A method for preparing liposomes

Abstract

Method of preparing liposomes comprising the steps of: a) dissolving liposome formulation components, comprising one or more lipids, in a single isotropic monophase solution of polar organic solvent and water; b) freeze drying the solution obtained in step (a), thereby forming a lipid containing formulation cake; and c) hydrating the lipid containing formulation cake, to obtain an aqueous liposome formulation. A kit for preparing cargo-encapsulating liposomes.

Description

[0001] A method for preparing liposomes

[0002] The present invention provides methods and kits for preparing liposomes, such as cargoencapsulating liposomes.

[0003] Cancer immunotherapies have emerged as promising strategies for the treatment of an extensive range of malignancies. In cancer vaccination, the host’s immune system is activated to generate and activate cancer specific T cells, which only destroy the malignant cells, inducing long-lasting anti-tumor immunity. There are a wide array of cancer immunotherapies currently being employed and clinically investigated, such as immune checkpoint blockades, cytokine therapy, adoptive T cell therapy, and cancer vaccines (1), (2).

[0004] In particular, therapeutic cancer vaccines are a topic of ongoing interest . Upon injection with a cancer vaccine, the antigen is taken up by dendritic cells (DCs) (professional antigen presenting cells (APCs)), leading to DC migration to the lymph node, where they present the peptide antigen on MHC class I and II molecules, leading to the activation and proliferation of tumor peptide-specific T cells traveling to a tumor site. If a T cell encounters their associated antigens, they then proceed to eliminate the tumor cells (3) .

[0005] Synthetic peptide-based cancer vaccines are a promising avenue of cancer vaccination. These tumor-specific peptide sequences are easy to produce, customizable, can be produced under GMP-conditions, and can be employed in combination therapy. Further, they do not require biological processing, giving rise to more control of dosage. However, certain peptide-based cancer vaccines can be poorly taken up by dendritic cells, leading to insufficient T cell activation and immune tolerance (4).

[0006] The immunogenicity of peptide-based cancer vaccines can be improved using peptide-loaded cationic liposomes (1). Liposomes are nanoparticles composed of lipid bilayers enclosing an aqueous core. These nanoparticles can encapsulate both hydrophilic and hydrophobic compounds. Liposomes can be made with various types of lipids and can possess a wide array of physiochemical properties (such as size and charge), all of which can affect their ability to increase T cell responses (5), (6).

[0007] The inventors have previously shown that cationic liposomal formulations composed of the lipids 1 ,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 1,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC) gave strong T cell responses both in vitro and in vivo (1) (7), and that encapsulation of peptides into these liposomes is critical for a functional T cell response and anti-tumor response (1). Similar formulations are now also used in Phase III clinical studies (8). These liposomes have a size below 300 nm, which is optimal for uptake by dendritic cells (7), and a positive surface charge, which promotes an antitumor immune response compared to anionic or neutral formulations.

[0008] There are various methods by which liposomes can be made (9), (10). These methods include the thin film dehydration-rehydration method, reverse phase evaporation, sonication, freezethawing, microfluidic mixing, and / or detergent removal. While these methods can successfully make liposomes, they come with a series of limitations. In particular, many of these methods are complex, requiring multiple steps and specialized equipment that makes liposome production labor-intensive. They can also lead to size heterogeneity, leading to a heterogenous end product, and low encapsulation efficiency, meaning that more product needs to be utilized. Moreover the equipment cost and maintenance is often prohibitive. Furthermore, the use of organic solvents means that the solvent needs to be adequately removed in order to ensure product safety, which is often labor-intensive, unlike solvents such as DMSO and ACN which have a lower freezing point and therefore are easier to lyophilize. There are also scale-up challenges caused due to differences in equipment, parameters and reproducibility, as well as limited stability. These limitations have made the incorporation of liposomes into the clinic problematic.

[0009] A standard method of making peptide-containing liposomes is the thin film dehydration- rehydration method (Figure 1). This method uses a rotary evaporator to create a lipid thin film, which is then hydrated with a peptide-containing solution and sequentially frozen, followed by rehydration of the lipid cake and high pressure homogenization via extrusion (9). However, this method is difficult to convert into a GMP-compliant production method due to the use of chloroform and / or methanol as a solvent, and the rotary evaporator and extruder are difficult to be utilized aseptically.

[0010] Brief summary of the disclosure

[0011] The inventors have developed a novel method of reproducibly making GMP-compliant peptide- loaded cationic liposomes (Figure 1). In this method, peptides, lipids and sugars are weighed out and dissolved in acetonitrile in water 1:1 (v / v), syringe filtered over a 0.22 pm filter, freeze- dried, filtered through one 0.22 pm syringe filter by a syringe pump, aliquoted, and freeze-dried for storage. This offers several advantages compared to the currently used methods. Namely, the inventors observe enhanced peptide encapsulation and recovery for a wide range of peptides. Further, the method satisfies safety and compliance requirements due to the lack of toxic solvents and its rapid reconstitution. Liposomes are made via a labor-efficient, single-step solution, where lyophilized products can be stably stored long-term.

[0012] Overall, the inventors have developed a GMP-compliant method to make reproducible liposomes. The present method simplifies the process of liposome formulation by eliminating the need for lipid film formation and extrusion, streamlining the process into a single-step solution preparation. Lipids, peptides, and cryoprotectants are dissolved in a single solution of polar organic solvent, such as DMSO and / or ACN, facilitating efficient incorporation of peptides into liposomes.

[0013] In the present method, the process of liposome formulation is not peptide dependent. In other words, the process is not dependent on the physicochemical properties of the peptide that is to be loaded into the liposome. Conversely, currently used methods are dependent on the peptide properties, for example hydrophobic vs hydrophilic and charge.

[0014] The present method achieves significantly more efficient peptide incorporation and recovery of peptide and lipid after formulation. This improvement ensures a higher yield of peptide- encapsulating liposomes, maximizing the efficacy and cost-effectiveness of the liposome preparation process. By combining multiple steps into one, the method simplifies the formulation process, reducing time and resources. The single-step solution preparation reduces labour requirements, making the process more efficient.

[0015] The present method avoids the use of toxic solvents (e.g. chloroform, methanol) and aligns with Good Manufacturing Practice (GMP) standards, ensuring safety and regulatory compliance.

[0016] In the present method, lyophilisation of the liposomes results in a dry product with extended shelf life and improved stability, overcoming issues related to storage and transportation of liquid formulations.

[0017] In the present method, liposomes are formed following rehydration of the freeze-dried cake. In other words, the solution placed in the freeze-drier for the first lyophilization step does not yet contain liposomes. The freeze drying step forms a lipid containing formulation cake; and it is only the hydration of the lipid containing formulation cake that produces an aqueous liposome formulation. Conversely, in a thin film dehydration-rehydration method a liposomal dispersion i.e. a solution containing liposomes, is produced prior to snap freezing and freeze-drying. Overall, this invention offers a streamlined and efficient approach to peptide-containing liposome preparation, addressing key challenges and offering numerous benefits for pharmaceutical and biotech companies.

[0018] In one aspect, the invention provides a method of preparing liposomes comprising the steps of: a) dissolving liposome formulation components, comprising one or more lipids, in a single isotropic monophase solution of polar organic solvent and water; b) freeze drying the solution obtained in step (a), thereby forming a lipid containing formulation cake; and c) hydrating the lipid containing formulation cake, to obtain an aqueous liposome formulation.

[0019] Suitably, step (a) comprises or consists of dissolving liposome formulation components, comprising one or more lipids, in a single isotropic monophase solution of dimethyl sulfoxide (DMSO) or acetonitrile (ACN) and water.

[0020] Suitably, the method may comprise of the steps of: a) dissolving liposome formulation components, comprising one or more lipids, in a single isotropic monophase solution of dimethyl sulfoxide (DMSO) or acetonitrile (ACN) and water; b) freeze drying the solution obtained in step (a), thereby forming a lipid containing formulation cake; and c) hydrating the lipid containing formulation cake, to obtain an aqueous liposome formulation.

[0021] In another aspect, the invention provides a method of preparing liposomes consisting of the steps of: a) dissolving liposome formulation components, comprising one or more lipids, in a single isotropic monophase solution of dimethyl sulfoxide (DMSO) or acetonitrile (ACN) and water; b) freeze drying the solution obtained in step (a), thereby forming a lipid containing formulation cake; and c) hydrating the lipid containing formulation cake, to obtain an aqueous liposome formulation.

[0022] In a further aspect, the invention provides a kit for preparing cargo-encapsulating liposomes, comprising a preformed mixture of (i) lipids, (ii) sugars, (iii) ACN and / or DMSO and (iv) water and instructions for using the preformed mixture for the preparation of cargo-encapsulating liposomes according to any preceding method.

[0023] Suitably, in the context of any aspect provided herein, the polar organic solvent may be dimethyl sulfoxide (DMSO) and / or acetonitrile (ACN).

[0024] Suitably, in the context of any method aspect provided herein, the method may additionally comprise a step of sterile filtration of the solution obtained in step (a). Suitably, in the context of any method aspect provided herein, the method may additionally comprise a step of filtering the aqueous liposome formulation obtained in step (c). Suitably, the filtering may use a syringe filter.

[0025] Suitably, in the context of any method aspect provided herein, the method may additionally comprise a step of freeze drying the aqueous liposome formulation, to obtain a liposome containing cake.

[0026] Suitably, in the context of any aspect provided herein, the polar organic solvent may be less than or equal to 50% ACN.

[0027] Suitably, in the context of any aspect provided herein, the polar organic solvent may be 50% ACN.

[0028] Suitably, in the context of any aspect provided herein, the lipids may be dioleoylic lipids, fluidic lipids and / or cholesterol-containing lipids.

[0029] Suitably, in the context of any aspect provided herein, the lipids may be 1 ,2-dioleoyl-3- trimethylammonium-propane (DOTAP), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac- glycerol) (DOPG), cholesterol-containing lipids, phospholipids, soy phosphatidylcholine (PC) and / or egg phosphatidylcholine (PC).

[0030] Suitably, in the context of any aspect provided herein, the lipids may be 1 ,2-dioleoyl-3- trimethylammonium-propane (DOTAP) and 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).

[0031] Suitably, in the context of any aspect provided herein, the liposomes may be cationic liposomes, anionic liposomes or neutral liposomes.

[0032] Suitably, in the context of any aspect provided herein, the liposomes may be cargoencapsulating liposomes.

[0033] Suitably, in the context of any aspect provided herein, the cargo may be biomacromolecules.

[0034] Suitably, in the context of any aspect provided herein, the liposomes may be peptide- encapsulating liposomes. Suitably, in the context of any aspect provided herein, the liposome formulation components may further comprise peptides.

[0035] Suitably, in the context of any method aspect provided herein, the peptides may be dissolved in (0.04%) ammonium hydroxide before addition to the single isotropic monophase solution in step (a).

[0036] Suitably, in the context of any aspect provided herein, the liposome formulation components may further comprise DNA and / or RNA.

[0037] Suitably, in the context of any aspect provided herein, the liposome formulation components may further comprise proteins.

[0038] Suitably, in the context of any aspect provided herein, the liposome formulation components may further comprise adjuvants, optionally immune adjuvants.

[0039] Suitably, in the context of any aspect provided herein, the liposome formulation components may further comprise sugars and / or cryoprotectants. Suitably, the sugars may be sucrose and / or trehalose.

[0040] Suitably, in the context of any method aspect provided herein, the method may not comprise the addition of a category 2B carcinogen.

[0041] Suitably, in the context of any method aspect provided herein, the method may not comprise the addition of tert-butyl alcohol, chloroform and / or methanol.

[0042] Suitably, in the context of any aspect provided herein, the liposomes may be 80-400 nm in diameter, optionally 80-300 nm in diameter, optionally 100-300 nm in diameter, preferably 100- 200 nm in diameter.

[0043] Suitably, in the context of any aspect provided herein, the liposome formulation components may comprise 10 mg / mL lipids DOTAP:DOPC [1:1] molar ratio, 1 mg / mL peptide, 10% (w / v) - 20% (w / v) sucrose. These concentrations and ratios may be in reference to the lipid form of the liposome.

[0044] Suitably, in the context of any aspect provided herein, the liposome formulation components may comprise 10 mg / mL lipids DOTAP:DOPC [1:1] molar ratio, 1 mg / mL peptide, 10% (w / v) sucrose. These concentrations and ratios may be in reference to the lipid form of the liposome. In another embodiment in the context of any aspect provided herein, the liposome formulation components may comprise 10 mg / mL lipids DOTAP:DOPC [1 :1] molar ratio, 1 mg / mL peptide, 20% (w / v) sucrose. These concentrations and ratios may be in reference to the lipid form of the liposome formulation.

[0045] Suitably, in the context of any aspect provided herein, the liposomes may be unilamellar particles.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] Various aspects of the invention are described in further detail below.

[0050] Brief description of the Figures

[0051] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

[0052] Figure 1. Comparison of the standard thin-film dehydration-rehydration method and new GMP-compliant method to make synthetic peptide loaded cationic liposomes. Procedure of (A) standard thin-film dehydration-rehydration method for incorporating synthetic peptides in lipid nanoparticles and (B) new GMP-compliant method.

[0053] Figure 2. Replacing the thin film layer generation done via rotary evaporation of lipids in the standard method by dissolution of formulation components in 50% acetonitrile in water. Standard method (thin-film dehydration-rehydration method) utilizes rotary evaporation of lipids dissolved in chloroform / methanol to make a thin film layer. New method of dissolution of formulation components utilizes 50% acetonitrile in water. (A) Physicochemical properties (size, polydispersity index (PDI), and zeta-potential (ZP)) as measured by DLS. (B) Peptide encapsulation efficiency (%) as determined by LIPLC. Formulation composition is 10 mg / mL lipids DOTAP:DOPC [1 :1] molar ratio and 1 mg / mL OVA24. Data represents mean ± SD (n=3). Statistics calculated via unpaired t-test.

[0054] Figure 3. Replacing extrusion in the standard method by filtration. Standard method (thin- film dehydration-rehydration method) utilizes extrusion to generate monodisperse liposomes. New method utilizes a 0.22 pm syringe filter. (A) Physicochemical properties (size, polydispersity index (PDI), and zeta-potential (ZP)) as measured by DLS. (B) Peptide encapsulation efficiency (%) as determined by UPLC. Formulation composition is 10 mg / mL lipids DOTAP:DOPC [1 :1] molar ratio and 1 mg / mL OVA24. Data represents mean ± SD (n=3). Significance calculated via one-way ANOVA with multiple comparisons.

[0055] Figure 4. Double filtration with syringe pump gives liposomes with similar physicochemical properties as using a 0.22 pm syringe filter in the new method or extrusion in the standard method. Standard method (thin-film dehydration-rehydration method) utilizes extrusion to generate monodisperse liposomes. New method looks at liposomes manually passing through 0.22 pm syringe filter or via syringe pump. Physicochemical properties (size, polydispersity index (PDI), and zeta-potential (ZP)) were measured by DLS. Formulation composition is 10 mg / mL lipids DOTAP:DOPC [1:1] molar ratio and 20% (w / v) sucrose. Data represents mean ± SD (n=2).

[0056] Figure 5. Physicochemical properties of OVA24-loaded liposomes prepared using new method with 20% w / v sucrose as a cryoprotectant and reconstitution enhancer. OVA24- loaded liposomes (1 mg / mL OVA24, 10 mg / mL lipids DOTAP:DOPC [1:1] molar ratio) were prepared by freeze-drying formulations dissolved in 50% ACN in water containing either no sucrose or 20% w / v sucrose. Physicochemical properties (particle size, polydispersity index, and zeta-potential) were determined after freeze-drying, filtration with a 0.22 pm syringe filter and second freeze-drying. Each bar represents mean ± SD (n=3). Significances calculated via unpaired t-test.

[0057] Figure 6. Physicochemical properties of OVA24-loaded liposomes prepared with various lipid compositions. OVA24-loaded liposomes (1 mg / mL OVA24) were prepared by freeze- drying formulations dissolved in 50% ACN in water containing 20% w / v sucrose. Molar ratios for liposomes used were 10 mg / mL DOTAP:DOPC [1:1], 10 mg / mL DOTAP:DOPE [1:1], 10 mg / mL DOTAP:DOPG [1:3], 10 mg / mL DOTAP:DOPC:DOPE [1:1:1] and 10 mg / mL DOTAP:DOPG [3:1], Physicochemical properties (particle size, polydispersity index, and zeta-potential) were determined after freeze-drying, filtration with a 0.22 pm syringe filter and second freeze-drying. Each bar represents mean ± SD (n=2).

[0058] Figure 7. Comparison of liposome properties and encapsulation of standard method to new method. Formulation composition of both methods is 10 mg / mL DOTAP:DOPC [1:1] molar ratio, 1 mg / mL peptide. New method uses 20% w / v sucrose as cryoprotectant. Each dot represents the average (A) physicochemical properties, (B) encapsulation efficiency and (C) peptide recovery of one liposomal peptide formulation (n>2). (D) Peptide properties used for encapsulation as a function of isoelectric point and grand average of hydropathy (GRAVY) index. Physicochemical properties measured by DLS. Encapsulation efficiency and peptide recovery measured by LIPLC. Significance calculated via unpaired t-tests.

[0059] Figure 8. In vivo CD8+ T cell priming after liposomal injection. (A) Naive C57BL / 6 received a prime boost vaccination at day 0 and 14 of either 4 nmol peptide (OVA24), peptide adjuvanted with 1 pg poly(l:C), or peptide encapsulated in liposomes made either via the standard method or via the new method. Formulation composition of liposomes is 10 mg / mL DOTAP:DOPC [1 :1] molar ratio, 1 mg / mL peptide, where new method uses 20% w / v sucrose as cryoprotectant. (B) Levels of SIINFEKL (Seq ID NO: 1) specific CD8+T cells in blood of C57BL / 6 mice were determined every couple of days until day 35 via MHC class I- SIINFEKL tetramer, seen by flow cytometry. (C) At day 35, splenocyte restimulation occurred with OVA24 peptide and splenocytes were analyzed for intracellular cytokines via flow cytometry. Data represented as mean ± SD (n=5).

[0060] Figure 9. Therapeutic liposome vaccination promotes tumor eradication. Left. Naive C57BL / 6 were injected subcutaneously with A) B16-OVA or B) at day 0. A) Mice were vaccinated intradermally at day 3 and 10 with 10 nmol peptide (OVA24) or peptide encapsulated in liposomes made either via the standard method or via the new method. Formulation composition of liposomes is 10 mg / mL DOTAPDOPC [1:1] molar ratio, 1 mg / mL peptide, where new method contains 20% (w / v) sucrose. Overall survival is displayed, right) Mice were vaccinated intradermally at day 8 and 15 with 5 nmol peptide (HPV16 E7) encapsulated in liposomes made via the new method or PBS. Formulation composition of liposomes is 10 mg / mL DOTAP:DOPC [1:1] molar ratio, 1 mg / mL peptide, where new method contains 20% (w / v) sucrose. At day 17 and 20, mice received an intravenous injection of 30 pg anti-CD8. Tumor growth is displayed.

[0061] Figure 10. Stability of OVA24-loaded liposomes at -20° C. Liposomes encapsulating OVA24 were stored for 26 weeks at -20° C. The physicochemical properties were measured via DLS. Each data point represents mean ± SD (n=3).

[0062] Figure 11. Peptide length, sequence, isoelectric point and GRAVY index for peptides used to make liposomes via GMP-compliant method. Peptides were synthesized at the peptide facility of the LUMC.

[0063] Figure 12. Freeze-dried lipid cakes of liposome formulations dissolved in 10%, 25% and 50% ACN in water. 10 mg / mL DOTAP:DOPC [1:1] molar ratio was dissolved in 10%, 25% or 50% ACN in MQ water and freeze-dried, resulting in small lipid cakes.

[0064] Figure 13. Physicochemical properties, liposome composition (molar ratio) and peptide encapsulation efficiency of liposomes prepared using the standard method or new in 10%, 25% and 50% ACN in water. Physicochemical properties (size, polydispersity index (PDI), and zeta-potential (ZP)) were measured by DLS. Liposome composition in molar ratio and peptide encapsulation efficiency (%) were determined by LIPLC. Formulation composition is 10 mg / mL lipids DOTAP:DOPC [1:1] molar ratio and 1 mg / mL OVA24. Data represents mean ± SD (n=3).

[0065] Figure 14. Physicochemical properties of liposomes prepared using the new method with filtration either before lyophilization, during rehydration or both. Physicochemical properties (size, polydispersity index (PDI), and zeta- potential (ZP)) were measured by DLS. Formulation composition is 10 mg / mL lipids DOTAP:DOPC [1:1] molar ratio, 1 mg / mL OVA24 and 20% (w / v) sucrose. Data represents mean ± SD (n=2).

[0066] Figure 15. Physicochemical properties of liposomes prepared with either sucrose or trehalose ranging from 0% w / v (no sugar) to 25% w / v. (A) Representation of lyophilized liposomes containing no sugar, 20% w / v sucrose or 20% w / v trehalose. (B) Physicochemical properties of liposomes freeze-dried without sugar or with sucrose ranging from 5% w / v to 25% w / v. Followed by filtration with 0.22 pm syringe filter and a second freeze-drying step. (C) Physicochemical properties of freeze-dried liposomes containing no sugar or 5% w / v to 25% w / v trehalose. Physicochemical properties are measured by DLS. Each bar represented the mean ± SD of three independent preparations. Significance between sugar concentrations was analyzed by two-way ANOVA.

[0067] Figure 16. Liposome composition (molar ratio) after the first freeze-drying step, after filtration with a 0.22 pm syringe filter, and after a second freeze-drying step of formulations containing 0% w / v to 25% w / v sucrose / trehalose measured by UPLC. Data represents mean ± SD (n=3)

[0068] Figure 17. Cryo-TEM of lipid nanoparticles prepared with the standard method (A) or the new method (B). Formulation composition is 10 mg / mL lipids DOTAP:DOPC [1:1] molar ratio and 1 mg / mL OVA24, formulation with the new method contains 20% (w / v) sucrose. Scale bar is 200 nm.

[0069] Figure 18. Cake appearance of liposomes after formulation without cryoprotectant and with 10%, 25%, or 50% ACN, or after formulation with 50% ACN and 10% w / v sucrose, 20% w / v sucrose or 20% w / v trehalose. Data represents visual inspection of at least 20 samples. Dark grey (blue in colour version of Figure) represents where the characteristics were visible.

[0070] Figure 19. Lipid recovery after the first freeze-drying step, after filtration with a 0.22 pm syringe filter, and after a second freeze-drying step. Formulations contained 0% w / v to 25% w / v sucrose / trehalose measured by UPLC. Data represents mean ± SD (n=3).

[0071] Figure 20. Speed of reconstitution of liposomes prepared using either the standard or the novel developed method. Formulation composition was 10 mg / mL lipids DOTAP:DOPC [1 :1] molar ratio and 1 mg / mL OVA24. Novel developed method included 20% w / v sucrose. Data represents mean ± SD (n=5).

[0072] Figure 21. In vivo CD8+ T cell priming after liposomal injection. (A) Naive C57BL / 6 received a prime boost vaccination at day 0 and 14 of either 4 nmol peptide (OVA24), peptide adjuvanted with 1 pg poly(l:C), or peptide encapsulated in liposomes made either via the standard method or via the novel developed method. Formulation composition of liposomes was 10 mg / mL DOTAP:DOPC [1 :1] molar ratio, 1 mg / mL peptide, where the novel developed method used 20% w / v sucrose as cryoprotectant. (B) Levels of SIINFEKL specific CD8+T cells in blood of C57BL / 6 mice were determined every couple of days until day 35 via MHC class I- SIINFEKL tetramer, seen by flow cytometry. (C) At day 35, splenocyte restimulation occurred with OVA24 peptide and splenocytes were analyzed for intracellular cytokines via flow cytometry. Data represented as mean ± SD (n=5).

[0073] Figure 22. Prophylactic liposome vaccination limits tumor growth. (A) Naive C57BL / 6 were vaccinated intradermally at day 0 and 14 with 10 nmol peptide (OVA24) or peptide encapsulated in liposomes made either via the standard method or via the novel developed method. Mice were challenged with B16-OVA tumor cells at day 28. Formulation composition of liposomes was 10mg / mL DOTAP:DOPC [1 :1] molar ratio, 1mg / mL peptide, where novel developed method used 20% w / v sucrose as cryoprotectant. (B) Overall survival and (C) tumor growth are displayed.

[0074] Figure 23. Cryo-TEM of lipid nanoparticles prepared with the standard method or the novel developed method. Formulation composition was 10 mg / mL lipids DOTAP:DOPC [1 :1] molar ratio and 1 mg / mL OVA24, with 20% w / v sucrose.

[0075] 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.

[0076] Various aspects of the invention are described in further detail below.

[0077] Detailed Description

[0078] In one aspect, the invention provides a method of preparing liposomes comprising the steps of: a) dissolving liposome formulation components, comprising one or more lipids, in a single isotropic monophase solution of polar organic solvent and water; b) freeze drying the solution obtained in step (a), thereby forming a lipid containing formulation cake; and c) hydrating the lipid containing formulation cake, to obtain an aqueous liposome formulation.

[0079] Liposomes are nanoparticles composed of lipid bilayers enclosing an aqueous core. These nanoparticles can encapsulate both hydrophilic and hydrophobic compounds. Liposomes can be made with various types of lipids and can possess a wide array of physiochemical properties (such as size and charge), all of which can affect their ability to increase T cell responses. Liposomes are also referred to as liposomal vesicles herein.

[0080] Liposomes can be unilamellar (i.e. have a single lipid bilayer), oligo-cellular or multilamellar. SUV, small unilamellar vesicle can range from about 20-100 nm. LUV, large unilamellar vesicle can range from about >100 nm. MLV, multilamellar vesicle can range from about >500 nm or they can be smaller, i.e. 500 nm or less.

[0081] The term "lipid bilayer" as used herein refers to a structure composed of two lipid layers, which are formed by the assembly of hydrophobic compounds.

[0082] The term "hydrophobic compound" as used herein refers to amphiphilic lipid compounds containing both polar and apolar regions. Hydrophobic compounds may be selected from phospholipids, sphingolipids, and sterols. A hydrophobic compound may be phosphatidylcholine, such as purified soybean phosphatidylcholine.

[0083] As used herein, and unless otherwise specified, the term "hydrophobic compound" means a compound with little or no water solubility. A hydrophobic compound may have an intrinsic water solubility (i.e., water solubility of the unionized form) of less than about 20 percent by weight, about 15 percent by weight, about 10 percent by weight, about 5 percent by weight, about 1 percent by weight, about 0.1 percent by weight or about 0.01 percent by weight. In other embodiments, a hydrophobic compound has an intrinsic water solubility of less than about 10 mg / mL, less than about 7 mg / mL, less than about 5 mg / mL, less than about 3 mg / mL, less than about 1 mg / mL or less than about 0.1 mg / mL.

[0084] The properties of the liposomal vesicles will depend, among other factors, on the nature of the constituent phospholipids. Thus, if it is intended to obtain liposomes with certain characteristics, the polar group load and / or the length and degree of saturation of its fatty acid chains must be taken into account. The bilayer(s) of liposomes most often comprise phospholipids, but may also comprise lipids including but not limited to fatty acids, fatty acid salts and / or fatty alcohols. The properties of the liposomes depend, among other factors, on the nature of the constituents.

[0085] Consequently, if liposomes with certain characteristics are to be obtained, the charge of its polar group and / or the length and the degree of saturation of its fatty acid chains must be taken into account.

[0086] If it is desired to modify the properties of liposomes, it is possible, for example, to incorporate cholesterol (CHOL) or other lipids into the membrane, modify the number of lipid bilayers or covalently bind natural molecules (for example proteins, polysaccharides, glycolipids, antibodies, enzymes) or synthetic molecules (for example polyethylene glycol) to its surface. Liposomal vesicles have application in different fields, for example they are useful as vehicles for the administration of drugs, cosmetic products, diagnostic agents and genetic material.

[0087] There are numerous combinations of phospholipids, optionally with other lipids or cholesterol, that can be present in an aqueous medium to obtain liposomes. Depending on the method of preparation and the lipids used, it is possible to obtain liposomal vesicles of different sizes, structures, and / or properties.

[0088] Another important parameter to consider with respect to the formation of liposomes is the rigidity of the lipid bilayer. The hydrated lipid that forms part of the bilayer may be in either a liquid-crystalline (fluid) or gel state. As the temperature increases, the gel state is converted into the liquid-crystalline state. This occurs at a temperature known as the transition temperature (Tc), which is specific to each lipid. The Tc is directly proportional to chain length and inversely proportional to the degree of unsaturation of the fatty acids and depends on the nature of the polar group.

[0089] There are numerous options to combine (phospho)lipids with an aqueous phase and obtain liposomes. Depending on the method of preparation and the lipids used it is possible to obtain liposomal vesicles of different size, structure and properties. A common step of the methods used to prepare liposomes includes evaporating the organic solvent in which the lipids are dissolved and subsequently dispersing the lipids in an aqueous solution (buffered or not). The preparation procedures differ in the way in which lipids are dispersed and can be classified into: a) hydration of a thin lipid layer; b) evaporation in reverse phase; and c) solvent injection. Hydration of a thin lipid layer is the original method of Bangham et al. From the organic solution of the bilayer constituent lipids, a lipid film is prepared by removal of the organic solvent, which can be carried out by evaporation (under reduced pressure in a rotary evaporator) or lyophilization. The dried lipid film deposited on the wall of the flask is hydrated by the addition of an aqueous solution and stirring at temperatures above Tc. When performing evaporation in reverse phase, a lipid film is first prepared by removal of the organic solvent. The system is purged with nitrogen and the lipids are redissolved in a second organic phase, usually composed of diethyl ether and / or isopropyl ether. The aqueous phase is added to the redissolved lipids. The system is kept under continuous nitrogen. The gel is formed by removal of the second organic solvent. Finally, with solvent injection, lipids dissolved in an organic solvent are slowly injected into a warm aqueous solution.

[0090] The liposomes prepared according to the process of this invention could also contain other lipid components such as sterols and / or derivatives (for example cholesterol); sphingolipids (for example sphingomyelin, gangliosides, cerebrosides); and / or stearylamine.

[0091] The process of the present invention allows active agents such as drugs, diagnostic material, cosmetics, food substances, genetic material, etc. to be encapsulated in liposomes efficiently and stably. Preferably, this procedure is used to incorporate pharmacologically active agents into liposomes. In principle, any drug can be encapsulated using the method of the present invention. The molar ratio of active substance : PLs will depend on the characteristics of the substance to encapsulate. The lower limit is determined by the least amount of substance that is practical for making liposomes given its intended use and can easily be determined by one skilled in the art. The upper limit is determined by the stability of the liposomes.

[0092] In certain embodiments, the liposomes are less than about 1 pm in diameter. In one embodiment, the liposomes are less than about 500 nm in diameter. Suitably, the liposomes may be about 80-400 nm in diameter, preferably about 100-200 nm in diameter. In one embodiment, the liposomes are less than about 300 nm in diameter. In one embodiment, the liposomes have an average size (Z-average) less than about 300 nm in diameter. In another embodiment, the liposomes are less than about 200 nm in diameter. In a further embodiment, the liposomes are less than about 100 nm in diameter. Liposomes with a size below 300 nm are optimal for uptake by dendritic cells. Methods for determining the size of liposomes are well known in the art. Generally, the size relates to the average size of the liposomes that are obtained by the method.

[0093] Polydispersity index (PDI) is used as a measure of liposome molecular weight distribution (for a liposome population). The larger the PDI, the more diverse the molecular weight. In some embodiments, the liposomes produced in the claimed method have a PDI below 0.5. In some embodiments, the liposomes produced in the claimed method have a PDI below 0.4. In some embodiments, the liposomes produced in the claimed method have a PDI below 0.3. In some embodiments, the liposomes produced in the claimed method have a PDI below 0.2. Zeta potential (ZP) (measured in mV) is a physical property which is exhibited by any particle in suspension, macromolecule or material surface. It can be used to optimize the formulations of suspensions, emulsions and protein solutions, predict interactions with surfaces, and optimise the formation of films and coatings. The term is widely used for quantification of the magnitude of the charge. In some embodiments, the liposomes produced in the claimed method have a ZP above about +10 mV, preferably above about +20 mV.

[0094] A liposome as provided herein also optionally contains (or has associated therewith) a ligand that facilitates the liposome's entry into a cell, e.g., a cell-specific ligand. A ligand may be a chemical moiety, such as a molecule, a functional group, or fragment thereof, which is specifically reactive with the cell of choice while being less reactive with other cells thus giving the liposome an advantage of transferring its cargo selectively into the cells of choice. By being “reactive” it is meant having binding affinity to a cell or tissue, or being capable of internalizing into a cell wherein binding affinity is detectable by any means known in the art, for example, by any standard in vitro assay, such as ELISA, flow cytometry, immunocytochemistry, surface plasmon resonance, etc. Usually a ligand binds to a particular molecular moiety — an epitope, such as a molecule, a functional group, or a molecular complex associated with a cell or tissue, forming a binding pair of two members. It is recognized that in a binding pair, any member may be a ligand, while the other being an epitope. Such binding pairs are known in the art. Exemplary binding pairs are antibody-antigen, hormone-receptor, enzyme-substrate, nutrient (e.g., vitamin)-transport protein, growth factor-growth factor receptor, carbohydrate-lectin, and two polynucleotides having complementary sequences. Fragments of the ligands are to be considered a ligand and may be used so long as the fragment retains the ability to bind to the appropriate cell surface epitope. Preferably, the ligands are proteins and peptides comprising antigen-binding sequences of an immunoglobulin. More preferably, the ligands are antigenbinding antibody fragments lacking Fc sequences. Such preferred ligands are Fab fragments of an immunoglobulin, F(ab)2 fragments of immunoglobulin, Fv antibody fragments, or singlechain Fv antibody fragments. These fragments can be enzymatically derived or produced recombinantly. In their functional aspect, the ligands are preferably internalizable ligands, i.e., the ligands that are internalized by the cell of choice for example, by the process of endocytosis. Likewise, ligands with substitutions or other alterations, but which retain the epitope binding ability, may be used. The ligands are advantageously selected to recognize pathological cells, for example, malignant cells or infectious agents. Ligands that bind to cell surface epitopes are preferred. One especially preferred group of ligands are those that form a binding pair with the tyrosine kinase growth factor receptors which are overexpressed on the cell surfaces in many tumors. Exemplary tyrosine kinase growth factors are the VEGF receptor, FGF receptor, PDGF receptor, IGF receptor, EGF receptor, TGF-alpha receptor, TGF-beta receptor, HB-EGF receptor, ErbB2 receptor, ErbB3 receptor, and ErbB4 receptor. EGF receptor vlll and ErbB2 (HER2) receptors are especially preferred in the context of cancer treatment using liposomes as these receptors are more specific to malignant cells, while scarce on normal ones. Alternatively, the ligands are selected to recognize the cells in need of genetic correction, or genetic alteration by introduction of a beneficial gene, such as: liver cells, epithelial cells, endocrine cells in genetically deficient organisms, in vitro embryonic cells, germ cells, stem cells, reproductive cells, hybrid cells, plant cells, or any cells used in an industrial process.

[0095] The ligand may be attached to the liposome by any suitable method available in the art. The attachment may be covalent or non-covalent, such as by adsorption or complex formation. The attachment preferably involves a lipophilic molecular moiety capable of conjugating to the ligand by forming a covalent or non-covalent bond, and referred to as an “anchor”. An anchor has affinity to lipophilic environments such as lipid micelles, bilayers, and other condensed phases, and thereby attaches the ligand to a lipid-nucleic acid microparticle. Methods of the ligand attachment via a lipophilic anchor are known in the art. Typically, an amount of a lipophilic anchor effective to provide ligand conjugation is included into the lipid component, e.g. lipid, prior to, or during, the liposome formation. Alternatively, the conjugate of an anchor and a ligand can be first formed, and then incorporated into liposomes by addition to the lipid prior to the liposome formation, or by addition of the conjugate to the aqueous suspension of liposomes after their formation. A particularly suitable mode of ligand attachment to liposomes is by using a ligand conjugated to a lipophilic anchor through an intermediate hydrophilic polymer linker. Thus, the ligand moves freely above the microparticle surface and can react even with hard-to- reach epitopes on the cell surface. Ligands conjugated to lipophilic anchors via a hydrophilic polymer intermediate linker advantageously become stably associated with preformed nucleic acid-lipid liposomes during co-incubation of the conjugated ligands and the liposomes in an aqueous medium. (U.S. Pat. No. 6,210,707, the contents of which are incorporated herein by reference). For additional discussion of targeting moieties and their incorporation into liposomes, see, e.g., U.S. Pat. No. 7,244,826; U.S. Pat. No. 7,507,407; and U.S. Pat. No. 6,794,128; the contents of each of these patents are incorporated herein by reference.

[0096] The liposomes can further comprise other components beneficial for its function of transporting cargo to cells. These can be viewed as transport-enhancing components, i.e. , an entity associated with the liposome that improves the delivery of a cargo to a living cell. These beneficial, transport-enhancing components, may include, without limitation, endosome-escape agents, nuclear localization factors, triggerable means for enhanced transfer into cytosol, pH- sensitive compounds, heat and radiation-triggerable release, and membrane fusion promoters such as membrane fusion-enhancing or membrane fusion-inducing compounds, intracellular nucleic acid release-enhancing or inducing components, transcription factors, and promotermodulating compounds.

[0097] Suitably, the liposomes may be unilamellar particles. The term “unilamellar particles”, "unilamellar liposome" or "UL" as used herein refers to a liposomal vesicle composed of a single lipid bilayer, which encloses a hydrophilic space. Said vesicle can be of any shape, including ellipsoids, discoids, pear-shaped vesicles, cup-shaped vesicles, budded vesicles, and spherical vesicles. Preferably, a unilamellar liposome is substantially spherical in shape.

[0098] ULs may be small unilamellar vesicles (SLIVs) having a diameter of up to about 100 nm, or large unilamellar vesicles (LU Vs) having a diameter of larger than about 100 nm up to about 1 pm. The diameter of a UL may be between about 50 to about 250 nm, between about 100 to about 200 nm, or between about 130 to about 150 nm.

[0099] The term "diameter" as used herein refers to the hydrodynamic diameter, which is to be understood as the size of a hypothetical hard sphere that diffuses in the same fashion as that of the particle being measured. Suitable methods for determining the hydrodynamic diameter of the herein disclosed UL, such as Dynamic Light Scattering (DLS), are known to a person skilled in the art.

[0100] Certain physicochemical properties of liposomes can be measured by Dynamic Light Scattering (DLS). Suitably, the average size (Z-average; nm) and polydispersity index (PDI) of the liposome particles are determined using dynamic light scattering (DLS). Suitably, the zetapotential (mV) of the liposome particles is determined by using laser Doppler electrophoresis (LDE). Suitably, the measurements are performed on a Zetasizer Nano (Malvern Instruments, Malvern, UK). Suitably, the samples are diluted 75-fld in PB (pH 7.4) prior to measurements.

[0101] “Liposome formulation components” (also referred to as the “formulation composition” herein) are the building blocks of liposomes. These components aid in the generation of liposomes, and / or are associated with liposomes once they are generated. In the present invention, the liposome formulation components comprise one or more lipids. The liposome formulation components may further comprise cargo, such as peptides, proteins or nucleic acids. The liposome formulation components may also comprise sugars and / or cryoprotectants. The liposome formulation components may further comprise adjuvants, optionally immune adjuvants. In the present invention, the liposome formulation components are dissolved in a single isotropic monophase solution of polar organic solvent and water. The term “dissolving” as used herein means the process where a solute in a gaseous, liquid, or solid phase dissolves in a solvent to form a solution.

[0102] Suitably, the method may additionally comprise a step of (sterile) filtration of the solution obtained in step (a). Suitably, the method may additionally comprise a step of filtering the aqueous liposome formulation. Suitably, the filtering may be sterile filtration. Suitably, the filtering may use a syringe filter.

[0103] The terms “filter”, “filtration”, or “filtering” refer to a physical separation process that separates solid matter and fluid from a mixture using a filter medium that has a complex structure through which only the fluid can pass. Filtering may be achieved using sterile filtration. “Sterile filtration” is a filtration technique used to remove or reduce microbial contaminants from liquids. It involves passing products through a specialized filter with extremely small pores, typically ranging from 0.1 to 0.45 pm in size. Filtration may occur through a syringe having a 0.22 pm filter (e.g., with a diameter 0 13 mm). Suitable syringe filters are known, e.g., PVDF filters (such as PVDF-filters with pore size of 0.22 pm (diameter 0 13 mm).

[0104] Filtration may be performed manually or using a syringe pump. The syringe pump may be used at speeds ranging from about 250 pL / min to about 2500 pL / min, for example using at least one tandem 0.22 pm syringe filter. In one embodiment, the aqueous liposome formulation is filtered through one or two 0.22 pm syringe filters by a syringe pump.

[0105] “Syringe pumps”, or “syringe drivers”, are motorised devices that accurately control the movement of fluid through a syringe by mechanically inserting or retracting the plunger.

[0106] Advantageously, the inventors have shown that filtration such as that described above (e.g. manual sterile filtration using a syringe with pore size of 0.22 pm (diameter 0 13 mm), or using two 0.22 (0 13 mm) syringe filters with a syringe pump) can be used as a substitute for high pressure homogenisation when preparing liposomes from liposome formulation components described herein.

[0107] Accordingly, the methods of preparing liposomes described herein may, in some embodiments, obtain an aqueous liposome formulation without performing high pressure homogenisation (also referred to as extrusion herein).

[0108] A filtration step may be incorporated into the methods described herein at any suitable stage. For example, filtration may be performed before step b) of the methods described herein (before freeze drying to form a lipid containing formulation case). In addition, or alternatively, filtration may be performed after the lipid formulation case has been hydrated.

[0109] The methods described herein comprise the step of b) freeze drying the solution obtained in step (a), thereby forming a lipid containing formulation cake. The lipid containing formulation cake is then hydrated, to obtain an aqueous liposome formulation.

[0110] Suitably, the method may additionally comprise a step of freezing drying the aqueous liposome formulation, to obtain a liposome containing cake.

[0111] The term “freeze drying” (also known as “lyophilising”) as used herein refers to a drying technique by which a product is solidified by freezing and the solvent that contains it (usually water) is evaporated by sublimation (a chemical phenomenon) upon heating. Freeze-drying is a process in which water is sublimated by the direct transition of water from solid (ice) to vapor, thus omitting the liquid state, and then desorbing water from the “dry” layer. It is widely used for the stabilization of high-quality food, biological materials, and pharmaceuticals, such as proteins, vaccines, bacteria, and mammal cells. In the process, the quality of the dried product (biological, nutritional, and organoleptic properties) is retained. This is due to the fact that freezing water in the material prior to lyophilization inhibits chemical, biochemical, and microbiological processes. The removal of water by sublimation results in the creation of highly porous structure of the freeze-dried products, and the rehydration of lyophilisates occurs immediately.

[0112] A freeze-drying program may involve:

[0113] 1) freezing at -40 °C for 8 hours;

[0114] 2) main drying for 20 hours at -40 °C with a chamber pressure of 0.100 mbar; and

[0115] 3) secondary drying for 20 hours with a gradual increase to 20 °C within four hours at 0.0010 mbar.

[0116] As used herein, a product is considered “dry” (or lyophilised) after freeze-drying when there is less than a 10% increase in pressure (mbar) in 30 seconds. Products that have been dried using freeze-drying are referred to as “cake” herein (e.g. a “liposome containing cake” or a “lipid containing formulation cake”). The terms “liposome containing cake” and “liposome containing lyophilised formulation” may be used interchangeably herein. The terms “lipid containing formulation cake” and “lipid containing lyophilised formulation” may be used interchangeably herein. The term “hydrating” as used herein refers to a hydration reaction which is a chemical reaction in which a substance combines with water. When water is used as the solvent, a dissolving process is called hydration.

[0117] In the present invention, the lipid containing formulation cake is hydrated and an aqueous liposome formulation is formed. The resulting solution contains liposome particles and therefore may be called a “dispersion”.

[0118] The term “a lipid containing formulation cake” as used herein means the layer of frozen material containing lipids, that results from the freeze-drying process.

[0119] A “liposome containing cake” is a layer of dry frozen material containing liposomes, that results from the freeze-drying process.

[0120] An aqueous solution is a solution in which the solvent is water. Formulations are complex mixtures of chemicals which have a specific use. The term “an aqueous liposome formulation” as used herein means a mixture or solution in water that contains liposomes.

[0121] As used herein, or unless otherwise specified, the terms "aqueous medium" or "aqueous media" include any water based medium, e.g., water, saline solution, a sugar solution, a transfusion solution, a buffer, and any other readily available water-based medium. Further, an aqueous medium may contain one or more water soluble organic solvents. In the case of a parenteral solution, an aqueous medium is preferably sterile and suitable for use as a carrier of an active agent. Examples of aqueous media include, but are not limited to, water for injection, saline solution, Ringer's solution, D5W, or other solutions of water-miscible substances such as dextrose and other electrolytes.

[0122] In another aspect, the invention provides a method of preparing liposomes consisting of the steps of: a) dissolving liposome formulation components, comprising one or more lipids, in a single isotropic monophase solution of dimethyl sulfoxide (DMSO) and / or acetonitrile (ACN) and water; b) freeze drying the solution obtained in step (a), thereby forming a lipid containing formulation cake; and c) hydrating the lipid containing formulation cake, to obtain an aqueous liposome formulation.

[0123] A method of preparing liposomes may consist of the steps of: a) dissolving liposome formulation components, comprising one or more lipids, in a single isotropic monophase solution of dimethyl sulfoxide (DMSO) and / or acetonitrile (ACN) and water; a1) sterile filtration of the solution obtained in step (a); b) freeze drying the solution obtained in step (a1), thereby forming a lipid containing formulation cake; c) hydrating the lipid containing formulation cake, to obtain an aqueous liposome formulation; d) filtering the aqueous liposome formulation obtained in step c); and e) freeze drying the aqueous liposome formulation, to obtain a liposome containing cake.

[0124] In another embodiment step d) of the method of the present invention comprises sterile filtering the aqueous liposome formulation obtained in step c).

[0125] In a further aspect, the invention provides a kit for preparing cargo-encapsulating liposomes, comprising a preformed mixture of (i) lipids, (ii) sugars, (iii) ACN and / or DMSO and (iv) water and instructions for using the preformed mixture for the preparation of cargo-encapsulating liposomes according to any method described herein.

[0126] A “preformed mixture” is a mixture or solution or dispersion that has been previously prepared and is provided in a kit for the desired process or method.

[0127] “Instructions for using the preformed mixture” provide guidance or steps for the use of the preformed mixture in a method of the invention for the preparation of liposomes.

[0128] Good Manufacturing Practice (GMP) is the minimum standard that a medicines manufacturer must meet in their production processes.

[0129] As used herein, and unless otherwise specified, the term "stable," when used in connection with a formulation, means that the active agent of the formulation, when prepared using the methods provided herein, remains solubilized for a specified amount of time and does not significantly degrade or aggregate or become otherwise modified (e.g., as determined by HPLC).

[0130] An isotropic solution is one such that the permittivity and permeability of the solution are uniform in all directions of the solution. “Monophase” refers to a type of matter or a compound that contains only one phase. A “single isotropic monophase solution” is a single solution that is uniform in all directions and contains only one phase. In the present invention, a “single isotropic monophase solution” indicates that all components (e.g. liposome formulation components) are dissolved initially in the same solvent.

[0131] A solvent is a substance that dissolves a solute, resulting in a solution. Polar solvents are solvents that have large dipole moments and possess charge separation. Polar solvents have a “positive” and a “negative” charge at different places in their structures and will dissolve other polar substances. Water is a polar solvent; other polar solvents include acetone, acetonitrile, dimethylformamide (DMF), dimelthylsulfoxide (DMSO), isopropanol, and methanol. An organic solvent is a liquid chemical composed of carbon and other elements. Organic solvents are carbon-based substances capable of dissolving or dispersing one or more other substances. A polar organic solvent is a carbon-based substance that dissolves a solute that has an electric dipole moment, with a negatively charged end and a positively charged end.

[0132] Examples of polar organic solvents include: ethanol, methanol, acetone, dimethyl sulfoxide (DMSO), acetonitrile; tetra hydrofuran (THF), dimethylformamide (DMF), dimethylacetamide (DMA), ethyl acetate, chloroform, dichloromethane (DCM), hexane, petroleum ether, diethyl ether, and isopropanol.

[0133] Suitably, the polar organic solvent may be dimethyl sulfoxide (DMSO) and / or acetonitrile (AON). Dimethyl sulfoxide is an organosulfur compound with the formula (CH3)2SO. This colorless liquid is the sulfoxide most widely used commercially. It is an important polar aprotic solvent that dissolves both polar and nonpolar compounds and is miscible in a wide range of organic solvents as well as water. Acetonitrile (ACN), often abbreviated MeCN, is the chemical compound with the formula CH3CN and structure H3C-C=N. This colourless liquid is the simplest organic nitrile. It is produced mainly as a byproduct of acrylonitrile manufacture. The freezing point of ACN is -46°C.

[0134] In some embodiments, the polar organic solvent is acetonitrile in water 1:1 (v / v). In some embodiments, the polar organic solvent may be less than or equal to 50% ACN. In some embodiments, the polar organic solvent may be more than or equal to 25% ACN. In some embodiments, the polar organic solvent may be 10% ACN. In some embodiments, the polar organic solvent may be 25% ACN. In some embodiments, the polar organic solvent may be 50% ACN. In some embodiments, the polar organic solvent may be between 10% and 50% ACN. In some embodiments, the polar organic solvent may be between 25% and 50% ACN. In some embodiments, the polar organic solvent may be between 50% and 70% ACN. In some embodiments, the polar organic solvent may be between 60% and 80% ACN. In some embodiments, the polar organic solvent may be between 80% and 95% ACN.

[0135] As used herein, and unless otherwise specified, "lipid" is understood to be a fatty acid, fatty acid salt, fatty alcohol, or phospholipid. Lipids may also be read to include sterols, including, but not limited to, cholesterol; sphingolipids, including, but not limited to, sphingomyelin; glycosphingolipids including, but not limited to, gangliosides, globocides and cerebrosides; and surfactant amines including, but not limited to, stearyl, oleyl and linoleyl amines. As used herein, and unless otherwise specified, the term "fatty acid" means a compound whose structure is a carboxylic group attached to a hydrocarbon chain having one or more carbon atoms. The hydrocarbon chain may be saturated or unsaturated (i.e. , alkyl, alkenyl or alkynyl hydrocarbon chains). Also, the hydrocarbon chain may be straight or branched. Moreover, in some embodiments, hydrogens in the hydrocarbon chain may be substituted.

[0136] As used herein, and unless otherwise specified, the term "fatty acid salt" means a compound formed from a reaction between a fatty acid and an inorganic / organic base. In addition, the term encompasses a compound formed from a reaction between a fatty alcohol and an inorganic / organic acid. Examples of such acids include, but are not limited to, sulfuric and phosphoric acid. The hydrocarbon chain of the fatty acid salt may be saturated or unsaturated (i.e., alkyl, alkenyl or alkynyl hydrocarbon chains). In addition, the hydrocarbon chain may be straight or branched. Moreover, in some embodiments, hydrogens in the hydrocarbon chain may be substituted.

[0137] As used herein, and unless otherwise specified, the term "fatty alcohol" means a compound whose structure is an alcohol group attached to a hydrocarbon chain having one or more carbon atoms. The hydrocarbon chain may be saturated or unsaturated (i.e., alkyl, alkenyl or alkynyl hydrocarbon chains). Also, the hydrocarbon chain may be straight or branched. Moreover, in some embodiments, hydrogens in the hydrocarbon chain may be substituted.

[0138] As used herein, and unless otherwise specified, the term "substituted" means a group substituted by one or more substituents including, but not limited to, alkyl, alkenyl, alkynyl, cycloalkyl, aroyl, halo, haloalkyl (e.g., trifluoromethyl), substituted or unsubstituted heterocycloalkyl, haloalkoxy (e.g., trifluoromethoxy), hydroxy, alkoxy, cycloalkyloxy, heterocylooxy, oxo, alkanoyl, aryl, substituted aryl, substituted or unsubstituted heteroaryl (e.g., indolyl, imidazolyl, furyl, thienyl, thiazolyl, pyrrolidyl, pyridyl, pyrimidyl and the like), arylalkyl, alkylaryl, heteroaryl, heteroarylalkyl, alkylheteroaryl, heterocyclo, aryloxy, alkanoyloxy, amino, alkylamino, arylamino, arylalkylamino, cycloalkylamino, heterocycloamino, mono- and di-substituted amino, alkanoylamino, aroylamino, aralkanoylamino, substituted alkanoylamino, substituted arylamino, substituted aralkanoylamino, carbamyl (e.g., CONH2), substituted carbamyl (e.g., CONH-alkyl, CONH-aryl, CONH-arylalkyl or instances where there are two substituents on the nitrogen), carbonyl, alkoxycarbonyl, carboxy, cyano, ester, ether, guanidino, nitro, sulfonyl, alkylsulfonyl, arylsulfonyl, arylalkylsulfonyl, sulfonamido (e.g., S02NH2), substituted sulfonamido, thiol, alkylthio, arylthio, arylalkylthio, cycloalkylthio, heterocyclothio, alkylthiono, arylthiono and arylalkylthiono. As used herein, and unless otherwise specified, the term "alkyl" means a saturated straight chain or branched non-cyclic hydrocarbon having 1-20 carbon atoms, preferably 1-10 carbon atoms and most preferably 1-4 carbon atoms. Representative saturated straight chain alkyls include -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, -n-hexyl, -n-heptyl, -n-octyl, -n- nonyl and - n-decyl; while saturated branched alkyls include -isopropyl, -sec-butyl, -isobutyl, - tert-butyl, - isopentyl, 2-methylbutyl, 3-methylbutyl, 2-methylpentyl, 3 -methylpentyl, 4- methylpentyl, 2- methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2,3-dimethylbutyl, 2,3- dimethylpentyl, 2,4-dimethylpentyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5- dimethylhexyl, 2,2-dimethylpentyl, 2,2-dimethylhexyl, 3,3-dimtheylpentyl, 3,3-dimethylhexyl, 4,4- dimethylhexyl, 2-ethylpentyl, 3-ethylpentyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2- methyl-2-ethylpentyl, 2- methyl-3-ethylpentyl, 2-methyl-4-ethylpentyl, 2-methyl-2-ethylhexyl, 2- methyl-3-ethylhexyl, 2- methyl-4-ethylhexyl, 2,2-diethylpentyl, 3,3-diethylhexyl, 2,2- diethylhexyl, 3,3-diethylhexyl and the like. An alkyl group can be unsubstituted or substituted. Unsaturated alkyl groups include alkenyl groups and alkynyl groups, which are discussed below.

[0139] As used herein, and unless otherwise specified, the term "alkenyl" means a straight chain or branched non-cyclic hydrocarbon having 2-20 carbon atoms, preferably 2-10 carbon atoms, most preferably 2-6 carbon atoms, and including at least one carbon-carbon double bond. Representative straight chain and branched (C2-Cio)alkenyls include -vinyl, -allyl, -1-butenyl, -2- butenyl, -isobutylenyl, -1-pentenyl, -2-pentenyl, -3 -methyl- 1-butenyl, -2-methyl-2-butenyl, -2,3- dimethyl-2-butenyl, -1-hexenyl, -2-hexenyl, -3-hexenyl, -1-heptenyl, -2-heptenyl, -3-heptenyl, - 1-octenyl, -2-octenyl, -3-octenyl, -1-nonenyl, -2-nonenyl, -3-nonenyl, -1-decenyl, -2-decenyl, -3- decenyl and the like. The double bond of an alkenyl group can be unconjugated or conjugated to another unsaturated group. An alkenyl group can be unsubstituted or substituted.

[0140] As used herein, and unless otherwise specified, the term "alkynyl" means a straight chain or branched non-cyclic hydrocarbon having 2-20 carbon atoms, preferably 2-10 carbon atoms, most preferably 2-6 carbon atoms, and including at lease one carbon-carbon triple bond. Representative straight chain and branched (C2-Ci0)alkynyls include -acetylenyl, -propynyl, -1- butynyl, -2-butynyl, -1-pentynyl, -2-pentynyl, -3-methyl-l-butynyl, -4-pentynyl, -1-hexynyl, -2- hexynyl, -5-hexynyl, -1-heptynyl, -2-heptynyl, -6-heptynyl, -1-octynyl, -2-octynyl, -7-octynyl, - 1- nonynyl, -2-nonynyl, -8-nonynyl, -1 -decynyl, -2-decynyl, -9-decynyl, and the like. The triple bond of an alkynyl group can be unconjugated or conjugated to another unsaturated group. An alkynyl group can be unsubstituted or substituted.

[0141] Suitably, the lipids may be dioleoylic lipids, fluidic lipids or cholesterol-containing lipids.

[0142] Dioleoylic lipids” have two oleoyl groups. “Fluidic lipids” wherein the lipid chains contain only one double bond (the more bonds the less fluidic). To make liposomes less fluidic (i.e. , more rigid) cholesterol can be added to the formulation (incorporation in the lipid membrane).

[0143] Cholesterol is a 27 carbon compound with a unique structure with a hydrocarbon tail, a central sterol nucleus made of four hydrocarbon rings, and a hydroxyl group. “Cholesterol-containing lipids” are lipids that contain cholesterol.

[0144] In one embodiment, at least one of the lipids is a phospholipid or a mixture of phospholipids. As used herein, and unless otherwise specified, "phospholipid" is understood to be an amphyphilic derivative of glycerol, in which one of its hydroxyl groups is esterified with phosphoric acid and the other two hydroxyl groups are esterified with long-chain fatty acids that can be equal to or different from each other and can be saturated or unsaturated. A neutral phospholipid is generally one in which the other phosphoric acid hydroxyl is esterified by an alcohol substituted by a polar group (usually hydroxyl or amino) and whose net charge is zero. A phospholipid with a charge is generally one in which the other phosphoric acid hydroxyl is esterified by an alcohol substituted by a polar group and whose net charge is positive or negative.

[0145] Examples of phospholipids include, but are not limited to, phosphatidic acid ("PA"), phosphatidylcholine ("PC"), phosphatidylglycerol ("PG"), phophatidylethanolamine ("PE"), phophatidylinositol ("PI"), and phosphatidylserine ("PS"), sphingomyelin (including brain sphingomyelin), lecithin, lysolecithin, lysophosphatidylethanolamine, cerebrosides, diarachidoylphosphatidylcholine ("DAPC"), didecanoyl-L-alpha-phosphatidylcholine, ("DDPC"), dielaidoylphosphatidylcholine ("DEPC"), dilauroylphosphatidylcholine ("DLPC"), dilinoleoylphosphatidylcholine, dimyristoylphosphatidylcholine ("DM PC"), dioleoylphosphatidylcholine ("DOPC"), dipalmitoylphosphatidylcholine ("DPPC"), distearoylphosphatidylcholine ("DSPC"), l-palmitoyl-2-oleoyl-phosphatidylcholine ("POPC"), diarachidoylphosphatidylglycerol ("DAPG"), didecanoyl-L-alpha-phosphatidylglycerol ("DDPG"), dielaidoylphosphatidylglycerol ("DEPG"), dilauroylphosphatidylglycerol ("DLPG"), dilinoleoylphosphatidylglycerol, dimyristoylphosphatidylglycerol ("DMPG"), dioleoylphosphatidylglycerol ("DOPG"), dipalmitoylphosphatidylglycerol ("DPPG"), distearoylphosphatidylglycerol ("DSPG"), 1 -palmitoyl-2-oleoyl-phosphatidylglycerol ("POPG"), diarachidoylphosphatidylethanolamine ("DAPE"), didecanoyl-L-alpha- phosphatidylethanolamine ("DDPE"), dielaidoylphosphatidylethanolamine ("DEPE"), dilauroylphosphatidylethanolamine ("DLPE"), dilinoleoylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine ("DM PE"), dioleoylphosphatidylethanolamine ("DOPE"), dipalmitoylphosphatidylethanolamine ("DPPE"), distearoylphosphatidylethanolamine ("DSPE"), 1 -palmitoyl-2-oleoyl-phosphatidylethanolamine ("POPE"), diarachidoylphosphatidylinositol ("DAPI"), didecanoyl-L-alpha-phosphatidylinositol ("DDPI"), dielaidoylphosphatidylinositol ("DEPI"), dilauroylphosphatidylinositol ("DLPI"), dilinoleoylphosphatidylinositol, dimyristoylphosphatidylinositol ("DM PI"), dioleoylphosphatidylinositol ("DOPI"), dipalmitoylphosphatidylinositol ("DPPI"), distearoylphosphatidylinositol ("DSPI"), 1-palmitoyl- 2- oleoyl-phosphatidylinositol ("POPI"), diarachidoylphosphatidylserine ("DAPS"), didecanoyl- L- alpha-phosphatidylserine ("DDPS"), dielaidoylphosphatidylserine ("DEPS"), dilauroy Iphosphatidy Iserine ("DLP S ") , dilinoleoy Iphosphatidy Iserine, dimyristoylphosphatidylserine ("DMPS"), dioleoylphosphatidylserine ("DOPS"), dipalmitoylphosphatidylserine ("DPPS"), distearoylphosphatidylserine ("DSPS"), l-palmitoyl-2- oleoyl-phosphatidylserine ("POPS"), diarachidoyl sphingomyelin, didecanoyl sphingomyelin, dielaidoyl sphingomyelin, dilauroyl sphingomyelin, dilinoleoyl sphingomyelin, dimyristoyl sphingomyelin, sphingomyelin, dioleoyl sphingomyelin, dipalmitoyl sphingomyelin, distearoyl sphingomyelin, and l-palmitoyl-2-oleoyl-sphingomyelin.

[0146] The phospholipids provided herein may be chiral or achiral. The chiral phospholipids provided herein may be D- or L-phospholipids, for example, L-a-phosphatidylcholine or L-3-phosphatidylcholine.

[0147] Suitably, the lipids may be 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1 ,2-dioleoyl- sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1 ,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), cholesterol-containing lipids, phospholipids, soy phosphatidylcholine (PC) and / or egg phosphatidylcholine (PC).

[0148] Suitably, the lipids may be 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 1,2- dioleoyl-sn-glycero-3-phosphocholine (DOPC).

[0149] Suitably, the liposome formulation components may comprise 10 mg / mL lipids DOTAP: DOPC [1 :1] molar ratio. In this context, “10 mg / mL lipids” refers to the total lipid concentration provided by DOTAP: DOPC in a 1 :1 molar ratio.

[0150] Suitably, the liposome formulation components may comprise 10 mg / mL lipids DOTAP: DOPC [1 :1] molar ratio and 1 mg / mL peptide. In this context, the “peptide” is suitably the cargo for the liposome.

[0151] Suitably, the liposome formulation components may comprise 10 mg / mL lipids DOTAP: DOPC [1 :1] molar ratio, 1 mg / mL peptide, and at least 10% (w / v) sucrose (or about 10% sucrose). Suitably, the liposome formulation components may comprise 10 mg / mL lipids DOTAP: DOPC [1 :1] molar ratio, 1 mg / mL peptide, and at least 20% (w / v) sucrose (or about 20% sucrose). Suitably, the liposome formulation components may comprise 10 mg / mL lipids DOTAP:DOPE [1 :1] molar ratio. In this context, “10 mg / mL lipids” refers to the total lipid concentration provided by DOTAP:DOPE in a 1:1 molar ratio.

[0152] Suitably, the liposome formulation components may comprise 10 mg / mL lipids DOTAP:DOPE [1 :1] molar ratio and 1 mg / mL peptide. In this context, the “peptide” is suitably the cargo for the liposome.

[0153] Suitably, the liposome formulation components may comprise 10 mg / mL lipids DOTAP:DOPE [1 :1] molar ratio, 1 mg / mL peptide, and at least 10% (w / v) sucrose (or about 10% sucrose). Suitably, the liposome formulation components may comprise 10 mg / mL lipids DOTAP:DOPE [1 :1] molar ratio, 1 mg / mL peptide, and at least 20% (w / v) sucrose (or about 20% sucrose). Suitably, the liposome formulation components may comprise 10 mg / mL lipids DOTAP:DOPC [1 :1] molar ratio. In this context, “10 mg / mL lipids” refers to the total lipid concentration provided by DOTAP:DOPC in a 1 :1 molar ratio.

[0154] Suitably, the liposome formulation components may comprise 10 mg / mL lipids DOTAP:DOPC [1 :1] molar ratio and 1 mg / mL peptide. In this context, the “peptide” is suitably the cargo for the liposome.

[0155] Suitably, the liposome formulation components may comprise 10 mg / mL lipids DOTAP:DOPC [1 :1] molar ratio, 1 mg / mL peptide, and at least 10% (w / v) sucrose (or about 10% sucrose). Suitably, the liposome formulation components may comprise 10 mg / mL lipids DOTAP:DOPC [1 :1] molar ratio, 1 mg / mL peptide, and at least 20% (w / v) sucrose (or about 20% sucrose). Suitably, the liposome formulation components may comprise 10 mg / mL lipids DOTAP:DOPG [1 :3] molar ratio. In this context, “10 mg / mL lipids” refers to the total lipid concentration provided by DOTAP:DOPG in a 1:3 molar ratio.

[0156] Suitably, the liposome formulation components may comprise 10 mg / mL lipids DOTAP:DOPG [1 :3] molar ratio and 1 mg / mL peptide. In this context, the “peptide” is suitably the cargo for the liposome.

[0157] Suitably, the liposome formulation components may comprise 10 mg / mL lipids DOTAP:DOPG [1 :3] molar ratio, 1 mg / mL peptide, and at least 10% (w / v) sucrose (or about 10% sucrose). Suitably, the liposome formulation components may comprise 10 mg / mL lipids DOTAP:DOPG [1 :3] molar ratio, 1 mg / mL peptide, and at least 20% (w / v) sucrose (or about 20% sucrose). Suitably, the liposome formulation components may comprise 10 mg / mL lipids DOTAP:DOPC;DOPE [1 : 1 ; 1] molar ratio. In this context, “10 mg / mL lipids” refers to the total lipid concentration provided by DOTAP:DOPC:DOPE in a 1:1:1 molar ratio.

[0158] Suitably, the liposome formulation components may comprise 10 mg / mL lipids DOTAP:DOPC:DOPE [1:1:1] molar ratio and 1 mg / mL peptide. In this context, the “peptide” is suitably the cargo for the liposome. Suitably, the liposome formulation components may comprise 10 mg / mL lipids DOTAP:DOPC:DOPE [1:1:1] molar ratio, 1 mg / mL peptide, and at least 10% (w / v) sucrose (or about 10% sucrose).

[0159] Suitably, the liposome formulation components may comprise 10 mg / mL lipids DOTAP:DOPC:DOPE [1:1:1] molar ratio, 1 mg / mL peptide, and at least 20% (w / v) sucrose (or about 20% sucrose).

[0160] Suitably, the liposome formulation components may comprise 10 mg / mL lipids DOTAP:DOPG [3:1] molar ratio. In this context, “10 mg / mL lipids” refers to the total lipid concentration provided by DOTAP:DOPG in a 3:1 molar ratio.

[0161] Suitably, the liposome formulation components may comprise 10 mg / mL lipids DOTAP:DOPG [3:1] molar ratio and 1 mg / mL peptide. In this context, the “peptide” is suitably the cargo for the liposome.

[0162] Suitably, the liposome formulation components may comprise 10 mg / mL lipids DOTAP:DOPG [3:1] molar ratio, 1 mg / mL peptide, and at least 10% (w / v) sucrose (or about 10% sucrose). Suitably, the liposome formulation components may comprise 10 mg / mL lipids DOTAP:DOPG [3:1] molar ratio, 1 mg / mL peptide, and at least 20% (w / v) sucrose (or about 20% sucrose). Suitably, the liposomes may be cationic liposomes, anionic liposomes or neutral liposomes.

[0163] Cationic formulations have a positive surface charge, which promotes an antitumor immune response compared to anionic or neutral formulations.

[0164] Cationic lipids capable of forming positively-charged liposomes are one of the most widely used nonviral vectors for gene delivery (Zhi et al, Bioconjugate Chemistry, 2013, 24: 478-519). Cationic lipids are amphiphilic molecules and generally contain a hydrophobic domain (e.g., aliphatic chains, steroid rings), a hydrophilic headgroup (e.g., amines, quaternary ammonium salts, guanidiniums, heterocycles), and a linker group (e.g., ether, ester, carbamate or amide bond) connecting the two domains. The hydrophilic headgroup enables the condensation of nucleic acids by electrostatic interactions with the negatively-charged phosphate groups of the genes, and further governs transfection efficiency. Cationic lipids are usually formulated as cationic liposomes with a neutral co-lipid like dioleoyl phosphatidyl ethanolamine (DOPE) or cholesterol to improve transfection. When mixed with negatively-charged DNA, the positively- charged liposomes spontaneously form uniquely compacted structures called lipoplexes.

[0165] “Cationic lipid” refers to a lipid containing a positive charge or being ionizable as a whole. Cationic lipids include, but are not limited to: N, N-dioleyl-N, N-dimethylammonium chloride (DODAC), N, N-distearyl-N, N-dimethylammonium bromide (DDAB), N- (1- (2, 3-dioleoyloxy) propyl) -N, N, N-trimethylammonium chloride (DOTAP), N- (1- (2, 3-dioleyloxy) propyl) -N, N, N- trimethylammonium chloride (DOTMA), N, N-dimethyl-2, 3-dioleyloxypropylamine (DODMA), 3- (didodecylamino) -N1, N1 , 4-tridodecyl-1-piperazineethylamine (KL10), N1- [2- (didodecylamino) ethyl ] -N1, n4, N4-tridodecyl-1, 4-piperazine diethylamine (KL22), 14, 25- ditridecyl-15, 18,21, 24-tetraaza-triacontane (KL25), 1, 2-dioleyloxy-N, any one of N- dimethylaminopropane (Dlin-DMA), 2-dioleyl-4-dimethylaminomethyl- [1,3] -dioxolane (Dlin-K- DMA), thirty-seven carbon-6, 9,28, 31-tetraen-19-yl 4- (dimethylamino) butyrate (Dlin-MC3- DMA) and 2, 2-dioleyl-4- (2-dimethylaminoethyl) - [1,3] -dioxolane (Dlin-KC2-DMA) and mixtures thereof.

[0166] For example, formulations containing DOTAP:DOPE (e.g. at a molar ratio of 1:1), DOTAP:DOPG (e.g. at a molar ratio of 1:1), DOTAP:DOPC:DOPE (e.g. at a molar ratio of 1:1:1), and DOTAP:DOPG (e.g. at a molar ratio of 3:1) were used herein to generate cationic liposomes.

[0167] “Anionic lipid” refers to lipid containing a negative charge. For example, anionic phospholipids include phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylinositol (PI), and its phosphorylated derivatives the phosphoinositides (e.g., phosphatidylinositol-4-phosphate [PI4P] and phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]).

[0168] “Neutral lipid” refers to any of a number of lipid substances, preferably phospholipids, that exist in the uncharged or neutral zwitterionic form at a selected pH. Such lipids include, but are not limited to: 1, 2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1, 2-dimyristoyl-sn-glycero- phosphocholine (DMPC), 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1, 2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC), 1, 2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1, 2-dicarballyl-sn-glycero-phosphocholine (DUPO), 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPO), 1 , 2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0Diether PC), 1-oleoyl-2-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OchemsPC), 1-hexadecyl-sn- glycero-3-phosphocholine (C16 Ly PC), 1, 2-dilinolacyl-sn-glycero-3-phosphocholine, 1, 2- dineoyl-sn-glycero-3-phosphocholine, 1 , 2-didodecanoyl-sn-glycero-3-phosphocholine, 1 , 2- dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1, 2-diphytanoyl-sn-glycero-3- phosphoethanolamine (ME 16. OPE), 1 , 2-distearoyl-sn-glycero-3-phosphoethanolamine, 1 , 2- dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1 , 2-didodecanoyl-sn-glycero-3- phosphoethanolamine, 1, 2-dioleoyl-sn-glycero-3-phospho-rac- (1-glycero) sodium salt (DOPG), Dioleoylphosphatidylserine (DOPS), Dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), Distearoylphosphatidylethanolamine (DSPE), Dipalmitoylphosphatidylethanolamine (DPPE), Dimyristoylphosphatidylethanolamine (DMPE), 1-stearoyl-2-oleoyl-Stearoylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, and Lysophosphatidylethanolamine (LPE), and combinations thereof. Neutral lipids may be of synthetic or natural origin.

[0169] For example, formulations containing DOTAP:DOPG (e.g., at a molar ratio of 1 :3), were used herein to generate anionic liposomes.

[0170] A "steroid lipid" may be selected from any one of cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and mixtures thereof

[0171] As used herein, and unless otherwise specified, "encapsulate" or "encapsulation" is understood to be the process of incorporating a cargo (e.g. an active agent) into liposomes. The encapsulated cargo can remain in the aqueous interior or associate with membranes. Encapsulating peptides into liposomes can be challenging due to their hydrophilic nature and susceptibility to degradation. Previously, several methods have been developed to encapsulate peptides into liposomes, but the known formulation procedures are suboptimal due to usage of organic solvents, labour intensive, difficult to translate to GMP, antigen alteration to enable peptide incorporation.

[0172] In a particular embodiment, the liposome formulation components used in the initial stage of the process of this invention do not contain a cargo (e.g. an active agent). In this way it is possible to obtain empty vesicles, which can be used as such or in which at least one cargo can be encapsulated (by active encapsulation). The active encapsulation method employed can be any of those known to a person skilled in the art, such as techniques by pH gradient or concentration gradient.

[0173] In another particular embodiment, the liposome formulation components employed in the first stage of the process of the present invention contain a cargo (e.g. an active agent). In this way it is possible to prepare, without additional steps, liposomes with at least one cargo (by passive encapsulation). In both cases the cargo to be encapsulated can be added in suspension or solution and once encapsulated it can remain in the aqueous interior and / or associated with membranes of the liposome.

[0174] A liposome may be “loaded” with cargo by the cargo residing in the aqueous interior of the liposome and / or associated with the liposome membrane.

[0175] Suitably, the liposomes may be cargo-encapsulating liposomes. Suitably, the liposomes may be peptide-encapsulating liposomes. Liposomes are lipid-based vesicles capable of encapsulating peptides for various applications in drug delivery, vaccine development, and biomedical research. Peptide-encapsulating cationic liposomal vaccine formulations are especially of interest for therapeutic T cell vaccines, including (personalized) cancer vaccines.

[0176] Suitably, the peptides may be dissolved in (0.04%) ammonium hydroxide before addition to the single isotropic monophase solution in step (a).

[0177] Encapsulation efficiency is the percentage of drug that is successfully entrapped into the micelle or nanoparticle.

[0178] In one embodiment, the method of preparation of cargo-encapsulating liposomes results in greater efficiency of incorporation of the cargo (“encapsulation efficiency”) into the liposome. In certain embodiments, the efficiency of incorporation of the cargo into the liposome is 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100%. In certain embodiments, the efficiency of incorporation is 90%, 95%, 98%, 99% or 100%.

[0179] A “cargo” is a molecule or molecules that are carried with or on, or are encapsulated by, a liposome.

[0180] In one embodiment, the cargo is an active agent. In one embodiment, the active agent is a hydrophobic drug.

[0181] In one embodiment, the cargo is added to or included in the liposome formulation components as a solid. In another embodiment, the cargo is added to or included in the liposome formulation components in an organic solvent. In another embodiment, the cargo is added to or included in the liposome formulation components in organic solvent which further comprises one or more fatty acid salts, fatty acids and / or phospholipids.

[0182] In one embodiment, the cargo is a water soluble membrane-impermeant agent such as a peptide, a protein, a nucleic acid, a nucleotide, a nucleoside, a carbohydrate or an analog thereof.

[0183] Suitably, the cargo may be a biomacromolecule. A biomacromolecule is a very large molecule important to biological processes, such as a protein or nucleic acid. Suitably, the liposome formulation components may further comprise peptides.

[0184] Suitably, the liposome formulation components may further comprise cancer peptides. Suitably, the liposome formulation components may further comprise anti-cancer peptides.

[0185] Suitably, the liposome formulation components may further comprise a nucleic acid. Suitably, the liposome formulation components may further comprise DNA and / or RNA.

[0186] A nucleic acid useful in embodiments of the present invention may be selected according to the biological or physiological effect desired to be produced, e.g., by its delivery into living cells. Such selection is well known to the skilled artisans in the fields of molecular biology and medicine. A nucleic acid is a polymeric material that is a nucleic acid or resembles in its structure and function a nucleic acid in that it exhibits a backbone of covalently linked repetitive molecular units (also referred to as monomers) and has a biological or physiological effect. A nucleic acid may include natural, modified or synthetic bases and backbone elements. A nucleic acid may be of natural or synthetic origin and may include a nucleic acid (i.e. , a polymer that comprises a plurality of nucleic acid bases attached to a backbone of covalently linked repetitive molecular units), DNA, RNA, natural and synthetic oligonucleotides (including antisense oligonucleotides, interfering RNA, small interfering RNA (siRNA), small hairpin RNA (shRNA)), double-stranded RNAs that are about 30 base pairs in length and can act as dicer enzyme substrates, nucleoprotein, peptide, nucleic acid, ribozyme, DNA-containing nucleoprotein, such as an intact or partially deproteinated viral particles (virions), oligomeric and polymeric anionic compounds other than DNA (for example, acid polysaccharides and glycoproteins), and the like. It is preferably DNA or RNA, and is more preferably DNA carrying a sequence of an expressible gene or siRNA. Antisense oligonucleotides are another preferred type of nucleic acids. To signify the process of transfer of an exogenous nucleic acid into a living cell the term “transfection” will be used without limitation to any particular kind of nucleic acid or to any particular function that may be performed in the cell by a nucleic acid so transferred. The transfection may be performed on cells in the body of a subject to be treated (in vivo) or on cells maintained outside a subject (in vitro or ex vivo). The terms “transfection” and “delivery” will be used interchangeably in this description. When it is advantageous for a particular application, liposomes may contain more than one kind of nucleic acid in respect to structure, function, or nucleotide sequences.

[0187] In the present invention, "nucleic acid" refers to DNA or RNA or modified forms thereof, which contain purine or pyrimidine bases (adenine "a", cytosine "C", guanine "G", thymine "T") present in DNA or purine or pyrimidine bases (adenine "a", cytosine "C", guanine "G", uracil "II") present in RNA. In the present invention, "RNA" refers to a ribonucleic acid which may or may not be naturally occurring. For example, the RNA can include modified and / or non-naturally occurring components, such as one or more nucleobases, nucleosides, nucleotides, or linkers. The RNA may include a cap structure, a chain terminating nucleoside, a stem loop, a polyadenylation sequence, and / or a polyadenylation signal. The RNA can have a nucleotide sequence encoding a polypeptide of interest. For example, the RNA may be messenger RNA (mrna). Translation of an mRNA encoding a particular polypeptide, for example, translation of an mRNA in vivo within a mammalian cell, can result in the encoded polypeptide. The RNA may be selected from the non-limiting group consisting of: small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), mRNA, single stranded guide RNA (sgRNA), cas9 mRNA, and mixtures thereof.

[0188] In the present invention, antisense oligonucleotides or small interfering RNAs (siRNAs) can inhibit the expression of target genes and proteins in vitro or in vivo.

[0189] Suitably, the liposome formulation components may further comprise proteins. Proteins are biopolymeric structures composed of amino acids. Proteins serve as structural support, biochemical catalysts, hormones, enzymes, building blocks, and initiators of cellular death.

[0190] Suitably, the liposome formulation components may further comprise adjuvants, optionally immune adjuvants.

[0191] In the present invention, an "adjuvant or adjuvant component" is typically a (e.g., pharmacological or immunological) agent or composition that can alter (e.g., enhance) the efficacy of other agents (e.g., drugs or vaccines). Generally, the term refers in the context of the present invention to a compound or composition that acts as a carrier or auxiliary substance for an immunogen and / or other pharmaceutically active compounds. It is to be interpreted in a broad sense and refers to a wide range of substances capable of increasing the immunogenicity of antigens incorporated into or co-administered with the adjuvant in question. In the present invention, an adjuvant will preferably enhance the specific immunogenic effect of the active agent of the present invention. Typically, "adjuvant" or "adjuvant component" have the same meaning and may be used interchangeably. Adjuvants can be classified, for example, as immunopotentiators, antigen delivery systems, or even combinations thereof. An “immune adjuvant” is defined as any substance that acts to accelerate, prolong, or enhance antigenspecific immune responses. Suitably, the liposome formulation components may further comprise sugars and / or cryoprotectants. A cryoprotectant is a substance used to protect biological tissue from freezing damage. Examples of suitable cryoprotectants include sugars such as trehalose or sucrose.

[0192] Examples of sugars that may be used in the methods provided herein include, but are not limited to, sucrose, glucose, fructose, lactose, maltose, mannose, galactose and trehalose. The sugar may act as a cryoprotectant and / or reconstitution enhancer.

[0193] Suitably, the sugars may be sucrose and / or trehalose. Suitably, the sugar may be sucrose.

[0194] In one embodiment, the liposome formulation components may contain up to 25% w / v of the sucrose or trehalose of the whole solution of step a).

[0195] In one embodiment, the liposome formulation components may contain at least 10% w / v sucrose or trehalose of the whole solution of step a).

[0196] In one embodiment, the liposome formulation components may contain at least 10% and up to 25% w / v sucrose or trehalose of the whole solution of step a).

[0197] In one embodiment, the liposome formulation components may contain about 20% w / v sucrose of the whole solution of step a).

[0198] In another embodiment, the liposome formulation components may contain about 10% w / v trehalose of the whole solution of step a).

[0199] As used herein, and unless otherwise specified, the term "harmful ingredient," when used in connection with pharmaceutical compositions, means an ingredient commonly used in a pharmaceutical composition that may cause clinical side effects such as, but not limited to, hemolysis, hypersensitive reaction, peripheral neuropathies, and / or decrease in the bioavailability of the active ingredient of the composition. Examples of harmful ingredients include, but are not limited to: toxic solvents, including organic solvents such as ethanol, methanol, 1-propanol, 2- propanol, acetone, ethyl acetate, methyl acetate, diethyl ether, dimethyl ether, diisopropyl ether, methyl tert-butyl ether ("MTBE"), tetrahydrofuran ("THF"), dichloromethane, chloroform, carbon tetrachloride, 1 ,2-dicholroethane, pentane, hexanes, heptane, petroleum ether, dioxane, ethylene glycol, diethylene glycol, diglyme, 1 ,2- dimethoxyethane, 1 -butanol, 2- butanol, 2-butanone, benzene, toluene, dimethylformamide ("DMF"), hexamethylphosphoramide ("HMPA"), N-methylpyrrolidone, glycerin, nitromethane, triethyl amine, xylenes, CREMOPHOR® EL, and polyethylene glycol ("PEG"); co-detergents or surfactants such as polysorbates (e.g., Tweens) or vitamin E; oils such as Castor oil or corn oil; proteins such as HSA; or any other biologic which is potential source of contamination.

[0200] Suitably, in some embodiments, the method may not comprise the addition of a category 2B carcinogen. Category 2B carcinogens are agents that are categorised as "possibly carcinogenic to humans".

[0201] Suitably, in some embodiments, the method may not comprise the addition of tert-butyl alcohol, chloroform and / or methanol.

[0202] In one embodiment, the liposomal preparation or liposomes produced by the claimed method or kit is suitable for parenteral administration to a patient suffering from one or more diseases or disorders.

[0203] In one embodiment, the patient is a human.

[0204] Method of treating a disease or disorder may also use liposomal compositions or liposomes provided by the methods outlined herein. The disease or disorder may include, but is not limited to, oncological disorders, proliferative disorders, central nervous system disorders, autoimmune disorders, and inflammatory diseases or disorders. The methods may be directed to the treatment of bacterial, viral or fungal infections.

[0205] Proliferative disorders (e.g. cancer) that may be treated include, but are not limited to, neoplasms, tumors (malignant and benign) and metastases, or any disease or disorder characterized by uncontrolled cell growth. The cancer may be a primary or metastatic cancer. Specific examples of cancers that can be prevented, managed, treated or ameliorated include, but are not limited to, cancer of the head, neck, eye, mouth, throat, esophagus, chest, bone, lung, colon, rectum, stomach, prostate, breast, ovaries, kidney, liver, pancreas, and brain. Additional cancers include, but are not limited to, the following: leukemias (e.g., acute leukemia, acute lymphocytic leukemia), acute myelocytic leukemias (e.g., myeloblasts, promyelocyte, myelomonocytic, monocytic, erythroleukemia leukemias and myelodysplasia syndrome), chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia), polycythemia vera, lymphomas (e.g., Hodgkin's disease, non-Hodgkin's disease), multiple myelomas (e.g., smoldering multiple myeloma, nonsecretory myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma and extramedullary plasmacytoma), Waldenstrom's macroglobulinemia, monoclonal gammopathy of undetermined significance, benign monoclonal gammopathy, heavy chain disease, bone and connective tissue sarcomas (e.g., bone sarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma, soft-tissue sarcomas, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, neurilemmoma, rhabdomyosarcoma, synovial sarcoma), brain tumors (e.g., glioma, astrocytoma, brain stem glioma, ependymoma, oligodendroglioma, nonglial tumor, acoustic neurinoma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma, pineoblastoma, primary brain lymphoma), breast cancer (e.g., adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, medullary breast cancer, mucinous breast cancer, tubular breast cancer, papillary breast cancer, Paget's disease, and inflammatory breast cancer), adrenal cancer (e.g., pheochromocytom and adrenocortical carcinoma), thyroid cancer (e.g., papillary or follicular thyroid cancer, medullary thyroid cancer and anaplastic thyroid cancer), pancreatic cancer (e.g., insulinoma, gastrinoma, glucagonoma, vipoma, somatostatin-secreting tumor, and carcinoid or islet cell tumor), pituitary cancers (e.g., Cushing's disease, prolactin-secreting tumor, acromegaly, and diabetes insipius), eye cancers (e.g., ocular melanoma such as iris melanoma, choroidal melanoma, and cilliary body melanoma, and retinoblastoma), vaginal cancers (e.g., squamous cell carcinoma, adenocarcinoma, and melanoma), vulvar cancer (e.g., squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget's disease), cervical cancers (e.g., squamous cell carcinoma, and adenocarcinoma), uterine cancers (e.g., endometrial carcinoma and uterine sarcoma), ovarian cancers (e.g., ovarian epithelial carcinoma, borderline tumor, germ cell tumor, and stromal tumor), esophageal cancers (e.g., squamous cancer, adenocarcinoma, adenoid cyctic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma), stomach cancers (e.g., adenocarcinoma, fungating (polypoid), ulcerating, superficial spreading, diffusely spreading, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma), colon cancers, rectal cancers, liver cancers (e.g., hepatocellular carcinoma and hepatoblastoma, gallbladder cancers such as adenocarcinoma), cholangiocarcinomas (e.g., pappillary, nodular, and diffuse), lung cancers (e.g., non-small cell lung cancer, squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large-cell carcinoma and small-cell lung cancer), testicular cancers (e.g., germinal tumor, seminoma, anaplastic, classic (typical), spermatocytic, nonseminoma, embryonal carcinoma, teratoma carcinoma, choriocarcinoma (yolk-sac tumor), prostate cancers such as but not limited to, adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma), penile cancers, oral cancers (e.g., squamous cell carcinoma), basal cancers, salivary gland cancers (e.g., adenocarcinoma, mucoepidermoid carcinoma, and adenoidcystic carcinoma), pharynx cancers (e.g., squamous cell cancer, and verrucous), skin cancers (e.g., basal cell carcinoma, squamous cell carcinoma and melanoma, superficial spreading melanoma, nodular melanoma, lentigo malignant melanoma, acral lentiginous melanoma), kidney cancers (e.g., renal cell cancer, adenocarcinoma, hypernephroma, fibrosarcoma, transitional cell cancer (renal pelvis and / or uterer)), Wilms' tumor, bladder cancers (e.g., transitional cell carcinoma, squamous cell cancer, adenocarcinoma, carcinosarcoma), myxosarcoma, osteogenic sarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma and papillary adenocarcinomas, follicular lymphomas, carcinomas with p53 mutations, hormone dependent tumors of the breast, prostate and ovary, precancerous lesions such as familial adenomatous polyposis, and myelodysplasia syndromes.

[0206] In therapeutic vaccination, specific immunotherapy, the host’s healthy immune system is activated to generate and activate de novo cancer-specific T cells, that will only destroy diseased malignant cells upon recognition and leave healthy cells unharmed. This approach aims to induce long-lasting anti-tumour immunity, potentially leading to tumour regression and improved patient outcomes. Using peptides for cancer vaccination offers several benefits such as specificity, customisability, safety, immunogenicity, easy of production and combination therapies. Peptides for cancer vaccination face challenges related to immune tolerance, as tumor-associated antigens (TAAs) targeted by peptide vaccines may be recognized as selfantigens, leading to immune tolerance or suppression. Additionally, peptides alone lack sufficient immunogenicity to elicit robust anti-tumor immune responses, necessitating the use of adjuvants to enhance immune activation. However, selecting appropriate adjuvants and formulating them with peptides is complex, and adjuvants may cause adverse reactions or systemic toxicity. Cationic liposomes that incorporate peptides are very promising for improving the immunogenicity of peptide based cancer vaccines.

[0207] Other specific diseases and disorders that may be treated include, but are not limited to, the following: allergic disorders, inflammation, asthma, arthritis, encephalitis, rheumatoid arthritis, osteoarthritis, psoriatic arthritis, inflammatory osteolysis, chronic or acute obstructive pulmonary disease, chronic or acute pulmonary inflammatory disease, inflammatory bowel disease, Crohn's Disease, gout, Bechet's Disease, Henoch-Schonlein purpura ("HSP"), septic shock, sepsis, meningitis, colitis, inflammation due to reperfusion, psoriasis, fibrosis including pulmonary fibrosis, Parkinson's disease, bradykinesia, muscle rigidity, Parkinsonian tremor, Parkinsonian gait, motion freezing, depression; defective long-term memory, Rubinstein-Taybi syndrome (RTS), dementia, sleep disorders, insomnia, postural instability, hypokinetic disorders, hyperkinetic disorders, synuclein disorders, multiple system atrophies, striatonigral degeneration, olivopontocerebellar atrophy, Shy-Drager syndrome, motor neuron disease with parkinsonian features, Lewy body dementia, Tau pathology disorders, progressive supranculear palsy, corticobasal degeneration, frontotemporal dementia; amyloid pathology disorders, mild cognitive impairment, Alzheimer disease, Alzheimer disease with parkinsonism, Wilson disease, Hallervorden-Spatz disease, Chediak-Hagashi disease, SCA-3 spinocerebellar ataxia, X-linked dystonia parkinsonism, Huntington disease, prion disease, chorea, ballismus, dystonia tremors, Amyotrophic Lateral Sclerosis ("ALS"), CNS trauma, myoclonus, and diseases or disorders associated with undesired immune reaction (e.g., organ rejection associated with an organ transplant).

[0208] Viral infections that may be treated include, but are not limited to, the following: human immunodeficiency virus ("HIV"), herpes simplex virus type 1, herpes simplex virus type 2, influenza viruses, influenza virus type A, influenze virus type B, parainfluenza virus, human papillomavirus ("HPV"), adenoviruses, rhinoviruses, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, dengue fever, yellow fever, West Nile virus, Japanese encephalitis virus, GB virus A, GB virus-B, GB virus-C, bovine viral diarrhea virus ("BVDV"), classical swine fever virus (i.e. , hog cholera virus), border disease virus, varicella zoster virus, smallpox, measles, rabies virus, arbovirus, cytomegalovirus, mumps virus, poliovirus, coxsackie B virus, Epstein-Barr virus, rubella virus, parvovirus B19, coronaviruses (e.g., SARS coronavirus), astrovirus, norovirus, rotavirus, and adenoviruses.

[0209] Fungal infections that may be treated include, but are not limited to, aspergillosis, blastomycosis, coccidioidomycosis, cryptococcosis, fungal sinusitis, histoplasmosis, hypersensitivity pneumonitis, mucormycosis, paracoccidioidomycosis, sporotrichosis, and Valley Fever.

[0210] Bacterial infections that may be treated include, but are not limited to, brucellosis, cholera, leprocy, leptospirosis, shigellosis, trench fever, tularemia, Q fever, Whitmore's disease, yersiniosis, yaws, vibrio vulnificus infections, streptococcus infections, staphylococcus infections and E. coli infections.

[0211] "Inhibiting expression of a target gene" refers to the ability of a nucleic acid to silence, reduce, or inhibit expression of a target gene. To test the extent of gene silencing, a test sample (e.g., a sample of cells in a medium expressing a target gene) is contacted with a nucleic acid that inhibits expression of the target gene. The expression of the target gene in the test sample or test animal is compared to the expression of the target gene in a control sample (e.g., a sample of cells in medium expressing the target gene) that has not been contacted or administered the nucleic acid. The expression of the target gene in the control sample can be assigned a value of 100%. In particular embodiments, inhibition of expression of the target gene is achieved when the level of expression of the target gene in the test sample is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0% relative to the level of expression of the target gene in a control sample or control mammal. A method for determining the expression level of the target gene includes, but is not limited to, dot blotting, northern blotting, in situ hybridization, ELISA, immunoprecipitation, enzyme action, and phenotypic assay.

[0212] “Transfection" refers to the introduction of a species (e.g., RNA) into a cell. Transfection may occur, for example, in vitro, ex vivo, or in vivo.

[0213] An "antigen" typically refers to a substance that is recognized by the immune system, preferably by the adaptive immune system, and is capable of triggering an antigen-specific immune response, for example by forming antibodies and / or antigen-specific T cells as part of the adaptive immune response. Typically, the antigen may be or may comprise a peptide or protein that can be presented by MHC to a T cell. In the sense of the present invention, an antigen may be the translation product of a provided nucleic acid molecule, preferably an mRNA as defined herein. In this context, fragments, variants and derivatives of peptides and proteins comprising at least one epitope are also understood as antigens.

[0214] "Delivery" refers to providing an entity to a target. For example, the drug and / or therapeutic and / or prophylactic agent is delivered to a subject, which is a tissue and / or cell of a human and / or other animal. Delivery can be intramuscularly, intradermally or subcutaneously. Preferably the delivery would be intradermally.

[0215] By "pharmaceutically acceptable carrier" is meant a diluent, adjuvant, excipient, or vehicle that is administered together with a therapeutic agent and which is, within the scope of sound medical judgment, suitable for contact with the tissues of humans and / or other animals without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable carriers that can be used in the pharmaceutical compositions of this invention include, but are not limited to, sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is an exemplary carrier when the pharmaceutical composition is administered intravenously. Physiological saline and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, maltose, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like. The composition may also optionally contain minor amounts of wetting agents, emulsifying agents, or pH buffering agents. Oral formulations may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. Specifically, for example, excipients include, but are not limited to, antiadherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (pigments), demulcents, emulsifiers, fillers (diluents), film formers or coatings, flavorants, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and water of hydration. More specific excipients include, but are not limited to, Butylated Hydroxytoluene (BHT), calcium carbonate, dicalcium phosphate, calcium stearate, croscarmellose sodium, crospovidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, phenyl paraben, retinol palmitate, shellac, silicon dioxide, carboxymethylcellulose sodium, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin a, vitamin E (alpha-tocopherol), Vitamin C, xylitol.

[0216] 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.

[0217] Aspects of the invention are demonstrated by the following non-limiting examples.

[0218] EXAMPLES

[0219] Materials and Methods

[0220] Peptides in Figure 11 were synthesized at the peptide facility of the Leiden University Medical Center (LUMC). The fluorescently labelled MHC-I SIINFEKL tetramer - Allophycocyanin (Kb- SIINFEKL-APC) was produced and purified at the peptide facility of the LUMC. The lipids 1 ,2- dioleoyl-3-trimethylammonium-propane (DOTAP), 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1 ,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG) and 1 ,2-di- (9Z0octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE) were purchased from Avanti Polar Lipid (Alabaster, AL, USA). Chloroform and Acetonitrile (ACN, HPLC gradient grade > 99.9%) were obtained from Biosolve BV (Valkenswaard, the Netherlands) and Vivaspin 2 centrifuge columns were acquired from Sartorius Stedim Biotech GmbH (Gottingen, Germany). The disaccharides sucrose and trehalose (dihydrate) were obtained from Sigma-Aldrich (Zijndrecht, the Netherlands). Sterile syringe PVDF-filters with pore size of 0.22 pm (diameter 0 13 mm) were bought from Santa Cruz Biotechnology Inc. (Texas, USA). Deionized water with resistivity of 18 MQcm was produced by a Millipore water purification system (MQ water). Phosphate buffer (PB) was composed of 2.3 mM NaH2PO4 2H2O and 7.7 mM

[0221] Na2HPO4 2H2O (10 mM PB, pH 7.4). 10 mM PB in MQ water was filtered through a 0.22 pm Millex-GP PES filter (0 24 mm, Millipore, Ireland) before use.

[0222] Cell culture

[0223] The D1 cell line is a long-term growth factor-dependent immature myeloid DC line of splenic origin derived from a female C57BL / 6 (H-2b) mouse. D1 cells were cultured at 37°C in a 5% CO2 atmosphere in IM DM (Gibco) supplemented with 8% FCS (Bodinco B.V.), 4 mM glutamax (Gibco), 100 lU / ml penicillin (Gibco), 50 pM p-mercaptoethanol (Gibco), and supplemented with GM-CSF supernatant (Winzler 1997).

[0224] Animals

[0225] Naive C57BL / 6 (Janvier Laboratories, France) female mice were purchased at 6-8 weeks and housed under specific pathogen-free conditions at the LUMC animal facility. Experiments were started when mice were 8-9 weeks old. All animal experiments were approved by and according to guidelines of the Dutch Animal Ethical Committee.

[0226] Preparation of peptide-loaded cationic liposomes

[0227] Thin Film dehydration-rehydration method

[0228] Peptide-loaded cationic liposomes composed of the lipids DOTAP and DOPC were produced using the thin film dehydration-rehydration method as previously described (11 , 12). In brief, both lipids (DOTAP: DOPC) were dissolved in chloroform and mixed at a 1 :1 molar ratio with 10 mg / mL total lipid concentration in a round bottom flask. Rotary evaporation was performed, resulting in a thin lipid film. The dry lipid film was hydrated with a 1 mg / mL peptide solution of MQ water that was added to the thin lipid film. The liposomal dispersion was snap frozen using liquid nitrogen followed by freeze-drying overnight in a Christ alpha 1-2 freeze-dryer (Osterode, Germany). After freeze-drying, the lipid cake was assessed for deformities and reconstituted with PB (pH 7.4) in three consecutive steps to reach the initial volume (30 minutes equilibration time between each step). Speed of reconstitution of the lipid cake was determined with a timer. Subsequently, the liposomes were extruded using Lipex extruder (Northern Lipids Inc., Canada) in which the liposomes were extruded four times through 400 nm and 200 nm polycarbonate filters (Nucleopore Millipore, Kent, United Kingdom). Concentration of the peptide loaded liposomes was done by centrifugation (931 G) in Vivaspin 2 centrifugation concentrators (molecular weight-cut-off of 300 kDA) by centrifugation at 2000 rpm and 4°C. Samples of liposomal fractions and flow-through were collected to determine the free and encapsulated peptide.

[0229] New GMP compliant method of making liposomes

[0230] DOTAP and DOPC can be weighted and dissolved in ACN:water mixtures with peptides and sugars, which was done for the preparation for larger batches of liposomal formulations. However, for small-scale test production and method optimization (to accurately include the lipids in the formulation and reduce required amounts of chemicals to prepare small batches), DOTAP and DOPC were dissolved in chloroform and mixed at a 1:1 molar ratio with a 10 mg / mL total lipid concentration into a freeze-drying vial and dried under a stream of nitrogen. Peptide corresponding to 1 mg / mL was added to the vial. Unless specified, dried lipids and peptide were hydrated using ACN:MQ 1:1 (v / v) containing 20% sucrose or trehalose. The vials were placed into the programmable Martin Christ Epsilon 2-6D freeze-dryer (Martin Christ, Germany), and freeze-dried using the program Liposomes sugar below. The lyophilized products were assessed for deformities and reconstituted at once with PB (pH 7.4); the speed of reconstitution was determined with a timer. The product was filtered either through one 0.22 (0 13 mm) syringe filter manually or through two 0.22 (0 13 mm) syringe filters using a syringe pump (NE-300, Pump Systems Inc.). For quality control, a Vivaspin 2 was used to determine encapsulated peptide as described above. The final product was aliquoted and freeze-dried again using the programmable freeze-dryer (program: Liposomes sugar).

[0231] Liposomes prepared using the novel freeze-drying method were lyophilized using the freeze- dryer. The freeze-drying program (Liposomes sugar) used was as follows: (1) freezing at -40°C for 8 hours followed by (2) main drying for 20 hours at -40°C with a chamber pressure of 0.100 mbar and at last (3) secondary drying for 20 hours with a gradual increase to 20°C within four hours at 0.0010 mbar. After the freeze-drying program has ended, the percentage of remaining moisture was checked with the pressure increase test build-in tool and the product was considered dry when there was less than 10% increase in pressure (mbar) in 30 seconds.

[0232] Liposome characterization

[0233] The average size (Z-average; nm) and polydispersity index (PDI) of the particles were determined using dynamic light scattering (DLS). The zeta-potential (mV) was determined by using laser Doppler electrophoresis. The measurements were performed on a Zetasizer Nano (Malvern Instruments, Malvern, UK). Prior to measurements, samples were diluted 75-fld in PB (pH 7.4).

[0234] Lipid and peptide content Both lipid and peptide content were determined using reversed phase ultra-high perform liquid chromatography (LIPLC, Waters Acquity LIPLC with Waters C18-1.8 pm (2.1 x 50 mm) column) as described before (11). A flow rate of 0.5 mL / min was used with initially 95% solvent A (A: water with 0.1% TFA) and 5% solvent B (B: ACN with 0.1% TFA), followed by a linear gradient to 100% solvent B in 7 minutes and back to 5% solvent B after 9 minutes. The total run time was 12 minutes.

[0235] DOTAP and DOPC calibration standards were prepared by evaporating solutions containing DOPC and DOTAP in concentration of 50 pg / mL of each lipid and dried vials were stored at - 80C as previously described (11). For each measurement a vial of DOTAP:DOPC calibration stock was dissolved in 1 mL MeOH:MQ 1 :1 (v / v) and volumes ranging from 5 pL - 50 pL were injected by the autosampler of the LIPLC. Detection of the lipids was measured using ELS detector (ELSD). Prior to measurement of the lipids, the liposomes were diluted 10-fold in MeOH:MQ 1 :1 (v / v) and injected (10 pL / injection). The lipids were quantified by integrating the area under the DOTAP and DOPC ELSD peaks of the samples and standards using MassLynx integration software (Waters). Using the concentration of the lipids, the lipid recovery was calculated according to Eq-1.

[0236] Eq-1. Lipid recovery (%) = ([C]lipids before / [C]lipids after) x 100%

[0237] Peptide calibration samples were made with 50 pg / mL peptide in MeOH:MQ 1:1 (v / v) and volumes ranging from 5 pL to 50 pL were injected into the UPLC system. Detection of peptides was achieved by measuring the absorbance at a wavelength of 214 nm with an Acquity TUV detector (Waters). The samples for the peptide measurements were diluted 10-fold in MeOH:MQ 1 :1 (v / v) prior to injection (20 pL / injection). Peptides were quantified by integrating the area under the UV peaks of the samples and standards using MassLynx integration software (Waters). The encapsulation efficiency of the liposomes was calculated by using equation Eq-2.

[0238] Eq-2. Encapsulation efficiency (%) - (([Ctotal peptide] — [Cfree peptide]) / [Ctotal peptide]) x 100%

[0239] Eq-3. Peptide recovery (%) = [peptide recovered in the final formulation] / [total peptide added initially] x 100%

[0240] Stability Study

[0241] OVA24 loaded liposomes were made according the method above (“New GMP compliant method”). The lyophilized products were stored at -20° C for up to a year. At various timepoints, the freeze-dried liposomes were reconstituted in PB (pH 7.4) to obtain a final peptide (OVA24) concentration of 1 mg / mL and final lipid concentration of 10 mg / mL, and were analyzed according to the method above (“Liposome characterization”).

[0242] In vivo Antigen-specific T cell response

[0243] Naive 8-9 weeks old C56BL / 6 female mice were injected intradermally at the tail base with 4 nmol of peptide-loaded cationic liposomes, OVA24 peptide only, or peptide with 1 pg poly(l:C) in 30 pL PBS. 14 days after initial injection, mice were injected again. Frequencies of antigen- specific CD8+T cells were determined in the peripheral blood of vaccinated mice, retrieved via tail puncture every few days.

[0244] 35 days after initial injections, mice were sacrificed and spleens were harvested and single cell suspensions were made. After an overnight pre-culture of 50,000 D1 cells with 5 pM OVA24, 1 million splenocytes from each mouse and 2 pg / mL brefeldin A (Sigma-Aldrich) were added and maintained at 37°C in a 5% CO2 atmosphere for 6 hours, followed by intracellular staining.

[0245] Prophylactic vaccination and B16-OVA tumor challenge

[0246] Naive 8-9 weeks old C56BL / 6 female mice were injected intradermally at the tail base on days 0 and 14 with 4 nmol of OVA24 peptide loaded cationic liposomes made via thin film dehydration- rehydration method or new GMP compliant method, or peptide only. 14 days post second injection, animals were subcutaneously injected in the right flank with 100,000 live B16-OVA cells in 100 pL PBS. Tumor sizes were measured 2-3 times a week and 1500 mm3(length x width x depth) was maintained as the humane endpoint.

[0247] Flow cytometric analysis and staining antibodies

[0248] Prior to flow cytometric analysis, red blood cells were first lysed. Staining with an MHC class I SIINFEKL tetramer was performed for 30 minutes at room temperature in FACS buffer (0.5% bovine serum albumin, 0.02% sodium azide in PBS). Subsequently, cells were washed and stained with antibodies to CD3 (PE, clone 17A2, Biolegend, San Diego, USA) and CD8a (FITC, clone 53-6.7, Biolegend, San Diego, USA) in FACS buffer for 30 minutes on ice. Live cells were distinguished by a fixable viability dye eFluor450 (eBioscience, San Diego, USA) stained in PBS.

[0249] For intracellular staining of splenocytes, cells were first stained with MHC class I SIINFEKL tetramers, surface markers and viability dye as mentioned above. Afterwards, cells were fixed for 30 minutes with fixation buffer (Biolegend, San Diego, USA), and were stained with IFN (PE-Cy7, clone XMG1.2, BD Biosciences, New Jersey, USA) and TNFa (BV605, clone MP6- XT22, Biolegend, San Diego, USA) through the use of intracellular staining permeabilization wash buffer (BioLegend, San Diego, USA). Data was acquired with the BD-LSR-II flow cytometry and analysed using FlowJo software (version 10.9.0).

[0250] Cryo-EM

[0251] CryoEM was performed at the electron microscopy facility of LUMC.

[0252] Cryo-EM imaging of liposomes

[0253] Quantifoil 2 / 2 grids were glow discharged in air at 0.2 mbar, 25 mA and 30 seconds using a Pelco Easyglow. To the glow discharged grids, 3 pl of sample was added and blotted away for 3 seconds using filter paper (Whatman no.4) at 85-95% humidity and room temperature using a Leica EM GP. The grid was subsequently plunged into liquid ethane / propane (2 / 1; v / v) at -196 °C. CryoEM images were acquired on a Talos Arctica (Thermo Fisher Scientific) operated by EPU software (Thermo Fisher Scientific) at 15000x nominal magnification on a Ceta camera (Thermo Fisher Scientific) at nominal magnification of 22000x, corresponding to a pixel size of 0.65 nm at specimen level, a defocus of -4 micron, and an electron dose of ~35 e / A2 / s with 1 second exposure time. Lamellarity of imaged liposomes was determined my manual visualization of liposomes using Imaged 1.54d.

[0254] Statistical analysis

[0255] Graphical representation and statistics was performed using GraphPad Prism 8.02 (San Diego, USA). Respective statistical details and tests are mentioned in the legends of the figures. Differences were considered significant when p-values were below 0.05.

[0256] Results

[0257] Different percentages acetonitrile (ACN) in water were investigated to determine the optimal amount of ACN to dissolve the lipids DOTAP and DOPC and model peptide OVA24. Solvents with more than 50% ACN in MQ could not be frozen in the freeze-dryer, and the peptide could not dissolve in concentrations of 25% ACN or lower (data not shown). Therefore, the peptide / lipid mix was first dissolved in 50% ACN, followed by dilution to 10% and 25% ACN using water (no observed flocculation or precipitation). Lyophilization of said formulations successfully resulted in small lipid cakes (Figure 12). There are various characteristics that lipid cakes can possess; upon inspection of said liposomes we observed cake shrinkage, cracked cakes, bubble formation and partial collapse (Figure 18). These small lipid cakes, upon rehydration, showed similar physicochemical properties in regards to size, polydispersity index and surface charge, to the standard thin film dehydration-rehydration method (Figure 2A, Figure 13). There was no difference in polydispersity index (PDI) and charge with the standard and the new method, while the liposome size was slightly higher with the new method (118.5 ± 3.6 nm vs 135.4 ± 3.3 nm).

[0258] To determine whether DOTAP and DOPC could be equally dissolved in ACN, the molar ratio of the lipids was determined via UPLC (Figure 13). The liposomal formulation had been made using a 10 mg / mL DOTAP:DOPC at a [1:1] molar ratio. It was observed that the molar ratio DOTAP:DOPC remained around 1 for both the standard method and the new method with ACN. Furthermore, the standard method and the new method resulted in similar peptide encapsulation efficiencies (Figure 2B, Figure 13); while the standard method had an encapsulation efficiency of 96.4 ± 2.2, the new method gave an encapsulation efficiency of up to 95.6 ± 1.4.

[0259] Therefore, the lipids DOTAP and DOPC, and the peptide OVA24 were found to be dissolvable at concentrations of ACN in water higher than 25% via a new GMP compliant method, which allowed for the formulation of liposomes with similar physicochemical properties and encapsulation efficiency as the standard method. The inventors investigated the use of filtration as a substitute for high-pressure extrusion, which is difficultly translatable to GMP production. OVA24-loaded liposomes were extruded or filtered, and their physicochemical properties and both lipid and peptide content were determined (Figure 3).

[0260] Both extrusion and filtration result a significant decrease in particle size and PDI (Figure 3A). Extrusion results in liposomes with a particle size of 135.4 ± 3.3 nm, PDI of 0.15 ± 0.02 and zeta- potential of 24,7 ± 3.5 mV. These physicochemical properties were significantly higher in the new method, at 206.8 ± 14.4, 0.25 ± 0.06, and 43.6 ± 1.7 respectively.

[0261] In addition, the lipid recovery and OVA24 encapsulation efficiency were determined (Figure 3B). The molar ratio is similar for both the extrusion and filtration method, as well as the lipid recovery for both DOTAP and DOPC. Meanwhile, extrusion resulted in a slightly lowered encapsulation efficiency of OVA24 compared to filtration.

[0262] Furthermore, the importance of timing of filtration was studied. Filtration was performed either before freeze-drying, after freeze-drying, or both before and after (Figure 14). It is seen that if filtration occurs before freeze-drying, the liposome size is significantly bigger and with a larger PDI. Meanwhile, there is no difference either in size or PDI between performing the filtration after freeze-drying or both before and after freeze-drying. The surface charge is similar for both filtration before freeze-drying and when filtration occurs twice. However, when filtration occurs solely after freeze-drying, the charge is significantly higher. Overall, it appears as though filtration has to occur after first reconstitution in order to maintain good physicochemical properties, though an additional filtration before freeze-drying minimally impacts the physiochemical properties.

[0263] Having determined the feasibility of using filtration as a substitute for extrusion, the combination of two syringe filters and an automated syringe pump were explored in order to render the filtration step GMP-compliant. Therefore, a syringe pump was used at speeds ranging from 250 pL / min to 2500 pL / min with the use of two tandem 0.22 pm syringe filters. Samples were taken at different fractions of the filtration. The inventors observed similar physiochemical properties regardless of the speed utilized (Figure 4). These properties were the same as what is seen with a single 0.22 pm syringe filter utilized manually. Furthermore, there was no difference in physiochemical properties throughout time when using a syringe pump.

[0264] Therefore, filtration can be used as a method of making monodisperse small liposomes and give similar results to the standard high-pressure homogenization protocol. Filtration can also be converted into a GMP-compliant method through the use of a syringe pump and one or two stacked syringe filters.

[0265] Example 3 - Sugars can be used as cryoprotectants of liposomes without affecting physicochemical properties and act as a reconstitution enhancer

[0266] Vaccine formulations often have an increased stability in dry form compared to liquid form. Therefore, liposomes can be freeze-dried in the presence of cryoprotectants, such as sucrose that will protect the particles from aggregation and drug leakage during the freezing process. To explore the importance of cryoprotectants in the new method, the inventors looked at the physicochemical properties of OVA24 liposomes when utilizing sugars as cryoprotectants. Lyophilization of the liposomal dispersion with sucrose or trehalose produced a smooth lipid cake without any cracks or collapsed parts (Figure 15).

[0267] Lyophilization without a cryoprotectant resulted in significantly bigger liposomes with larger PDIs and higher surface charge than when the liposome had a cryoprotectant ranging from 5% to 20% w / v sucrose (Figure 15). Both sucrose and trehalose resulted in liposomes with similar physiochemical properties and DOTAP:DOPC molar ratios when at least 10% w / v was used (Figures 15 and 16). In regards to total lipid recovery, at least 15% sugar was found to be ideal to get at least 90% recovery (Figure 19). Moreover, adding sugars significantly enhanced the ease of reconstitution of the lipid cake (Figure 20) (seconds instead of >1h) and resulted in good quality lipid cakes after lyophilization. The lipid cake that was formed with a cryoprotectant was observed to no longer partially collapse (as was the case for lipid cakes made without cryoprotectant). At 10% w / v sucrose, the lipid cakes displayed cake shrinkage and cracking: this was not observed at 20% w / v / sucrose or 10% w / v trehalose (Figure 18). For further experiments 20% w / v sucrose was chosen for the preparation of peptide-containing liposome formulations. Upon analysis with cryo-TEM, the new method, with 20% w / v sucrose used as cryoprotectant, gave unilamellar liposomes, while the standard method generated more heterogenous multilamellar liposomes. The lipid cakes that contained cryoprotectant displayed no cake collapse, shrinkage or cracking, unlike those that did not contain a cryoprotectant (Figure 18). Overall, the lipid cake characteristics were deemed acceptable. Moreover, the addition of sugars results in much improved reconstitution and improved the quality of the lyophilized lipid cake, without cryoprotectant the reconstitution is sluggish (>1h) (Figure 20), which underscores the necessity of a cryoprotectant.

[0268] Example 4 - New method is reproducible for multiple lipids and for peptides with different

[0269] To display the versatility of the novel GMP-compliant method, the inventors prepared liposomes with different lipid compositions. The inventors saw that, regardless of the lipid formulation used, they were able to formulate peptide-loaded liposomes with sizes below 300 nm and PDI below 0.3 (Figure 6). Formulations containing DOTAP:DOPE [1:1], DOTAP:DOPG [1 :1], DOTAP:DOPC:DOPE [1:1:1], and DOTAP:DOPG [3:1] resulted in cationic liposomes. The formulation containing DOTAP:DOPG [1:3] resulted in anionic liposomes. Therefore, the new method successfully generated both anionic and cationic liposomes with consistent sizes and PDIs.

[0270] The new method for liposome production was also found to be utilizable for liposomes of various peptide properties. When comparing peptides that were encapsulated in this study with the new method and in previous studies (12), the inventors have seen that in both cases they were able to encapsulate peptides with varying GRAVY indexes and isoelectric points (Figure 7D). The peptides tested resulted in liposomes with similar charges and PDI when utilizing the standard versus the new method (Figure 7A). The surface charge of the liposomes was, on average, higher in peptides encapsulated via the new method compared to the standard method. Furthermore, both the encapsulation efficiency and the peptide recovery were significantly increased in the new method (Figure 7B and C respectively).

[0271] Therefore, this new method allows the formation of peptide-loaded cationic liposomes smaller than 200 nm and with a PDI of less than 0.3. This method can successfully load peptides of wide ranges of hydrophobicity and isoelectric points with an increased encapsulation efficiency and peptide recovery, compared to the standard method.

[0272] Example 5 - Liposomes made via the new method induce T cell responses and survival in vivo

[0273] To determine the functionality of the liposomes generated via the new GMP-compliant method in vivo, the inventors administered 4 nmol of OVA24 liposomes or peptide only in naive mice intradermally (Figure 8A; Figure 21A). In mice vaccinated with OVA24 liposomes, SIINFEKL specific CD8+T cells in blood were observed compared to mice vaccinated with peptide alone or peptide in combination with poly(l:C) as an adjuvant (Figure 8B; Figure 21 B). Although SIINFEKL specific CD8+T cells in blood were slightly reduced in response to the liposomes made via the new method compared to those made by the standard method, data shows the response obtained using the standard method is less reproducible. The blood SIINFEKL specific CD8+T cell response produced via the new method shows much lower variability than the standard method (standard deviation error bars, Figure 21 B). Therefore, the data shows there was no significant difference in antigen-specific CD8+T cell responses between liposomes made via the standard method and those made via the new method, i.e. the liposomes made via the two methods show similar immunogenicity.

[0274] Upon restimulation of the splenocytes of the injected mice, the new method leads to higher number of cytokines released by CD8+T cells compared to the standard method. Overall, these data indicate that the new GMP-complaint method leads to antigen specific T cell responses in vivo to at least similar levels as the standard method.

[0275] In a prophylactic tumor B16-OVA setting, it was found that OVA24 liposomes made via the new method lead to similar survival and tumor growth compared to those made via the standard method (Figure 9A; Figure 22), which was significantly different than the peptide alone. In a prophylactic B16-OVA tumor model, the inventors compared the therapeutic efficacy of liposomes prepared using the novel GMP-compliant method (“new method”) with those prepared using the conventional formulation method (“standard method”). Both groups showed comparable tumor growth and survival outcomes (Figure 22B), indicating that the new method is at least equally effective in eliciting protective anti-tumor responses. Although one additional mouse had to be sacrificed in the group receiving the new formulation, this was not related to reduced vaccine efficacy. Instead, it was due to inherent limitations of the B16-OVA tumor model, which is known to frequently develop necrotic or ulcerating lesions regardless of size. In this specific case, one mouse had a borderline tumor size of -1000 mm3, and the other had a small but necrotic and bleeding tumor that necessitated early euthanasia. These findings underscore the robustness of the new method for in vivo cancer vaccination and highlight the importance of careful interpretation of survival data in aggressive tumor models like B16. Furthermore, in a TC-1 therapeutic setting (HPV-peptide), liposomes made through the new method lead to increased survival and decreased tumor growth (Figure 9B) compared to the untreated condition.

[0276] Example 6 - Liposomes made via the new method display Iona term storaae

[0277] To investigate the thermal stability of the freeze-dried liposomes with sucrose, the stability of the lyophilized products containing 20% w / v sucrose stored at -20°C was measured. After six months of storage at -20°C, there was a slight decrease in liposome size (Figure 10). However, liposome PDI, surface charge, encapsulation efficiency and molar ratio remained unperturbed. Therefore, it appears that liposomes made in the new method can be stored at -20°C for at least 6 months.

[0278] Discussion

[0279] Cationic liposomes induce excellent anti-tumor responses both in vitro and in vivo. However, the lack of a GMP-compliant liposomal preparation method means that the usability of these cationic liposomes in the clinic is limited. Therefore, in the current study the inventors have developed an aseptic GMP-compliant liposome manufacturing method.

[0280] The inventors have compared the novel method to the standard thin film dehydration- rehydration method of preparing cationic peptide liposomes. They have substituted the use of a rotary evaporator, which comes with risks of contamination, concerns of the lack of reproducibility of the lipid film, and the use of toxic solvents such as chloroform and methanol, and substituted it with the dissolution of lipids and peptides in 50% acetonitrile in water followed by freeze-drying. While chloroform, used in the standard method, has a very low freezing point (-63.5°C) that cannot be reached with the shelf temperature of many freeze-dryers, ACN, with a freezing point of -45°C can be used in most programmable freeze-dryers. Furthermore, ACN is often utilized in GMP processes to dissolve and purify peptides, making it GMP-compliant. However, concentrations higher than 50% ACN did not successfully permit freezing of the solvent, and concentration of 25% or lower of ACN did not allow for fully dissolution of the peptide. Therefore, further optimization of the method utilized 50% ACN. The inventors thus found that using 50% ACN and a freeze-dryer allowed for the formation of peptide-loaded cationic liposomes of homogenous size and charge, similar what to was seen in the standard method, without changing the encapsulation efficiency and molar ratio of said liposomes. Therefore, ACN in combination with lyophilization can be used as a substitute to rotary evaporation.

[0281] Secondly, the inventors substituted high-pressure homogenization (referred to as extrusion) with disposable syringe filters. It is difficult to ensure (because of cleaning) and maintain sterility when utilizing extrusion, while syringe filters are disposable and packaged individually and sterile. Filtration lead to a broader size distribution compared to extrusion. However, it was still within the requirements for peptide-loaded liposomes of under 200 nm and with a PDI under 0.3 so that it can still be considered homogenous. Moreover, filtration gave high lipid recovery and encapsulation efficiency, indicating that filtration is a viable alternative to extrusion.

[0282] In order to maintain sterility, the inventors explored whether the liposomes formed would remain unaltered regardless of when filtration occurred. They distinguished no difference in size and PDI if filtration occurred solely after first reconstitution or if it occurred before freeze-drying and after the first reconstitution. However, the size and PDI was higher if filtration solely occurred before freeze-drying, possibly due to a fusion of phospholipid membranes, which would lead to increased heterogeneity. Overall, filtration has to occur after first reconstitution in order to maintain good physicochemical properties of liposomes, though an additional filtration before freeze-drying does not negatively impact the physiochemical properties and can thus be used if required by GMP.

[0283] The filtration step was then expanded by using liposomes pushed through two stacked syringe filters by a syringe pump, as double filtration may be required for GMP compliance. In order to ensure that a solution remains sterile during filtration, the process must be short (e.g., 4 hours for 100 vials / doses). Therefore, the inventors used a syringe pump with speeds ranging from 250 pL / min to 2500 pL / min, all of which fit in the speeds required for GMP in the GMP facility of LLIMC. Using a syringe pump, the inventors observed that the physicochemical properties of the liposomes remained unchanged between manual filtration with a single 0.22 pm filter and stacked filters used by syringe pump, regardless of which speed was utilized. The characteristics of the liposomes remained unperturbed by the timing by which a sample was collected, indicating that the liposomes pushed through the syringe filters via a syringe pump are comparable between aliquots. Therefore, when using a syringe pump, reproducible liposomes can be made at speeds that allow for scalability under GMP.

[0284] Furthermore, the inventors explored the importance of cryoprotectants in the formulation. Cryoprotectants are thought to be important in liposomal formulations as they prevent aggregation due to the replacement of water molecules associated with polar head groups of the hydrated phospholipid bilayer by sugar molecules, thereby reducing surface tension at the liposome surface during freeze-drying. The inventors saw that cryoprotectants such as sucrose could preserve the liposomal size, charge and PDI. This was the case only for percentages of at least 10% sucrose: below that, the size and PDI were increased. The presence of cryoprotectants did not affect the molar ratio. Overall, an addition of a cryoprotectant resulted in cationic liposomes of sizes below 200 nm and PDI below 0.3, indicating the importance of adding a cryoprotectant to the formulation. Moreover, addition of sugars results in much improved reconstitution and improved the quality of the lyophilized lipid cake.

[0285] Upon visualization of liposomes made via the standard method and the novel method, the inventors observed that the novel method resulted in more unilamellar liposomes while the standard method gave multilamellar liposomes (Figure 23). While both uni- and multilamellar liposomes are used, unilamellar liposomes are more homogenous, and therefore more suitable for clinical production as reproducibility of a process is highly important in GMP production and might provide some advantages in cancer vaccinations where reproducibility is key.

[0286] In addition, the inventors looked at the stability of liposomes made via the new method when stored at -20°C. They observed that the physiochemical properties of the liposomes remained unchanged. They also observed that the encapsulation efficiency and molar ratio remained unperturbed during storage. Therefore, liposomes produced in the novel method can be stored, once lyophilized, at -20°C for at least 6 months.

[0287] The inventors then tested the versatility of the liposomes by utilizing various lipid formulations. They observed that it was possible to make both cationic and anionic liposomes with different lipid formulations and concentrations, while maintaining adequate physicochemical properties. This indicates that the method can be versatilely used for multiple types of liposomes, opening up the possibility of use beyond synthetic peptide cancer vaccines.

[0288] Subsequently, the inventors tested the reproducibility of the liposomes by incorporating peptides of different hydrophobicity and isoelectric points. They observed that, similarly to the thin film dehydration-rehydration method (12), they could incorporate a wide array of different peptides. The liposomes generated with either method gave similar sizes and PDI. However, the charge was higher in the cationic liposomes made via the novel method as compared to the standard method, possibly due to less formulation loss (especially cationic lipid) during the procedure. The encapsulation efficiency and peptide recovery was also increased in the novel method, possibly as there are less steps in which the peptide can be lost. Higher encapsulation efficiency indicates a potential advantage in vaccination due to more homogeneity in the liposomes in a sample, while higher peptide recovery is important to decrease costs and increase output and efficiency of the process. Overall, the inventors have observed that the new method of liposomal preparation could be utilized for a variety of peptides and led to homogeneously sized liposomes with high peptide recovery and encapsulation efficiency. In addition, the inventors looked at whether the liposomes made via the novel method were functional in vivo. They observed that the antigen-specific CD8+T cell responses were similar for liposomes made by the standard thin film dehydration-rehydration method and the new method (Figure 21). Both these methods surpassed vaccination with peptide alone in the number of CD8+T cell responses, as well as peptide with an adjuvant. When looking into these T cell responses, the inventors saw that the novel method was at least comparable to the standard method in terms of polyfunctionality of the T cells. These results highlight the importance of proper vaccine formulations in order to adequately stimulate the immune system. Lastly, the inventors also looked at the effect of the liposomes made with the new GMP- compliant method in a tumor setting. They saw that in a prophylactic B16-OVA tumor setting, the liposomes made either via the standard method or with the new method displayed similar tumor growth and survival (Figure 22). Furthermore, liposomes made via the novel method also successfully led to a decrease in tumor burden and increase in survival in a therapeutic TC-1 (HPV tumor) setting. Overall, these results indicate that the novel method can effectively be used for cancer vaccination in vivo.

[0289] In this work, the inventors report a novel method of producing peptide loaded cationic liposomes via a pharmaceutical freeze-drying process. This method is reproducible, adaptable to GMP, and labor efficient. This method can successfully load peptides of varying properties into liposomes with high peptide recovery and encapsulation efficiency, essential for peptide cancer vaccines. Altogether, this novel GMP-compliant method opens opportunities to develop new (personalized) peptide-based cancer and T cell vaccines suitable for human use.

[0290] 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.

[0291] 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.

[0292] 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.

[0293] 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.

[0294] Sequences

[0295] References (1) Varypataki et al., Efficient Eradication of Established Tumors in Mice with Cationic

[0296] Liposome-Based Synthetic Long-Peptide Vaccines, Cancer Immunol Res (2017) 5 (3): 222-233.

[0297] (2) Waldman et al., A guide to cancer immunotherapy: from T cell basic science to clinical practice, Nature Reviews Immunology volume 20, pages651-668 (2020).

[0298] (3) Maeng et al., Strategies for developing and optimizing cancer vaccines, F1000Research 2019, 8(F1000 Faculty Rev):654.

[0299] (4) Nelde et al., The Peptide Vaccine of the Future, REVIEW | SPECIAL ISSUE: IMMUNOPEPTIDOMICSI VOLUME 20, 100022, 2021.

[0300] (5) Joshi et al., Targeting tumor antigens to dendritic cells using particulate carriers, Journal of Controlled Release, Volume 161, Issue 1, 10 July 2012, Pages 25-37. (6) Ma et al., The role of surface charge density in cationic liposome-promoted dendritic cell maturation and vaccine-induced immune responses, Nanoscale., Issue 5, 2011.

[0301] (7) Varypataki,., Cationic Liposomes Loaded with a Synthetic Long Peptide and Poly(l:C): a Defined Adjuvanted Vaccine for Induction of Antigen-Specific T Cell Cytotoxicity, AAPS J 17, 216-226 (2015). https: / / doi.org / 10.1208 / s12248-014-9686-4.

[0302] (8) Zhen et al., Paclitaxel loading in cationic liposome vectors is enhanced by replacement of oleoyl with linoleoyl tails with distinct lipid shapes, Sci Rep 11 , 7311 (2021).

[0303] (9) Lombardo et al., Methods of Liposomes Preparation: Formation and Control Factors of Versatile Nanocarriers for Biomedical and Nanomedicine Application, Pharmaceutics 2022, 14(3), 543;

[0304] (10) Akbarzadeh et al., Liposome: classification, preparation, and applications, Nanoscale Res Lett 8, 102 (2013). https: / / doi.org / 10.1186 / 1556-276X-8-102.

[0305] (11) Heuts et al., Quantification of Lipid and Peptide Content in Antigenic Peptide- loaded Liposome Formulations by Reversed-phase UPLC using UV Absorbance and Evaporative Light Scattering Detection, Journal of Pharmaceutical Sciences, VOLUME 111, ISSUE 4, P1040-1049, APRIL 2022.

[0306] (12) Heuts et al., Cationic Liposomes: A Flexible Vaccine Delivery System for Physicochemically Diverse Antigenic Peptides, Pharm Res 35, 207 (2018).

Claims

Claims1. A method of preparing liposomes comprising the steps of: a) dissolving liposome formulation components, comprising one or more lipids, in a single isotropic monophase solution of polar organic solvent and water; b) freeze drying the solution obtained in step (a), thereby forming a lipid containing formulation cake; and c) hydrating the lipid containing formulation cake, to obtain an aqueous liposome formulation.

2. The method of claim 1, wherein the polar organic solvent is dimethyl sulfoxide (DMSO) and / or acetonitrile (ACN).

3. The method of claim 1 or claim 2, wherein the method additionally comprises a step of sterile filtration of the solution obtained in step (a).

4. The method of any preceding claim, wherein the method additionally comprises a step of sterile filtering the aqueous liposome formulation obtained in step c).

5. The method of claim 3 or claim 4, wherein the filtering uses a syringe filter.

6. The method of any preceding claim, wherein the method additionally comprises a step of freeze drying the aqueous liposome formulation, to obtain a liposome containing cake.

7. The method of any preceding claim, wherein the polar organic solvent is less than or equal to 50% ACN.

8. The method of claim 7, wherein the polar organic solvent is 50% ACN.

9. The method of any preceding claim, wherein the lipids are dioleoylic lipids, fluidic lipids and / or cholesterol-containing lipids.

10. The method of claim 9, wherein the lipids are 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), 1 ,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), cholesterol-containing lipids, phospholipids, soy phosphatidylcholine (PC) and / or egg phosphatidylcholine (PC).

11. The method of claim 9 or claim 10, wherein the lipids are 1 ,2-dioleoyl-3- trimethylammonium-propane (DOTAP) and 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).

12. The method of any preceding claim, wherein the liposomes are cationic liposomes, anionic liposomes or neutral liposomes.

13. The method of any preceding claim, wherein the liposomes are cargo-encapsulating liposomes.

14. The method of claim 13, wherein the cargo is a biomacromolecule.

15. The method of any preceding claim, wherein the liposomes are peptide-encapsulating liposomes.

16. The method of any preceding claim, wherein the liposome formulation components further comprise peptides.

17. The method of claim 16, wherein the peptides are dissolved in (0.04%) ammonium hydroxide before addition to the single isotropic monophase solution in step (a).

18. The method of any preceding claim, wherein the liposome formulation components further comprise DNA and / or RNA.

19. The method of any preceding claim, wherein the liposome formulation components further comprise proteins.

20. The method of any preceding claim, wherein the liposome formulation components further comprise immune adjuvants, optionally wherein the immune adjuvant is a toll like receptor (such as imiquimod, CpG, Poly(l:C), Pam(3)Cys or MPI_A.

21. The method of any preceding claim, wherein the liposome formulation components further comprise sugars and / or cryoprotectants.

22. The method of claim 21 , wherein the sugars are sucrose and / or trehalose.

23. The method of any preceding claim, wherein the method does not comprise the addition of a category 2B carcinogen.

24. The method of any preceding claim, wherein the method does not comprise the addition of tert-butyl alcohol, chloroform and / or methanol.

25. A method of preparing liposomes consisting of the steps of: a) dissolving liposome formulation components, comprising one or more lipids, in a single isotropic monophase solution of dimethyl sulfoxide (DMSO) and / or acetonitrile (ACN) and water; b) freeze drying the solution obtained in step (a), thereby forming a lipid containing formulation cake; and c) hydrating the lipid containing formulation cake, to obtain an aqueous liposome formulation.

26. The method of claim 25, wherein THF, ethanol, DMF and DMA are excluded.

27. The method of any preceding claim, wherein the liposomes are 80-400 nm in diameter, preferably 100-200 nm in diameter.

28. The method of any preceding claim, wherein the liposome formulation components comprise 10 mg / mL lipids DOTAP:DOPC [1:1] molar ratio, 1 mg / mL peptide, 10% (w / v) sucrose.

29. The method of any preceding claim, wherein the liposome formulation components comprise 10 mg / mL lipids DOTAP:DOPC [1:1] molar ratio, 1 mg / mL peptide, 20% (w / v) sucrose30. The method of any preceding claim, wherein the liposomes are unilamellar particles.

31. A kit for preparing cargo-encapsulating liposomes, comprising a preformed mixture of:(i) lipids,(ii) sugars,(iii) ACN and / or DMSO, and(iv) water and instructions for using the preformed mixture for the preparation of cargoencapsulating liposomes according to the method of any preceding claim.

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