 3000 liquid chromatography system. Liquid chromatography was performed using a gradient of 0 to 90% acetonitrile in water containing 0.1 % TFA on a 50 x 4.6 mm Phenomenex Gemini 3 pm C18 column.
SDS-PAGE
Samples for SDS-PAGE were mixed with reducing Laemmli sample buffer and, without heating first, loaded on a 10% SDS poly acrylamide gel. Electrophoreses was performed at 200 V. The gels were stained with 0.5 M CuCh for 15 minutes and washed with water for 3 x 5 minutes. The stained gels were photographed on a black background. Fluorescently labeled polypeptides were visualized on gels using a Thypoon FLA 9500 Fluorescent Image Analyzer Scanner (GE Healthcare). The percentage of dye attached to the polypeptide with respect to the free dye was determined using Fiji Imaged software.
The gels used to analyze the purification process of ELP-E and ELP-K were stained with Coomassie Brilliant Blue R-250 (Bio-Rad).
UV-VIS spectroscopy
Polypeptide concentrations were determined based on the absorbance at 280 nm. The absorption was measured on an Agilent 8453 device at 10 °C. The theoretical extinction coefficient for ELP (E = 1490 L mol-1 cm-1) was calculated using the ProtParam tool of ExPasy.36 The labeling factor (number of dye molecules per polypeptide molecule) was determined by measuring the absorption at 494 nm for FITC-ELP ( EFITC of 70000 L mol'1 cm'1) or at 555 nm for TRITC-ELP (ETRITC = 65000 L mol'1 cm-1), for FITC and TRITC, respectively. Extinction coefficients for ELP-E and ELP-K of 6990 L mol'1 cm'1 and for ELP-OVA323 of 1490 L mol'1 cm'1 were used. PLS and SLS
All SLS and DLS measurements were performed on a Malvern Zetasizer Nano-S instrument using BRAND UV cuvettes micro. The sample (75 pL) was equilibrated to the desired temperature for 3.5 minutes. Three measurements were performed, each consisting of 12 runs of 10 seconds and averaged. For DLS measurements and for SLS measurements used to determine the CMT, the attenuator was automatically set by the Zetasizer software. The CMCs were determined by diluting a stock solution to various concentrations. Each of these diluted samples was measured with SLS, with the attenuator set to 11 . The count rates were normalized to the count rate of PB and plotted as a function of log [ELP], The shape of the autocorrelation functions was used to determine whether particles were detected. One trendline was fitted through the data points for which particles were detected and another through those of non-particulate ELPs. The CMC was determined as the concentration corresponding to the intercept of these trendlines. The DLS measurements for the heating/cooling cycles were performed on a 0 °C sample that had been placed in the 37 °C cell of the instrument. Measurements were started immediately without incubation time at 37 °C. The measurement duration was 1 second at a ~5 second interval with an attenuator of 8.
Zeta potential
Zeta potential measurements were performed on a Malvern Zetasizer Nano-ZS using a Malvern Zetasizer nano series Universal Dip Cell kit. 1 mL of a 2.5 pM polypeptide solution in PB was incubated at 37 °C before starting the measurements.
TEM
A 20 pM solution of ELP in milliQ, milliQ and 1 % uranyl acetate (each 10 pL) were placed on a piece of parafilm and incubated at 37 °C for 2 minutes. A preheated copper grid was subsequently placed on top of the ELP, milliQ and staining droplets, every time removing excess liquid by blotting with filter paper. The grid was incubated at 37 °C for 1-2 hours and imaged on a JEOL TEM 1010 electron microscope with an accelerating voltage of 100 kV.
AFM
Samples for AFM were prepared by drop-casting 20 pL of 37 °C 2 pM ELP or ELP dimer on a silicon oxide wafer (Siegert Wafer) with a 285 nm thermal oxide layer or on a mica disc (V1 grade; Muscovite). The samples were dried at 37 °C for 30 minutes. AFM images were recorded using a JPK NanoWizard Ultra Speed microscope and the obtained data was processed using the JPK SPM Data Processing software. All experiments were performed using a silicon probe (Olympus, OMCL-AC160TS) with a nominal resonance frequency of 300 kHz. The images were all scanned and recorded (with a resolution of 512x512 pixels) in intermittent contact mode in air at room temperature. Mass spectrometry
The mass of ELP-E, ELP-K and ELP-OVA323 was determined as described for ELP below. The mass of ELP, FITC-ELP and TRITC-ELP was determined by ESI-QTOF on a Nanoacquity LIPLC system (Waters) connected to a Synapt G2Si mass spectrometer (Waters) using an Acquity LIPLC M-Class column (300 pm x 50 mm), packed with BEH C4 material (particle diameter = 1.7 pm; pore size = 300 A). 5 pl sample was injected and the components were separated using a gradient of 10 to 90% acetonitrile (Biosolve) in milliQ, both containing 0.1% formic acid (Sigma Aldrich). Electro-spray ionization (ESI) was used via Nano-spray source with ESI emitters (New Objectives) fused silica tubing 360 pm OD x 25 pm ID tapered to 5 ± 0.5 pm (5 nL/cm void volume). The following settings in positive resolution mode were used: source temperature of 80 °C, capillary voltage 4.5 kV, nano flow gas of 0.25 bar, purge gas 250 L/h, trap gas flow 2.0 mL/min, cone gas 100 L/h, sampling cone 25 V, source offset 25, trap CE 32 V, scan time 3.0 seconds, mass range 400-2400 m/z. Lock mass acquiring was performed with a mixture of Leu Enk (556.2771) and Glu Fib (785.84265), lockspray voltage 3.5 kV, Glufib fragmentation was used as calibrant. Masslynx software was used for acquisition and Ent3 software for polymer envelope signal deconvolution. The MaxEnt 1 software was used for mass deconvolution of the charge state envelopes.
CD
CD spectra were measured on a JASCO J-815 CD spectrometer connected to a Peltier temperature controller using a quartz cuvette with a path length of 1 mm. The reported spectra are averages of 10 spectra that were measured in quick succession, using a range of 190 to 260 nm with 1 nm intervals and a bandwidth of 1 nm. The polypeptide concentration was 25 pM. The sample was incubated at 37 °C for 90 seconds prior to starting the measurement. The measured ellipticity values were corrected for the polypeptide concentration and the number of amino acid residues.
Coiled coil association efficiency
The efficiency of coiled coil association of E-OVA323 to the 10% ELP/ELP-K was studied using E-OVA323-TMR. Coiled coil micelles, plain ELP micelles mixed with E-OVA323-TMR and free E- OVA323-TMR samples were prepared containing 2 pM E-OVA323-TMR and 20 pM polypeptide. First, 175 pL of each sample was incubated at 37 °C for 5 minutes. Subsequently, unbound E- OVA323-TMR was removed using a centrifugal filter unit (Amicon Ultra-0.5 mL) at 10000 ref and 37 °C for 5 minutes. The resulting solutions were washed five times with 175 pL 37 °C 10 mM PB pH 7.8 using the same centrifugation protocol. The samples were diluted with 300 pL PB and the fluorescence was measured using fluorescence spectroscopy.
Fluorescence spectroscopy Fluorescence spectra were measured on an FS920 steady state spectrometer from Edinburgh Instruments using an excitation of 541 nm (slit size of 5 nm) and emission of 572 nm (slit size of 1 nm). A series of E-OVA323-TMR concentrations ranging from 62.5 nM to 2 pM was measured to confirm the linear correlation between fluorescent signal and TMR concentration.
BMDCs
BMDCs were cultured as described in literature.37 Bone marrow was taken from the hind legs of wild-type C57BL/6 or TIM4-/- mice. The bone marrow cells were suspended using a 70 pm cell strainer (Greiner Bio-One). The cells were incubated for 10 days at 37 °C and 5% CO2 in Iscove’s Modified Dulbecco’s Medium (IMDM; Lonza) containing 2 mM L-glutamine, 8% (v/v) FCS, 100 U/rnL penicillin/streptomycin (Lonza), 50 pM p-mercaptoethanol (Sigma) and 20 ng/mL GM- CSF (PeproTech). The medium was refreshed every 2-3 days. For the first 9 days, the cells were cultured in 95 mm Petri dishes (Greiner Bio-One) and for the last day in 96 well F-bottom plates (Greiner Bio-One).
DC uptake and maturation
Fluorescently labeled micelles in PB were diluted 10 times with medium and added to the DCs in 96 well plates (for flow cytometry analysis) and an S well plate (Ibidi GmbH; for confocal microscopy). Hoechst dye (final concentration of 0.01 mg/mL) was added to each of the wells of the 8 well plate. Each well contained 50000 cells in 200 pL. For the negative control BMDCs were treated with medium only. The cells were incubated at 37 °C with 5% CO2 for 4 hours. The cells attached to the bottom of the 8 well plate were washed with medium ten times and imaged by confocal microscopy. The cells in the 96 well plates were isolated by centrifugation and transferred with 100 pL 4 mM EDTA to U-bottom 96 well plates. Excess EDTA was removed by centrifugation and the cells were labeled with live/dead APC-Cy7, CD11cPeCy7, and CD86APC (Invitrogen) in FACS buffer (PBS containing 1 % FCS and 2 mM EDTA) for 20 minutes at 4 °C. Next, the cells were spun down, washed with FACS buffer, spun down again, resuspended in FACS buffer and analyzed by flow cytometry.
T-cell isolation
T-cells were obtained as described in literature.38 Spleens from donor mice and were mashed with a syringe plunger and suspended in PBS through a 70 pm cell strainer. The buffer was removed by centrifugation and the red blood cells were lysed for 1 minute with 0.15 M NH4CI, 1 mM KHCO3, 0.1 mM Na2EDTA (pH 7.3). CD4+ T-cells were isolated by negative selection using sheep-anti-rat IgG Dynabeads (Dynal, Invitrogen) and an excess amount of anti-B220 (RA3-6B2), anti-CD11 b (M1/70), anti-MHCH (M5/114) and anti-CD8 (YTS169) mAb in Magnetic-activated cell sorting (MACS; Miltenyi Biotec) buffer. The cells were spun down and resuspended in 1 pM carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes) in PBS. Next, the cells were labeled for 10 minutes at 37 °C and 5% CO2 before removing excess CFSE by centrifugation. A suspension of 250000 cells/mL in medium was prepared and stored at 4 °C.
T-cell activation
Various micelle solutions and peptide solutions of OVA323 (Invivogen) or E-OVA323 in PB were diluted 10 times with medium and added to the DCs in the 96 well plates. Each well contained 10000 cells. OVA323 concentrations were 270 nM, 90 nM, 30 nM, 10 nM, 3.3 nM and 1.1 nM, which corresponds to total polypeptide concentrations of 2.7 pM, 900 nM, 300 nM, 100 nM, 33 nM and 11 nM, respectively. The control samples (ELP, ELP/ELP-K and ELP/OVA323) were only included at a total polypeptide concentration of 2.7 pM. As a negative control, BMDCs were treated with medium only. Cells were incubated at 37 °C with 5% CO2 for 4 hours, spun down and mixed with 50000 T-cells suspended in medium. Next, the cells were incubated for 3 days at 37 °C with 5% CO2. After centrifugation the cells were resuspended in FACS buffer containing CD4efluor, Thy1.2PeCy7, CD25APC, CD67Pe and live/dead APC-Cy7 (Invitrogen). After incubation at 4 °C for 20 minutes, the buffer and excess antibodies were removed by centrifugation and the cells were taken up in FACS buffer. The cells were analyzed by flow cytometry.
Flow cytometry
Flow cytometry was performed on a CytoFLEX S Beckman Coulter device. Analysis of the data was done using FlowJo software.
Confocal microscopy
BMDCs were visualized on an SP8 LIGHTNING Confocal Microscope using a 63x lens. The colocalization percentage was determined using Imaged software by setting the lower and upper thresholds at 15 and 255, respectively and subsequently running the 3D MultiColoc plugin of 3D Image suite.
LPS content determination
The resulting endotoxin levels in the cell assays were at least 40 times lower than the acceptable concentration of endotoxins in commercial subunit vaccines.39 The endotoxin levels were determined using HEK-blue TLR4 reporter cells (InvivoGen) expressing either the human (ELP) or murine (ELP-K and ELP-OVA323) TLR4-MD2 receptor complex. Suspensions of 25 thousand cells in 100 pL were mixed with 100 pL polypeptide or LPS samples at various concentrations in a 96 well plate (Greiner Bio-One). The plate was incubated at 37 °C overnight, resulting in the activation of the TLR4 pathway, which was detected by secretion of embryonic alkaline phosphatase (SEAP). The SEAP levels were measured with a QUANTI-Blue assay. First, 20 pL of the cell supernatants were incubated with 180 pL QUANTI-Blue solution (rep-qb1 , InvivoGen) for 2 hours at 37 °C. Next, the optical density (OD) at 650 nm was measured with a Tecan i- control 1.7.1.12 plate reader. The endotoxin levels in the covalent, coiled coil and hybrid micelles were <5 Ell/rng.
Statistics
Flow cytometry data was analyzed in Graphpad Prism 8 for Windows. Groups were compared with an ordinary One-way ANOVA and Tukey’s multiple comparison test.
Results
Expression and purification of ELPs
ELP consists of a hydrophobic block at the N-terminus and a hydrophilic block at the C-terminus, facilitating assembly into well-defined micelles.31 ELP-E, ELP-K and ELP-OVA323 were also expressed in Escherichia Coli (E. Coli) and purified with five cycles of inverse transition cycling (ITC). The purity of samples at various stages was studied by SDS PAGE and Coomassie Blue (ELP-E and ELP-K) or copper chloride (ELP-OVA323) staining (Figure 7). Copper chloride staining was used, because ELP-OVA323 does not contain enough positively charged amino acids required for efficient Coomassie Blue staining.32 Neither the ELP-E nor the ELP-K gel shows a clear difference between the protein composition before and after induction, possibly because these polypeptides contain only a few positively charged amino acids required for efficient staining. As a result, both desired polypeptides in the lysate lanes appear as a light blue colored band, in contrast to the dark blue background caused by other proteins. The observed bands match with the expected mass for both polypeptides of 43.1 kDa. For ELP-OVA323, a new band was observed after induction, corresponding to the expected mass (41.5 kDa). For all of these ELP fusion proteins, five cycles of ITC were applied to remove impurities. ELP-E and ELP- OVA323 were purified more efficiently in the first two cycles than ELP-K. The non-ELP moieties may have some affinity for E. coli proteins, encapsulating these into the ELP aggregates and thereby complicating the purification. Conjugation of peptide K might be most problematic due to the amphiphilic character, causing ELP-K to bind various proteins. During the purification of ELP- OVA323 an impurity of approximately 70 kDa was especially difficult to remove. However, ELP- E and ELP-K and ELP-OVA323 were at least 95% pure after five cycles of ITC, as confirmed by HPLC (Figure 7). Polypeptide yields were 30 mg/L (ELP-E), 38 mg/L (ELP-K) and 55 mg/L (ELP- OVA323) respectively, in line with previous reports.31 33 The exact mass of ELP-E, ELP-K and ELP-OVA323 was verified by ESI-TOF mass spectrometry (Figure 6).
Self-assembly behavior of ELP-E and ELP-K
The inverse transition behavior of ELP-E and ELP-K was studied with SLS measurements to analyze the effect of extending ELPs with a coiled coil peptide on the self-assembly in solution. The ELPs were dissolved in phosphate buffer (PB) and light scattering was recorded as a function of temperature. The onset of ELP-E assembly occurred at a higher temperature as compared to ELP, while ELP-K assembly was already observed at a lower temperature (Figure 8).
The CMT is defined as the temperature at which the polypeptide starts to aggregate, resulting in an increase in scattering. Extension of ELP with peptide E resulted in a 4 °C increase in CMT (22 vs. 26 °C). In addition, the scattering intensity plateau is higher for ELP-E than ELP. This can be due to either an increased number of micelles formed by ELP-E or the formation of larger micelles. However, DLS revealed no difference in size for ELP and ELP-E micelles (Table 2), thus the increased scattering of ELP-E at the plateau can be contributed to an increase in particle number. These results indicate that the soluble peptide E domain increases the CMT, while it shifts the equilibrium towards micelle formation above the CMT. The multiple polar residues in peptide E could complicate the hydrophobic collapse of ELP, preventing self-assembly in the 22-26 °C range, while once assembled peptide E possibly stabilizes the micelles due to homodimerization at high local concentration,25 consequently shifting the equilibrium to form more micelles.
Figure imgf000075_0001
[polypeptide] = 10 pM for DLS and 2.5 pM for zeta potential; measured in 10 mN PB pH 7.8 at 37 °C. Size distributions are shown in Figure 30. Values represent average values ±SD (n=3). 1hydrodynamic radius; 2 polydispersity index. N.d. = not determined
Table 2: Physicochemical properties of micelles
ELP-K exhibited a different inverse transition behavior than ELP and ELP-E as ELP-K tends to assemble into large aggregates. The critical aggregation temperature (CAT) of ELP-K was ~16 °C (Figure 8). Peptide K is prone to homodimerization and is also known to interact with membranes, burying the hydrophobic face of the helix in the bilayer while the lysine side chains “snorkel” towards the polar/nonpolar interface.25 Therefore it is likely that peptide K also has an affinity for the hydrophobic domain of ELP micelles. This might result in intra-micellar and inter-micellar interactions between peptide K and the ELP core inducing severe aggregation.
Next, the critical micelle concentration (CMC) of ELP-E and critical aggregation concentration (CAC) of ELP-K were determined using SLS. The CMC of ELP-E was 0.11 pM (Figures 9 -10), which is comparable to plain ELP (CMC = 0.15 pM). In contrast, the CAC of ELP-K was considerably lower (56 nM), confirming the strong influence of peptide K on ELP assembly (Figures 9 - 10).
Coiled coil formation on micelle surface
Before using ELP micelles as an adjuvant, micelle formation was studied as a function of ELP to coiled coil peptide ratio. For this, ELP and ELP-E or ELP-K were mixed in the absence and presence of the complementary water-soluble peptide K or E (Figure 11 and 12).
All ELP/ELP-E mixtures assembled into well-defined micelles with average sizes in the range of 50-60 nm at 37 °C (Figure 11). Addition of equimolar peptide K before micellization did not change the hydrodynamic diameter and polydispersity index (Table 3).
Figure imgf000076_0001
[polypeptide] = 20 pM; T = 37 °C; measured in 10 mN PB pH 7.8. All reported averages are based on three DLS measurements. *Peptide K added to ELP/ELP-E or peptide E added to ELP/ELP.
Table 3: Composition and size of coiled coil displaying ELP micelles
Thus, modification of ELP micelles with coiled coil peptides does not affect its self-assembly behaviour. Circular dichroism (CD) spectroscopy could not be used to prove E/K coiled coil formation, since the ELP domain dominated the CD spectrum (Figure 13). Dual labelling of ELP- E and peptide K confirmed binding of the latter to the micelles (see Figure 22). In summary, successful E/K coiled coil formation enables antigen loading of micelles. Furthermore, antigen loading can be controlled by adjusting the ELP/ELP-coiled coil peptide ratio.
However, the formation and stability of mixed ELP micelles displaying coiled coils at their surface showed to be strongly dependent on the primary amino acid sequence of the coiled coil peptide domain. ELP/ELP-K (9:1) mixtures formed stable micelles at 37 °C with a hydrodynamic diameter comparable to plain ELP micelles (54 nm and 61 nm, respectively). However, increasing the percentage of ELP-K induced significant aggregation (Figure 12, top panel). ELP-K apparently interacts with neighbouring micelles, possibly due to homocoiling25 or the general affinity of peptide K for hydrophobic structures. Interestingly, equimolar addition of peptide E in the mixtures prevented aggregation up to 40% ELP-K (Figure 3.5, bottom panel). The presence of peptide E prevents massive aggregation of ELP/ELP-K micelles most likely because heterodimer coiled coil formation is favoured over peptide K homodimerization. These results show that it is possible to formulate stable ELP/ELP-K mixed micelles, as long as peptide E is added before micellization.
ELP/ELP-K (9:1) micelles were somewhat larger and more polydisperse as compared to ELP/ELP-E or plain ELP micelles (Table 2). Transmission electron microscopy (TEM) revealed the formation of spherical micelles with comparable size distributions for all formulations (Figure 15). The zeta potential was near neutral for all formulations due to the absence of charged amino acids in ELP, the major component in these assemblies. As expected, ELP-E micelles had a more negative zeta potential (-10.9 mV), because peptide E contains multiple glutamates.
Micelles displaying OVA323 on their surface
Extension of ELP with either peptide E or K resulted in a change in inverse transition behaviour (Figure 8). In contrast, covalent conjugation of OVA323 to the hydrophilic segment of ELP did not have a noticeable effect on the inverse transition behaviour (Figure 16). This is because the OVA323 epitope contains only 17 residues, is fully water-soluble and does not homo-oligomerize.
The CMC of ELP-OVA323 was comparable to ELP (0.10 pM vs. 0.15 pM; Figures 9 and 10). Likewise, size and surface charge of ELP-OVA323 micelles are identical to ELP micelles (Table 4). Thus, covalent conjugation of the OVA323 epitope to ELP did not alter its self-assembly behavior.
Figure imgf000077_0001
Figure imgf000078_0001
[polypeptide] = 10 pM for DLS and 2.5 pM for zeta potential; measured in 10 mM PB pH 7.8 at 37 °C. Size distributions are shown in Figure 31 and 32. Values represent average values ±SD (n=3). 1hydrodynamic radius; 2 polydispersity index.
Table 4 Physicochemical properties of micelles
Covalent, coiled coil and hybrid micelles (Figure 1) were prepared by mixing the individual polypeptide and peptide solutions in the correct ratio and heating the resulting formulations to 37 °C. The resulting assemblies were analyzed with DLS and zeta potential measurements revealing comparable size and surface charge of these micelles, albeit with a higher polydispersity compared to ELP or ELP-OVA323 micelles (Table 4). The surface charge of each micelle formulation was near neutral, which is expected as each formulation contains non-charged ELP as the main component. Next, the morphology of the micelles was investigated with AFM (Figure 17; and Figures 18 to 20) and transmission electron microscopy (TEM) (Figure 21).
AFM studies showed that the covalent, coiled coil and hybrid samples all assembled into spherical particles with an average height of 23 nm, 15 nm and 11 nm respectively (Figure 17). These observed sizes are much smaller than the hydrodynamic diameter observed with DLS (~57 nm). Typically, hydrodynamic diameters are generally larger than imaged diameters by AFM.34 For this technique, a sample of these dynamic micelles is dried on a silicon oxide surface, which might result in flattening of the assemblies. This effect might be more pronounced for the coiled coil and hybrid micelles, explaining the measured particle height differences. The observed large assemblies are most likely clustered micelles as a result of sample preparation. TEM imaging confirmed the size range (-10-50 nm) observed with AFM and DLS (Figure 21).
Coiled coil formation on the micelle corona was proven using coiled coil micelles mixed with tetramethyl rhodamine (TMR) labeled E-OVA323. These micelles were thoroughly washed and subsequently the remaining unbound E-OVA323 peptide was quantified (Figure 22). A 3-fold higher amount of E-OVA323 remained on coiled coil micelles compared to ELP micelles confirming that E-OVA323 is conjugated to the micelles via E/K coiled coil formation. This is in line with previous studies concerning coiled coil-mediated antigen conjugation to the surface of liposomes.35
ELP-K facilitates uptake of micelles in dendritic cells
The effect of displaying OVA323 at the surface of covalent, coiled coil and hybrid micelles on the uptake by APCs was investigated. BMDCs were exposed to the micelle formulations, each containing 8% FITC-labeled ELP (FITC-ELP). To examine the potential immune-stimulating role of peptide K in BMDC uptake,17 18 ELP/ELP-K (9:1) micelles were also included in this study. In addition, the coiled coil sample contained TMR-labeled E-OVA323 (15% labeled). The use of different fluorescent dyes for E-OVA323 and ELP enabled us to study the antigen uptake independently from the micelle uptake. Free E-OVA323-TMR peptide was included as a control for micelle-independent uptake. BMDCs were incubated for 4 hours with the various samples and analyzed with flow cytometry, showing that micelle uptake is concentration dependent (Figure 23).
At 90 and 30 nM OVA323, all micelle formulations containing ELP-K (/.e., ELP/ELP-K, coiled coil and hybrid) showed a significantly higher uptake compared to both micelle formulations without ELP-K (/.e., ELP and covalent) (Figure 24). At all concentrations, plain ELP micelles are taken up less efficiently. Peptide K likely binds to the plasma membrane of BMDCs25, forcing the micelles in close proximity and ultimately facilitating cell uptake.
For the coiled coil micelles, it was studied whether the antigen is colocalized with the ELP micelles upon internalization. For comparison, the uptake of free TMR-labeled E-OVA323 peptide was also studied (Figure 25). E-OVA323 was not internalized by the BMDCs, while conjugation to ELP-K containing micelles via coiled coil formation ensured efficient antigen uptake. Most of the OVA (i.e. , TMR signal) positive cells were also ELP (i.e., FITC signal) positive: 93%, 80% and 59% for 270 nM, 90 nM and 30 nM OVA323, respectively. These double positive cells confirm successful antigen and micelle uptake, whereas the TMR positive FITC negative cells seem to have merely taken up the antigen. This could be due to the internalization of some of the antigen by the BMDCs without ELP uptake.
Micelle uptake in BMDCs was confirmed with confocal microscopy (Figure 13). No cell uptake was observed for ELP micelles or covalent micelles, which is not fully in line with the (relatively low number of) ELP positive cells measured in these samples with flow cytometry. The micelles may not have been internalized in these ELP positive cells, but merely adhered to the outside of a cell membrane. Cells were thoroughly washed before confocal microscopy imaging and noninternalized micelles and were therefore not observed. In contrast, micelles containing ELP-K (i.e., ELP/ELP-K, coiled coil and hybrid) were internalized efficiently inside cells. Thus, plain ELP micelles do not effectively enter the BMDCs at this concentration. These results indicate that ELP- K is essential for BMDC uptake due to its ability to interact with cell membranes.25 Confocal imaging also confirmed that free E-OVA323 peptide is not readily taken up by BMDCs. However, E-OVA323 conjugated to ELP-K containing micelles via coiled coil formation was taken up efficiently, supporting the flow cytometry data. Furthermore, BMDCs pulsed with coiled coil micelles revealed colocalization of ELP and E-OVA323, confirming the stability of the coiled coil complex upon cell uptake (Figure 26).
Peptide-K induces DC maturation
After antigen uptake, DC maturation is the first step to effectively stimulate T helper cells. Therefore, expression of the costimulatory molecule CD86, a marker for DC maturation, was quantified. ELP-K containing micelles induced more CD86 expression as compared to micelles without this polypeptide (Figure 27). This shows that peptide K may stimulate the immune response by inducing both increased maturation and more efficient uptake of antigens. Peptide K is known to interact with membranes and potentially induces membrane disruption,25 which could in turn result in enhanced BMDC maturation.36
T-cell proliferation by ELP micelles displaying OVA323
After antigen uptake and subsequent maturation, DCs can induce antigen-specific T-cell proliferation. Therefore, CD4+ T-cells were isolated from OT-II transgenic mice, which exclusively contain T-cells with OVA323-specific T-cell receptors. These T-cells were labeled with carboxyfluorescein diacetate N-succinimidyl ester (CFSE) and cocultured with BMDCs previously exposed to the micelle formulations. Finally, subsequent T-cells proliferation was quantified with flow cytometry.
Besides the covalent, coiled coil and hybrid micelles, several control groups were included in this study. OVA323 peptide, E-OVA323 peptide, ELP-OVA323 micelles and a mixture of ELP micelles with free OVA323 peptide were used as control groups. ELP micelles, ELP/ELP-K micelles and cell medium were used as negative controls. As expected, T-cell proliferation was dependent on the concentration of OVA323, and ELP micelles without OVA323 did not induce T-cell proliferation (Figure 28). Interestingly, ELP-OVA323 did not induce OT-II proliferation at any of the tested concentrations (Figure 29).
Potentially, the covalent conjugation of ELP to the OVA323 epitope had a strong negative effect on T-cell growth, most likely due to the need for internal processing before the epitope can be presented on MHCII. In contrast, E-OVA323, which also requires internal processing, induced a comparable level of T-cell proliferation as OVA323. This study reveals that internal processing of the antigen is sensitive to the conjugation method (i.e., covalent vs coiled coil). Interestingly, treatment of BMDCs with ELP-OVA323 micelles also resulted in less divided T-cells compared to the covalent micelle group. Since the covalent group comprises more micelles per antigen, this result indicates that ELP micelles function as an adjuvant. The lower antigen loading per micelle may also be favorable. The covalent micelles had a similar effect on T-cell proliferation as free OVA323. The positive influence of increased uptake by the micelles may have compensated for the negative influence of antigen processing issues related to covalently attaching ELP to OVA323. The coiled coil and hybrid groups did have significantly stronger effects on T-cell proliferation compared to their free peptide counterparts E-OVA323 and OVA323, respectively. These groups also outperformed the covalent group, suggesting the presence of peptide K provides an additional advantage over plain ELP micelles. Taken together, these results indicate the presence of ELP-K stimulates division of T-cells. This is likely caused by the increased uptake of ELP-K-containing formulations as well as the higher level of DC maturation.
Discussion
The fusion proteins ELP-E and ELP-K were successfully expressed and purified. Both peptide E and peptide K had a distinct effect on the inverse transition behavior of ELP. ELP-E formed micelles similar to ELP, while ELP-K assembled into large aggregates. The CMT of ELP-E was higher compared to ELP, while the CAT of ELP-K was lower. Above the CMT, ELP-E had a higher particle concentration than ELP. ELP-K was incorporated into micelles by co-assembly with ELP. Aggregation of these ELP/ELP-K mixed micelles could be prevented in the presence of an equimolar amount of peptide E.
The model antigen OVA323 was expressed as a fusion protein (ELP-OVA323) and the selfassembly behavior was comparable to unmodified ELP. Covalent micelles were compared to coiled coil micelles and hybrid micelles. All micelle formulations had a comparable diameter and zeta potential. The presence of ELP-K stimulated the uptake in DCs, as confirmed by flow cytometry and confocal microscopy studies. Furthermore, coiled coil-associated OVA323 was colocalized with the micelles, which increases its immunogenicity.12-14 Moreover, the colocalization of antigen and micelle confirms that coiled coil complexes remain intact at the ELP micelles surface in the presence of DCs. ELP-K also induced increased DC maturation, in agreement with a previous study using peptide-K modified polymersomes.18 Furthermore, BMDCs treated with micelles containing ELP-K (coiled coil and hybrid micelles) induced the highest level of CD4+ T-cell proliferation. A similar effect of peptide K on CD4+ T-cell proliferation was observed for liposomes with coiled coil-associated OVA323.35 Interestingly, conjugation of OVA323 to ELP micelles negatively affected T-cell expansion. In summary, peptide-K containing micelles enhance BMDC uptake and maturation, as well as subsequent CD4+ T-cell proliferation. These remarkable properties are possibly caused by interactions of peptide K with the cell membrane,25 resulting in enhanced stimulation of the BMDCs.
Based on the adjuvant effect of ELP-K, the stability of the coiled coil complex and the well-defined character of the micelles, these ELP micelles with coiled coil attached cargo are a promising vaccine delivery system to activate the cellular arm of the immune response. References
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Example 2 - ELP micelle platform for birch pollen allergy immunotherapy
There is growing concern about the toxicity of colloidal aluminum salts (alum) used as adjuvants in subcutaneous allergen immunotherapy (SCIT). Additionally, the Th2-skewed response of alum may not be suitable for SCIT applications. Therefore, alternative adjuvants and delivery systems are being explored to replace alum in SCIT. In this example, temperature-sensitive elastin-like polypeptide (ELP) micelles are studied as an adjuvant for the birch pollen allergen Bet v 1. The fusion protein ELP-Bet v 1 was expressed and purified by a combination of immunoaffinity chromatography and inverse transition cycling. Bet v 1 displaying micelles, denoted “ELP/ELP- Bet v T’, were obtained by mixing ELP-Bet v 1 fusion proteins with ELPs in a 1 :9 ratio at T > 24 °C. These assemblies were characterized using dynamic light scattering (DLS), atomic force microscopy (AFM) and transmission electron microscopy (TEM). At T = 37 °C rapid assembly of the polypeptide unimers was observed into spherical micelles with a ~50 nm diameter. The IgE binding affinity for Bet v 1 on the micelle corona was comparable to soluble Bet v 1 , revealing correct protein folding and accessibility of the antigen. Important for future SCIT applications, the mixed ELP/ELP-Bet v 1 micelles showed to be hypoallergenic (10-fold), as determined by measuring mediator release from rat basophilic leukemia (RBL) cells, expressing the human IgE receptor and sensitized with human birch pollen specific IgE. An increased protective antibody response with an earlier onset for ELP/ELP-Bet v 1 compared to alum- adsorbed Bet v 1 was observed in an in vivo study using naive mice. Finally, ELP/ELP-Bet v 1 micelles did not induce a Th2 skewing immune response as evidenced by the induction of background to low levels of IL-4, IL-5 and IL-13 cytokines. In summary, the hypoallergenic character and strong humoral immune response combined with the absence of a Th2 skewing T- cell response make ELP-based nanoparticles a promising candidate to replace alum in SCIT applications
Bet v 1 -displaying ELP micelles with the fusion protein ELP-Bet v 1 and 9 equivalents of plain ELP were prepared (Table 5) and by subsequently heating the resulting mixture These “ELP/ELP- Bet v 1” micelles were characterized using static light scattering (SLS), dynamic light scattering (DLS), atomic force microscopy (AFM) and transmission electron microscopy (TEM). Next, the folding and accessibility of Bet v 1 was examined using circular dichroism (CD) and an IgE binding assay. Then, the hypoallergenic character was studied by performing a rat basophil leukemia (RBL) assay. Finally, ELP/ELP-Bet v 1 was compared to alum-adsorbed Bet v l in a mouse immunogenicity study.
Figure imgf000085_0001
Table 5: Names and sequences of proteins and polypeptides used in this study
Materials and Methods
Chemicals and reagents
Ultrapure water was obtained using a Milli-Q® system.
Mice
Six to eight weeks old female BALB/c mice were purchased from ENVIGO (The Netherlands). The animals were housed under specific, pathogen free conditions at the animal facility of the Academic Medical Center. All experiments were approved by the Animal Ethics Committee of the AMC.
Plasmid
The plasmid coding for ELP-Bet v 1 was constructed from the pET52b-ELP plasmid described in Example 1 . pET52-ELP-Bet v 1 was constructed by cloning the Bet v 1 gene (BaseClear, Leiden, the Netherlands) into the Acc651 and Notl sites.
Expression of Bet v 1
Bet v 1 (Bet v 1.0101) was purchased from the Department of Molecular Biology of the University of Salzburg, where it had been expressed in E. coli and purified according to previously established purification protocols.
Expression of ELP, ELP-Bet v 1 and ELP-K
ELP, ELP-Bet v 1 and ELP-K were expressed as described for ELP in Example 1 . Purification of ELP and ELP-K
ELP and ELP-K were purified as described in Example 1.
Purification of ELP-Bet v 1
The lysate was first purified using immunoaffinity chromatography at 4 °C. A Bet v 1 specific monoclonal antibody, 5H8H943, was coupled to cyanogen bromide-activated Sepharose 4B (GE Healthcare) according to manufacturer’s instructions. The lysate was loaded on the column that had been equilibrated with 10 mM PB. The column was washed with 5 column volumes of PB and eluted with 100 mM glycine pH 2.5 (Merck). The fractions were immediately neutralized with 1 M Tris pH 8.8. The elution fractions containing ELP-Bet v 1 were combined, concentrated and rebuffered to 10 mM PB. The protein was further purified by one cycle of inverse transition cycling as described above for the purification of ELP. The final ELP-Bet v 1 solution was dialyzed against 10 mM PB pH 7.8.
SDS PAGE
Protein analysis by SDS-PAGE was carried out as described for ELP in Example 1 . The gels were stained with Coomassie Brilliant Blue-R250 (Bio-Rad).
UV-VIS spectroscopy
All protein and polypeptide concentrations were determined with UV-VIS based on the absorbance at 280 nm as described in Example 1. The extinction coefficient used for both Bet v 1 and ELP-Bet v 1 was 10430 L mol'1 cm-1.
Mass spectrometry
The mass of ELP-Bet v 1 was determined as described for ELP in Example 1 .
PLS and SLS
DLS and SLS measurements were done as described for ELP in Example 1 . The measurements of the dilution into Tyrode’s buffer were done by first heating a cuvette containing 495 pL of Tyrode’s buffer (containing 9.5 g/L Tyrode's salts, 0.1% (w/v) BSA and 0.5 g/L NaHCOs) at 37 °C for 5 minutes inside the DLS device. Then 5 pL of cold 100 pM ELP or ELP/ELP-Bet v 1 was injected into the buffer and the measurement was started immediately.
Zeta potential
The zeta potential of the samples was determined as described for ELP in Example 1.
TEM
Grid preparation and imaging was done as described for ELP in Example 1. AFM
AFM samples were prepared and measured as described for ELP in Example 1 . CD
CD spectra were measured as described in Example 1.
LPS content determination
LPS levels were determined as described for ELP in Example 1.
IqE binding of ELP-Bet v 1
IgE binding of ELP-Bet v 1 and ELP/ELP-Bet v 1 was determined by ImmunoCap IgE inhibition assay in triplicate. The samples were diluted in 10 mM PB, 280 mM sucrose, pH 7.4 to 540 nM Bet v 1 concentration. A pool of 36 birch pollen allergic patient sera was diluted to 12 kU/mL IgE and added 1 :1 (v/v) to the samples followed by incubation at room temperature for 1 hour. Uncomplexed IgE was measured on a Phadia 250 with rBet v 1 caps (t215), following the manufacturer’s instructions. Inhibition values were calculated using the uninhibited signal (PBS + serum) and maximally inhibited signal (PBS) as references. Bet v 1 and ELP were used as controls.
RBL assay
The assay was performed using a rat basophil (RBL-2H3) cell line, transfected with the human high-affinity IgE receptor (FCERI), as previously reported.2930 In short, 1 x 105 RBL-2H3 cells per well were seeded in flat-bottom 96-well, Nunclon Delta-treated microplates (Thermo Fisher Scientific, Waltham, MA, USA) and passively sensitized with human sera derived from birch pollen allergic patients (n=10) in a final dilution of either 1 :10 or 1 :20. Prior to the sensitization step sera were incubated with P3X63Ag8.653 cells (ATCC CRL-1580™, Manassas, VA, USA) in order to neutralize the complement system. To trigger the p-hexosaminidase release, the cells were stimulated for 1 hour at 37 °C, 7% CO2, with the respective antigen in concentrations ranging from 80 pg/mL to 0.024 fg/mL. For detection of p-hexosaminidase activity, the fluorogenic substrate, 4-methyl umbelliferyl-N-acetyl-beta-D-glucosaminide (Sigma-Aldrich) was used and measured at an excitation and emission wavelength of 360 nm and 465 nm, respectively. The data were corrected for spontaneous release (untreated cells) and normalized to the maximal enzyme release caused by cell lysis (10% Triton X-100, Sigma-Aldrich).
Immunogenicity of ELP-Bet v 1 nanoparticles
For the first in vivo immunogenicity experiment mice were immunized subcutaneously at days 0, 7 and 14 with ELP-Bet v 1 and ELP/ELP-Bet v 1 containing 36 pg Bet v l (n=5-6) or the equivalent amount of Bet v 1 adsorbed to alum. ELP in phosphate buffered sucrose (10 mM PB pH 7.8, 280 mM sucrose) was used as negative control group (n=5). In the second experiment the ELP-Bet v 1 group was replaced by a phosphate buffered sucrose alum group as negative control. Serum immunoglobulin levels were measured in serum samples taken via puncture of the vena saphena at days -1 , 6, 13 and 20. At day 28, 29 and 30 the animals received 100 pg/mL birch pollen extract in PBS intranasally under 3% (v/v) isoflurane anesthesia to further boost antibody production. After sacrificing the mice on day 31 , blood and lung draining lymph nodes were collected to analyze Bet v 1 specific lgG1 , lgG2a, and IgE levels, and the production of IL- 4, IL-5, IL-13, IL-10, IL-17A and IFN-y, respectively.
Analysis of serum Bet v 1 specific Immunoglobulin levels.
Bet v 1 specific IgE, lgG1 and lgG2a antibodies in serum, collected at the different time points, were analyzed as described previously.44 Briefly, NUNC Maxisorp plates were coated overnight with 5 pg Bet v 1 . The next day, the plates were blocked with FCS, followed by incubation with the serum samples. After washing, bound immunoglobulins were detected with horse radish peroxidase conjugated specific antibodies against mouse IgE, IgG 1 (Opteia, BD, San Diego, CA, USA) and lgG2a (eBioscience), according to the manufacturer’s instructions. Serum samples of all groups were diluted 10-fold for IgE detection. Serum samples for lgG1 and lgG2a detection were diluted 100-fold except for the ELP/ELP-Bet v 1 and alum-adsorbed Bet v 1 groups which were diluted between 100- and 10000-fold depending on the measured time point.
Ex vivo Re-stimulation of lung draining lymph node cells.
Lung draining lymph node cell suspensions were plated in a 96 well round bottom plate at a density of 2 ■ 105 cells per well and were re-stimulated for 4 days with Bet v 1 . Expression levels of cytokines IL-4, IL-5, IL-9, IL-13, IFN-y and I L-17A were determined in the supernatant by ELISA (eBioscience).
Statistics
For the RBL test, the data was normalized based on the minimum and maximum values of the Bet v 1 series for each patient. For calculation of the antigen concentration necessary for half maximal release, the average of the maximal and minimal values of each curve were used. A repeated-measures one-way ANOVA followed by Tukey’s post-hoc analysis test was performed to determine significant differences among the treatment Normal distribution was confirmed via QQ plot. lgG1 and lgG2a levels were first Iog10 transformed and then analyzed with two-way ANOVA followed by Tukey’s multiple comparison test. IgE and cytokine levels were Iog10 transformed and subseguently analyzed with a one-way ANOVA followed by Tukey’s multiple comparison test. Significant differences are denoted with asterisks based on their P-values: *P < 0.05, **P < 0.01 , ***P < 0.001 and ****p < 0.0001.
Results Expression and purification of ELP-Bet v 1
ELP-Bet v 1 was expressed in Escherichia Coli (E. Coli). After induction the cells produced a protein of approximately 60 kDa, which is illustrated by SDS PAGE (Figure 33) by the extra band in the lane of the sample after induction (+) with respect to the sample before induction (-). The gel further shows the purification process of this protein, which consists of immunoaffinity chromatography and inverse transition cycling (ITC). The lysate containing ELP-Bet v 1 was loaded on a Bet v 1 specific monoclonal antibody column. Most proteins in the lysate did not bind to the column (FT) and were washed out (W1-3). ELP-Bet v 1 was eluted at pH 2.5 (E1-4) and the elution fractions were immediately neutralized using Tris buffer pH 8.8. The combined elution fractions still contained some impurities. After dialysis to 10 mM phosphate buffer (PB) pH 7.8 and concentration of the resulting solution (E1-4), NaCI was added (3 M) to precipitate the protein (P1), which was collected by centrifugation at room temperature. The supernatant did not contain any substantial amount of protein (S1). The pellet was resuspended in cold 10 mM PB and centrifuged again at 4 °C. All protein impurities and some of the ELP-Bet v 1 remained in the pellet (P2). The P2 lane shows a strong ELP-Bet v 1 band. However, since the P1 and P2 samples were not loaded on the gel in amounts comparable to the other samples, the actual amount of target protein lost in this step is estimated by comparing the ELP-Bet v 1 band in E1-4 with the same band in the supernatant of the cold centrifuging step (S2). The bands have similar intensity and therefore, no substantial amount of ELP-Bet v 1 was lost in P2. S2 contains pure ELP-Bet v 1 protein. The yield was 7 mg per liter culture, which is a factor 4 to 8 lower than the yields of ELP-based polypeptides reported in Example 1. The exact mass of ELP-Bet v 1 was confirmed by mass spectrometry (Figure 33) and the purity by analytical RP-HPLC (Figure 34). The absence of endotoxins was confirmed by a HEK-blue TLR4 reporter assay (Figure 35).
Self-assembly behavior of ELP-Bet v 1
Conjugation of Bet v 1 to ELP increased the critical micelle temperature (CMT) from 22 °C to 28 °C (Figure 36). ELP/ELP-Bet v 1 had a CMT of approximately 24 °C. The inverse transition temperature of ELP/ELP-Bet v 1 is in between those of ELP and ELP-Bet v 1 , even though the major component in this mixture is ELP. This indicates that conjugated Bet v 1 suppresses the ELP self-assembly, even of the ELP unimers.
A similar influence of conjugated Bet v 1 to ELP assembly was observed for the critical micelle concentration (CMC) of ELP-Bet v 1 and the ELP/ELP-Bet v 1 mixture (Figure 36). The CMC of ELP-Bet v 1 is 1.1 pM, which is 7-fold increased relative to the CMC of ELP (0.15 pM). The CMC of ELP/ELP-Bet v 1 (0.22 pM) matches the expectation based on the 9:1 ratio of the individual polypeptides. In summary, the presence of Bet v 1 in the fusion protein increases both the minimal concentration required for micellization and the minimal temperature.
Physicochemical properties of ELP/ELP-Bet v 1 micelles At body temperature (37 °C), both ELP-Bet v 1 and the ELP/ELP-Bet v 1 mixture assembled into micelles with a size slightly larger size and polydispersity index as compared to ELP (Table 6). ELP/ELP-Bet v 1 micelles had a near-neutral zetapotential as expected the formulation consists of 90% neutral ELP. The ELP-Bet v 1 micelles however had a negative zetapotential of -12.0 mV, which is consistent with the net charge of ELP-Bet v 1 of -5.
Figure imgf000090_0001
[polypeptide] = 10 pM for DLS and 2.5 pM for zeta potential; measured in 10 mN PB pH 7.8 at 37 °C. Values represent average values ±SD (n=3). 1 hydrodynamic radius; 2polydispersity index.
Table 6 Physicochemical properties of micelles used in this study.
The ELP/ELP-Bet v 1 micelles were visualized with both TEM (Figure 38) and AFM (Figure 39). Both techniques show the micelles are spherical in shape. The observed diameter by TEM imaging could not be measured accurately because it changed depending on how the sample was focused. The height of the micelles as measured with AFM was similar to ELP micelles (15 vs 17 nm. The difference between AFM-determined height and DLS-determined hydrodynamic diameter is expected, as hydrodynamic diameters are usually larger than imaged diameters/heights by either electron microscopy or AFM. In addition DLS is strongly biased towards particles with the largest diameter.25
Towards ELP and ELP/Bet v 1 based vaccines in an in vivo environment
Particle analysis with DLS as described above was performed after heating a polypeptide solution to 37 °C. However, preheating samples for injection in vivo preclinical and clinical studies would be inconvenient. Effective treatments with room temperature formulations would require the micelles to form upon injection after adjusting to the body temperature. These conditions were simulated in vitro by injecting cold concentrated solutions into a 100-fold larger volume of 37 °C Tyrode’s buffer (TB) containing bovine serum albumin (BSA) and a variety of salts. The hydrodynamic diameter was measured immediately after mixing, revealing that both ELP and ELP/ELP-Bet v 1 rapidly assembled into micelles (Figure 40).
For in vivo studies an isotonic vaccine is preferred. However, in the previous characterization studies of the ELP-based micelles, hypotonic PB (10 mM, pH 7.6) was used. In order to make the samples suitable for in vivo use, the buffer was supplemented with 280 mM sucrose and the hydrodynamic diameters were measured. All micelle formulations appeared to be somewhat smaller in this isotonic buffer (Table 7) compared to the hypotonic 10 mM PB (Table 6). However, the Pdl values are similar for both buffers. Possibly the presence of sucrose further drives the hydrophobic collapse of the hydrophobic ELP block, making the micelle more compact.
Figure imgf000091_0001
[ELP] and [ELP-Bet v 1] = 10 pM; [ELP/ELP-Bet v 1] = 0.10 mM; T = 37 °C; measured with DLS in 10 mM PB pH 7.8 with 280 mM sucrose.
Table 7 Sizes of micelles in phosphate buffered sucrose.
IgE binding to ELP/ELP-Bet v 1 does not lead to efficient mediator release
To determine whether Bet v 1 in ELP/ELP-Bet v 1 nanoparticles is still recognized by IgE, the binding capacity of the nanoparticles for IgE were compared with the IgE binding capacity of soluble Bet v 1 using an IgE ImmunoCAP inhibition assay (Figure 41). Results show that IgE binds ELP/ELP-Bet v 1 egually well compared to ELP-Bet v 1 and free Bet v 1 . Only the highest concentration ELP-Bet v 1 and the highest three concentrations of ELP/ELP-Bet v 1 are above the respective CMCs (19 pg Bet v 1/mL and 0.39 pg Bet v 1/mL, respectively).
However, IgE binding capacities are also similar between groups in this higher concentration range, so these results confirm that Bet v 1 is located on the corona of the micelles and not buried within the core. Furthermore, The IgE-binding epitopes remain intact when Bet v 1 is conjugated to ELP, which means the Bet v 1 domain of ELP-Bet v 1 is properly folded. The correct folding of Bet v 1 on the micelle surface is further confirmed by the CD spectrum of ELP-Bet v 1 , which resembles the average of the spectra of ELP and Bet v 1 , as shown in Figure 42. Correct folding of allergens ensures the presentation of B-cell, T-cell and IgE-binding epitopes.26 While SCIT requires B-cell and/or T-cell epitopes, IgE-binding epitopes could lead to adverse effects.2627 Therefore, it is difficult to say whether properly folded Bet v 1 in AIT is desirable. However, incorrectly folded Bet v 1 would be more difficult to standardize and may lead to batch-to-batch differences of the induced immune response.
Even though attaching Bet v 1 to ELP micelles did not reduce its binding affinity to IgE, it does reduce the allergenicity, as was determined using an RBL assay (Figure 43). Each curve shows that an increase in Bet v 1 concentration initially results in more IgE crosslinking, in turn causing more mediator release. At the maximum value of mediator release, the basophil surface is saturated with crosslinked IgEs bound to Bet v 1. Further increasing the Bet v 1 concentration causes IgE molecules to bind individual Bet v 1 proteins, which usually results in lower release of mediator molecules. The left side of the mediator release peak exhibits relevant information about the allergenicity of the antigen and is generally applied to compare allergen variants.28-30 However, in our case the Bet v 1 concentrations on the left side of the maximum correspond to ELP concentrations below the CMC. This means that with this assay we can only study the effect of attaching ELP to Bet v 1 as soluble fusion proteins, not the effect of the micelle. This explains why the curves of ELP-Bet v 1 and ELP/ELP-Bet v 1 are similar in this region. Both these curves shifted to the right with respect to the curves of Bet v 1 and plain ELP micelles mixed with free Bet v 1 (Figure 43). This means higher antigen concentrations were required to induce the same levels of mediator release. The concentration required to induce half the maximum mediator release (left of the peak) is on average a 10-fold higher for ELP/ELP-Bet v 1 than for Bet v 1 (Figure 44).
Full IgE binding capacity of an antigen seems to be conflicting with a hypoallergenic character. However, a similar set of results has been reported by others. This study involves cat allergen Fel d 1 displayed on virus-like particles, which reportedly bound IgE but failed to activate human mast cells.31 In another study, NPs displaying trimers of mite allergen Der p 2 were also found to be hypoallergenic with the ability to induce strong humoral responses.28 Both studies name biophysical reasons such as steric hindrance by the NP as explanations for the reduced IgE crosslinking.28’31 While the same rationale applies to IgE binding capacity, sterics should logically be more problematic for antigens to bind two IgE molecules than one, which could explain full IgE binding capacity in combination with reduced IgE crosslinking.
ELP particles induce strong humoral but weak T-cell responses in naive mice
Two independent immunogenicity experiments were conducted in naive mice using the same experimental set-up (Figure 45). The first study included ELP-Bet v 1 as an extra control group (Figures 46 and 47), while the second study included alum alone as an additional control (Figure 48 and 49).
The first study shows that ELP-Bet v 1 micelles did not induce significant levels of IgG 1 or lgG2a. However, both studies indicate that ELP/ELP-Bet v 1 induced IgG 1 and lgG2a responses up from day 13 (Figure 48a-b; Figure 46a-b). In both studies, alum-adsorbed Bet v l induced only background levels of lgG1 , except for the endpoint in the second study where the lgG1 levels were similar to the ELP/ELP-Bet v 1 group. Correspondingly, the lgG2a levels in mice injected with alum-adsorbed Bet v 1 were all similar to the buffer group in the first study, while these were significantly higher up from day 13 in the second study. At day 13 in the second study, lgG2a levels induced by ELP/ELP-Bet v 1 were significantly higher than the levels induced by alum- adsorbed Bet v 1 (18.6-fold), while these levels were similar at the endpoint. Collectively, these data indicate that alum induces weak antigen specific humoral immune responses when administered via the subcutaneous route, while the humoral immune response induced by ELP micelles is stronger, with an earlier onset. Notably, the results of both studies were different, despite the same set-up. Similar differences between the results of two such immunogenicity studies with the same design were observed by the Ferreira group.3233 Additionally, the IgG responses to alum in that study were also detected only after all immunizations and challenges with birch pollen had been administered.
In the first study, none of the groups showed a significant IgE response, while in the second study, only alum-adsorbed Bet v 1 induced significant levels of IgE. (Figure 49c; Figure 46c). Murine IgGi is considered the equivalent of human lgG4.5 Clinical studies have shown that AIT-induced lgG4 antibodies can block IgE binding and subsequent IgE-mediated allergic responses, and is therefore considered a biomarker of AIT efficacy.334-36 In mouse models, both lgG1 and lgG2a have shown correlation with decreased allergic symptoms.3637 For example, in a birch pollen allergy therapeutic mouse model, it was shown that increasing levels of lgG2a correlated to reduction in airway hyperreactivity.38 The question remains, which antibody isotype contributes the most to the protective effect of SCIT. Our ELP/ELP-Bet v 1 nanoparticles induced both isotypes more strongly than alum-adsorbed Bet v 1 which is an important advantage over alum in SCIT.
Next, the type of immune response induced by alum adsorbed Bet v 1 and ELP/ELP-Bet v 1 was compared by measuring cytokine expression levels of re-stimulated cells isolated from the lung draining lymph nodes (Figure 49, Figure 47). As expected, alum-adsorbed Bet v 1 showed significant expression of Th2 cytokines IL-4, IL-5 and IL-13. Furthermore, alum-adsorbed Bet v 1 showed expression of regulatory T-cell (Treg) related IL-10 but not Th1 cytokine IFN-y and Th17- related cytokine IL-17A. In the pilot experiment, the induction of IL-4 by ELP/ELP-Bet v 1 was not significant compared to the ELP control group (Figure 47a). However, the IL-4 expression in the ELP/ELP-Bet v 1 group from the follow-up study was similar to the alum-adsorbed Bet v 1 group (Figure 49a)). In the second study, ELP/ELP-Bet v 1 induced only background levels of IL-5, IL- 13 and IL-10 (Figure 49b-d), while these levels were significant in the screening study (Figure 47b-d). The Treg-associated IL-10 levels in the ELP/ELP-Bet v 1 group were similar to the alum- adsorbed Bet v 1 group, while Th2 cytokines IL-5 and IL-13 were significantly lower (2.6-fold and 1.8-fold, respectively) for ELP/ELP-Bet v 1 compared to alum-adsorbed Bet v l ELP-Bet v 1 induced a similar cytokine profile as ELP/ELP-Bet v 1 (Figure 47).
Although alum in SCIT is able to induce a regulatory skewed immune response after repeated administrations, it is also known as a driver of Th2 immune responses.39 Our murine in vivo data confirmed that alum is a typical Th2 adjuvant. In contrast, the ELP/ELP-Bet v 1 micelles showed a weak, Th2-skewed immune response, indicated by variable and low IL-4, IL-5 and IL-13 expression. Moreover, other pro-inflammatory cytokines such as IFN-y and IL-17A were not induced, suggestive of an overall weak T-cell response. Interestingly, IFN-y is associated with lgG2a induction but in the alum-adsorbed Bet v 1 and ELP/ELP-Bet v 1 groups IFN-y was not detected, despite measuring significant lgG2a levels. A possible explanation is that IFN-y producing T-cells necessary for lgG2a isotype switching were short-lived and therefore not detectable at the study endpoint. In addition, the overall weak pro-inflammatory T-cell response could be related to the non-immunogenic character of ELP that was used in our design and because Bet v 1 requires pollen derived factors to stimulate DCs and induce T-cell polarization.40
IL-10 produced by regulatory T- and B-cells is able to suppress the ongoing, Th2 associated allergic inflammation during SCIT.41 Besides a generally weak pro-inflammatory immune response the ELP/ELP-Bet v 1 micelles also induced Treg associated IL-10. These IL-10 levels were 1.4-fold lower than those induced by alum-adsorbed Bet v 1. This could be linked to the much weaker pro-inflammatory response of ELP/ELP-Bet v 1 , because Th2 cells can also produce IL-10.42 During SCIT, allergen-specific regulatory T-cells producing IL-10 have been shown to be crucial to suppress Th2 associated allergic inflammation.6 More investigation is needed to determine the IL-10 source induced by our ELP/ELP-Bet v 1 micelles and whether these immunosuppressive responses are sufficiently strong to suppress Th2 associated allergic inflammation in an in vivo SCIT model. Additionally, further in vitro studies could be performed to translate the murine data to the human situation. Firstly, human monocyte derived DCs stimulated with ELP/ELP-Bet v 1 micelles could be cocultured with T-cells to study T-cell differentiation patterns. In addition, experiments with human B-cells could provide more pre-clinical data regarding the capacity of our ELP/ELP-Bet v 1 micelles to induce favorable human humoral responses associated with successful SCIT.
Conclusion
In this study ELP/ELP-Bet v 1 micelles were investigated as a possible candidate to replace alum in SCIT. Spherically shaped ELP/ELP-Bet v 1 micelles were produced, the ~50 nm size of which are suitable as an antigen delivery vehicle and as non-Th2 adjuvant. Furthermore, characterization of these particles showed that the CMT is 24 °C, which is below body temperature of both mice and humans ensuring the presence of micelles inside the body. This in vivo micellization is further supported by the CMC, which is a 465-fold lower than the concentration used for the immunogenicity study. Moreover, the data shows that rapid micelle formation occurs when a cold concentrated solution of the polypeptides is diluted into Tyrode’s buffer at 37 °C.
Bet v 1 was correctly folded and accessible on the corona of ELP/ELP-Bet v 1 micelles, as evidenced by the CAP inhibition assay. Despite being fully recognized by IgE antibodies, their IgE cross-linking capacity was reduced, as measured by the higher antigen concentrations required to get the same level of mediator release. These results are considered ideal for a SCIT candidate, since correctly folded of Bet v 1 seems more likely to induce blocking antibodies that can recognize natural Bet v 1 after exposure to birch pollen, while the hypoallergenic character of ELP/ELP-Bet v 1 can reduce adverse effects.
The ELP/ELP-Bet v 1 micelles induced earlier and stronger lgG1 and lgG2a antibody levels compared to alum adsorbed Bet v 1. Although alum-based SCIT is able to induce a regulatory skewed immune response after repeated administrations, it is also known as a driver of Th2 immune responses.14 The murine in vivo data confirmed alum as a typical Th2 adjuvant. In contrast, the ELP/ELP-Bet v 1 micelles lacked a Th2 skewing effect, indicated by the low IL-5 and IL-13 expression. Moreover, T regulatory cytokine IL-10, Th1 cytokine IFN-y and IL-17A were also low and, in case of IFN-y and IL-17A, did not differ from the control groups, suggestive of an overall weak T helper cell response.
In summary, the ELPs not only facilitate the manufacturing of this vaccine but also induce specific immune responses that may improve the efficacy of allergy specific immunotherapy. Compared to alum, the strong humoral response might reduce the treatment frequency and duration whereas the weak Th2 skewing effect and hypoallergenic character of ELP based nanoparticles contribute to the safety of SCIT. These findings support the ELP technology as a promising platform to develop novel, alum-free SCIT vaccines.
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The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.
All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive.
Each feature disclosed in this specification (including any accompanying claims, abstract and drawings), may be replaced by alternative features serving the same, equivalent, or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.
The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.
Sequences
Figure imgf000098_0001
Figure imgf000099_0001
Figure imgf000100_0001

Claims

Claims
1. A composition comprising a micelle, wherein the micelle comprises one or more elastin-like polypeptides (ELP) and one or more immunomodulators.
2. The composition of claim 1, wherein the immunomodulator is covalently bound to the ELP to form a ELP-immunomodulator fusion protein.
3. The composition of claim 1, wherein the immunomodulator is non-covalently bound to the ELP.
4. The composition of any one of the preceding claims, wherein the immunomodulator comprises at least one epitope selected from the group consisting of: an allergenic epitope, a viral epitope, a bacterial epitope, a parasitic epitope, a disease-associated epitope, and a tumour-associated epitope.
5. The composition of claim 4, wherein the immunomodulator comprises an allergenic epitope.
6. The composition of claim 5, wherein the immunomodulator comprises an allergenic epitope from, or derived from, a Type I allergen.
7. The composition of claim 6, wherein the immunomodulator comprises an allergenic epitope from, or derived from, Bet v 1.
8. The composition of any one of the preceding claims, wherein at least one of the ELPs is covalently bound to a first coiled coil forming peptide.
9. The composition of claim 8, wherein the immunomodulator is covalently bound to a cognate coiled coil forming peptide.
10. The composition of claim 8 or 9, wherein: a) the first coiled coil forming peptide comprises peptide K and/or peptide E; b) the first coiled coil forming peptide comprises peptide K and the cognate coiled coil forming peptide comprises peptide E; or c) the first coiled coil forming peptide comprises peptide E and the cognate coiled coil forming peptide comprises peptide K.
11. The composition of any one of the preceding claims, wherein: a) the micelle comprises an average hydrodynamic diameter of from about 30 to about 70 nm; b) the micelle comprises an average polydispersity index of from about 0.001 to about 0.25; and/or c) the micelle comprises an average zeta potential of from about -15 mV to about 15 mV.
12. A composition comprising a micelle, wherein the micelle comprises one or more elastin-like polypeptides (ELP) and one or more immunomodulators, wherein the immunomodulator is covalently bound to the ELP to form an ELP-immunomodulator fusion protein.
13. A composition comprising a micelle, wherein the micelle comprises: a) one or more elastin-like polypeptides (ELP) covalently bound to a first coiled coil forming peptide; and b) one or more immunomodulators covalently bound to a cognate coiled coil forming peptide, wherein the immunomodulator is non-covalently bound to the ELP via the coiled coil forming peptides.
14. A composition comprising a micelle, wherein the micelle comprises a peptide K covalently bound to elastin-like polypeptide (ELP) and an immunomodulator covalently bound to a further ELP.
15. A pharmaceutical formulation comprising a composition according to any one of claims 1 to 14, further comprising a pharmaceutically acceptable carrier, excipient, diluent and/or adjuvant.
16. Use of a micelle comprising one or more elastin-like polypeptides (ELP):
(i) for delivery of an immunomodulator;
(ii) for forming of an immunogenic vaccine; and/or
(iii) as an adjuvant.
17. The use according to claim 16, wherein an immunomodulator is covalently bound to the ELP to form a ELP-immunomodulator fusion protein.
18. The use according to claim 16, wherein an immunomodulator is non-covalently bound to the ELP.
19. The use according to claim 18, wherein a first coiled coil forming peptide is covalently bound to the ELP and the immunomodulator is covalently bound to a cognate coiled coil forming peptide, wherein the immunomodulator is non-covalently bound to the ELP via the coiled coil forming peptides.
20. The use according to claim 16, wherein the micelle comprises peptide K covalently bound to an ELP and an immunomodulator covalently bound to a further ELP.
21. A pharmaceutical formulation according to claim 15 for use as a medicament.
22. A pharmaceutical formulation according to claim 15 for use in preventing and/or treating an allergic disease.
23. A pharmaceutical formulation according to claim 15 for use in preventing and/or treating cancer.
24. A pharmaceutical formulation according to claim 16 for use in preventing and/or treating an infectious disease.
25. A method for inducing an immune response specific for an immunomodulator in a subject, the method comprising administering to a subject a therapeutically effective amount of a pharmaceutical formulation according to claim 15.
26. A method of preventing and/or treating an allergic disease, the method comprising administering to a subject a therapeutically effective amount of a pharmaceutical formulation according to claim 15.
27. A method of preventing and/or treating cancer, the method comprising administering to a subject a therapeutically effective amount of a pharmaceutical formulation according to claim 15.
28. A method of preventing and/or treating an infectious disease, the method comprising administering to a subject a therapeutically effective amount of a pharmaceutical formulation according to claim 15.
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