WO2024102332A1 - Compositions vaccinales comprenant un néo-antigène de kras - Google Patents

Compositions vaccinales comprenant un néo-antigène de kras Download PDF

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WO2024102332A1
WO2024102332A1 PCT/US2023/036862 US2023036862W WO2024102332A1 WO 2024102332 A1 WO2024102332 A1 WO 2024102332A1 US 2023036862 W US2023036862 W US 2023036862W WO 2024102332 A1 WO2024102332 A1 WO 2024102332A1
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composition
neoantigen
kras
immune response
cell
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PCT/US2023/036862
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English (en)
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Olga HRYTSENKO
Rajkannan RAJAGOPALAN
Liliana PORTALES CERVANTES
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Himv Llc
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  • the present application relates to vaccine compositions comprising one or more neoantigen(s), wherein the one or more neoantigen(s) is associated with KRAS, a pharmaceutically acceptable carrier, and optionally an adjuvant and/or T-helper epitope, and methods of using such compositions in the treatment of cancer.
  • Neoantigens are emerging as a very strong option to advance personalized cancer medicine, as they have tremendous potential to effect cancer treatments that provide truly individualized immunotherapies; however, suitable delivery platforms are still required (Mullard 2016).
  • Neoantigens are the result of mutations in the somatic DNA of tumors and, as such, represent a form of personalized therapy.
  • shared tumor antigens which are selectively expressed or over-expressed in tumors in many individuals (but still may be expressed in normal cells)
  • neoantigens contain tumor-specific and/or patient-specific mutations and have the potential to uniquely mark a tumor for destruction while avoiding selftolerance.
  • neoantigens contain predicted epitopes (B cell and T cell) that are unique to each patient.
  • Neoantigens, and the neoepitopes contained therein may or may not be immunogenic when injected as a vaccine, therefore selecting the appropriate formulation for immunization is crucial for ensuring optimal immunogenicity.
  • each peptide pool is unique to each patient, the process of identifying and then formulating the neoantigens and/or neoepitopes into an appropriate vaccine formulation within a reasonable time frame is a significant consideration in respect of their ultimate use in patient therapy.
  • Each peptide pool will contain different peptides with different properties which may require optimization, particularly if the vaccine formulation is not sufficient to handle weakly immunogenic antigens.
  • the present disclosure describes vaccine compositions for enhancing immunogenicity against neoantigens, including neoantigens which are weakly immunogenic.
  • the present application describes vaccine compositions comprising one or more neoantigen(s), wherein the one or more neoantigen(s) is associated with KRAS, a pharmaceutically acceptable carrier, and optionally an adjuvant and/or T-helper epitope, and methods of using such compositions in the treatment of cancer.
  • a vaccine composition comprising: one or more neoantigen(s), wherein the one or more neoantigen(s) is associated with KRAS, a pharmaceutically acceptable carrier, and optionally an adjuvant and/or T-helper epitope.
  • the one or more neoantigen(s) is a KRAS neoantigenic peptide or a polynucleotide encoding a KRAS neoantigenic peptide.
  • the polynucleotide is a ribonucleic acid (RNA), RNA derivative, deoxyribonucleic acid (DNA), or DNA derivative.
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • the polynucleotide comprises or encodes a messenger RNA (mRNA), an antisense RNA, an interfering RNA, a catalytic RNA, or a ribozyme.
  • mRNA messenger RNA
  • antisense RNA an antisense RNA
  • interfering RNA an interfering RNA
  • catalytic RNA a catalytic RNA
  • ribozyme a messenger RNA (mRNA)
  • mRNA messenger RNA
  • antisense RNA an antisense RNA
  • interfering RNA interfering RNA
  • catalytic RNA a catalytic RNA
  • ribozyme ribozyme
  • the polynucleotide comprises an mRNA.
  • the KRAS neoantigenic peptide is 5 to 50 or 9 to 11 amino acids in length.
  • the KRAS neoantigenic peptide comprises one or more neoepitope(s).
  • the one or more neoepitope(s) comprises at least one of an MHC class I T-cell neoepitope, an MHC class II T-cell neoepitope, or a B-cell neoepitope, or a combination thereof.
  • the KRAS neoantigenic peptide comprises the amino acid sequence KLVVVGADGV (SEQ ID NO: 9), KLVVVGAVGV (SEQ ID NO: 10), YKLVVVGAV (SEQ ID NO: 12), KLVVGAVGV (SEQ ID NO: 14), or LVVVGAVGV (SEQ ID NO: 16), or a fragment or variant thereof.
  • the KRAS neoantigenic peptide is a weakly immunogenic antigen.
  • the carrier is a hydrophobic carrier.
  • the composition comprises a low dose amount of the one or more neoantigen(s), wherein the low dose amount is about 50% of the dose amount in an aqueous-based vaccine formulation.
  • the composition further comprises an amphipathic compound. [0023] In some embodiments, the composition is water-free or substantially free of water.
  • the composition comprises less than about 10%, 9%. 8%, 7%, 6%, 5%, 4%. 3%, 2%, 1%. 0.5%, 0.1%, 0.05% or 0.01% water on a weight/weight basis of the total weight of the carrier.
  • one or more of the neoantigen(s) is sufficiently miscible in the hydrophobic carrier, or is made sufficiently miscible in the hydrophobic carrier.
  • the one or more neoantigen(s) is made sufficiently miscible in the hydrophobic carrier by the presence of the amphipathic compound.
  • the amphipathic compound is closely associated with one or more the neoantigen(s) to make the one or more neoantigen(s) miscible in the hydrophobic carrier.
  • the amphipathic compound forms a complex with the one or more neoantigen(s).
  • the complex comprises a sheet or vesicular structure, wherein the sheet or vesicular structure partially or completely surrounds the one or more neoantigen(s).
  • the one or more neoantigen(s) is freeze-dried and reconstituted in the hydrophobic carrier.
  • the hydrophobic carrier is an oil or a mixture of oils.
  • the hydrophobic carrier is mineral oil or a mannide oleate in mineral oil solution.
  • the amphipathic compound is a lipid selected from a phospholipid, a mixture of phospholipids, a cholesterol, or a derivative thereof, or a combination thereof.
  • composition(s) may further comprise an ionizable aminoglycoside.
  • the ionizable aminoglycoside is one or more of chitosan, cationic alginate, cationic gelatin, cationic dextran, diethylaminoethyl (DEAE)-dextran hydrochloride, aminated cellulose, aminated sucrose, aminated trehalose, N-acetyl-D- glucosamine, D-(+)-glucosamine hydrochloride, trehalose-6,6-dibehenate (TDB) with Dimethyldioctadecylammonium (DDA), heptakis(6-deoxy-6-amino)-0-cyclodextrin heptahydrochloride, and glycyrrhizic acid ammonium salt, or derivatives thereof.
  • DDA Dimethyldioctadecylammonium
  • DDA Dimethyldioctadecylammonium
  • the ionizable aminoglycoside is chitosan.
  • the ionizable aminoglycoside is chitosan, wherein the chitosan has a molecular weight of about 60 kDa to 150 kDa, about 100 kDa to 120 kDa, or about 100 kDa.
  • the ionizable aminoglycoside is chitosan, wherein the chitosan has a degree of deacetylation (DD) of about 15- 95%. or about 25%.
  • DD degree of deacetylation
  • the ionizable aminoglycoside is chitosan, wherein the chitosan is added in a concentration of about 0.5 mg/mL to about 3 mg/mL, or about 1 mg/mL to about 2 mg/mL.
  • the composition(s) may further comprise an adjuvant, a T-helper epitope, or a combination thereof.
  • the adjuvant is a polyEC polynucleotide adjuvant, CpG oligonucleotide, a lipid-based adjuvant, a lipid A mimic or analog, or any combination thereof.
  • the T-helper epitope is PADRE (pan-DR epitope) comprising the amino acid sequence AKXVAAWTLKAAA (SEQ ID NO: 6); Tetanus toxoid peptide F21E comprising the amino acid sequence FNNFTVSFWLRVPKVSASHLE (SEQ ID NO: 7); or modified Tetanus toxin peptide A16L comprising the amino acid sequence AQYIKANSKFIGITEL (SEQ ID NO: 1).
  • PADRE pan-DR epitope
  • T-helper epitope comprising the amino acid sequence AKXVAAWTLKAAA (SEQ ID NO: 6); Tetanus toxoid peptide F21E comprising the amino acid sequence FNNFTVSFWLRVPKVSASHLE (SEQ ID NO: 7); or modified Tetanus toxin peptide A16L comprising the amino acid sequence AQYIKANSKFIGITEL (SEQ ID NO: 1).
  • composition described herein comprising:
  • a hydrophobic carrier comprising mannide oleate in mineral oil solution
  • the hydrophobic carrier comprises Montanide® ISA 51.
  • the T-helper epitope comprises the amino acid sequence AQYIKANSKFIGITEL (SEQ ID NO: 1).
  • the polyLC polynucleotide adjuvant is a single-stranded polyEC may be a sequence of repeating dldC, optionally comprising ICICICICICICICICICICIC (SEQ ID NO: 5).
  • composition described herein comprising: a) an amphipathic compound, b) the polynucleotide encoding a KRAS neoantigenic peptide, c) an ionizable aminoglycoside, and d) wherein the carrier comprises a continuous phase of a hydrophobic substance.
  • the amphipathic compound comprises one or more of a phospholipid, cholesterol or a cholesterol derivative, or a combination thereof.
  • the phospholipid is one or more of phosphoglycerol, phosphoethanolamine, phosphoserine, phosphocholine, phosphoinositol, phosphatidylcholine or lecithin.
  • the phospholipid comprises dioleoyl phosphatidylcholine (DOPC), l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), dioleoyl phosphatidylethanolamine (DOPE), l,2-dipalmitoyl-sn-glycero-3-succinate (DGS), or a combination thereof.
  • DOPC dioleoyl phosphatidylcholine
  • DPPC dioleoyl phosphatidylcholine
  • DOPE dioleoyl phosphatidylethanolamine
  • DVS dioleoyl-sn-glycero-3-succinate
  • the amphipathic compound comprises DOPC and cholesterol.
  • the ionizable aminoglycoside is one or more of chitosan, cationic alginate, cationic gelatin, cationic dextran, diethylaminoethyl (DEAE)-dextran hydrochloride, aminated cellulose, aminated sucrose, aminated trehalose, N-acetyl-D- glucosamine, D-(+)-glucosamine hydrochloride, trehalose-6,6-dibehenate (TDB) with Dimethyldioctadecylammonium (DDA), heptakis(6-deoxy-6-amino)-0-cyclodextrin heptahydrochloride, and glycyrrhizic acid ammonium salt, or derivatives thereof.
  • DDA Dimethyldioctadecylammonium
  • DDA Dimethyldioctadec
  • the ionizable aminoglycoside is chitosan.
  • the ionizable aminoglycoside is chitosan and wherein the chitosan has a molecular weight of about 60 kDa to 150 kDa, about 100 kDa to 120 kDa, or about 100 kDa.
  • the ionizable aminoglycoside is chitosan and wherein the chitosan has a degree of deacetylation (DD) of about 15- 95%, or about 25%.
  • DD degree of deacetylation
  • the ionizable aminoglycoside is chitosan and wherein the chitosan is added in a concentration of about 0.5 mg/mL to about 3 mg/mL, or about 1 mg/mL to about 2 mg/mL.
  • the polynucleotide is a ribonucleic acid (RNA), RNA derivative, deoxyribonucleic acid (DNA), or DNA derivative.
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • the polynucleotide comprises or encodes a messenger RNA (mRNA), an antisense RNA, an interfering RNA, a catalytic RNA, or a ribozyme.
  • mRNA messenger RNA
  • antisense RNA an antisense RNA
  • interfering RNA an interfering RNA
  • catalytic RNA a catalytic RNA
  • ribozyme a messenger RNA (mRNA)
  • mRNA messenger RNA
  • antisense RNA an antisense RNA
  • interfering RNA interfering RNA
  • catalytic RNA a catalytic RNA
  • ribozyme ribozyme
  • the polynucleotide comprises an mRNA.
  • the KRAS neoantigenic peptide is 5 to 50 amino acids in length.
  • the KRAS neoantigenic peptide comprises one or more neoepitope(s).
  • the one or more neoepitope(s) comprises at least one of an MHC class I T-cell neoepitope, an MHC class II T-cell neoepitope, or a B-cell neoepitope, or a combination thereof.
  • the KRAS neoantigenic peptide comprises the amino acid sequence KLVVVGADGV (SEQ ID NO: 9), KLVVVGAVGV (SEQ ID NO: 10), YKLVVVGAV (SEQ ID NO: 12), KLVVGAVGV (SEQ ID NO: 14), or LVVVGAVGV (SEQ ID NO: 16) or a fragment or variant thereof.
  • the one or more neoantigen(s) is a weakly immunogenic antigen.
  • the concentration ratio of the amphipathic compound and the polynucleotide molecule is between about 33000: 1 to about 3300: 1, between about 26400: 1 to about 6600: 1, or about 13200: 1.
  • the carrier comprises an oil or a water-in-oil emulsion.
  • composition described herein wherein e) the one or more lipids comprise DOPC and cholesterol,
  • the negatively charged molecule is a polynucleotide.
  • the carrier comprises an oil or a water-in-oil emulsion, and h) the ionizable aminoglycoside is chitosan.
  • the oil is a natural oil or a synthetic oil, optionally selected from a vegetable oil, mineral oil, a nut oil, soybean oil, peanut oil, and combinations thereof
  • the carrier is a mannide oleate in mineral oil solution.
  • the carrier is Montanide® ISA 51.
  • the composition is capable of inducing an antibody immune response and/or a cell-mediated immune response to one or more KRAS neoantigenic peptides in a subject.
  • the composition does not induce an antibody immune response and/or a cell-mediated immune response to a wild-type version of the one or more KRAS neoantigenic peptides in a subject.
  • the composition boosts a neoantigen-specific antibody immune response and/or a cell-mediated immune response in a subject that has previously been primed by an earlier immunization or other exposure to the one or more KRAS neoantigenic peptides.
  • the composition is capable of an enhanced cell-mediated immune response that is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6- fold, at least 7-fold, at least 8-fold, at least 9-fold or at least 10-fold greater than when the one or more neoantigen(s) is formulated in an aqueous-based vaccine formulation.
  • the enhanced cell-mediated immune response is provided by a single administration with the composition.
  • the enhanced cell-mediated immune response is provided by a multiple administrations with the composition.
  • the enhanced cell-mediated immune response is provided by a starting dose(s)/regimen followed by one or more maintenance dose(s)/regimen.
  • the enhanced cell-mediated immune response is provided by a low dose amount of the one or more neoantigen(s) in the composition, wherein the low dose amount is about 50% of the dose amount in the aqueous-based vaccine formulation.
  • the administering comprises only a single administration of the composition to the subject. [0081] In some embodiments, the administering comprises administering one or more starting dose(s) followed by one or more maintenance dose of the composition to the subject.
  • the administering comprises administering a single priming administration and at least one or more boosting administration(s) of the composition to the subject.
  • the administering comprises administering at least one booster administration of the composition to the subject following an earlier immunization with a different composition comprising the one or more neoantigen(s) or other exposure to the one or more neoantigen(s).
  • the composition comprises a low dose amount of the one or more neoantigen(s), wherein the low dose amount is about 50% of the dose amount in the aqueous-based vaccine formulation.
  • the method is a method for the treatment and/or prevention of cancer.
  • the method further comprising administering to the subject an agent that interferes with DNA replication and/or an immune response checkpoint inhibitor.
  • the agent that interferes with DNA replication comprises cyclophosphamide and the immune response checkpoint inhibitor comprises an inhibitor of Programmed Death-Ligand 1 (PD-L1), Programmed Death 1 (PD-1), CTLA-4, PD-L2.
  • PD-L1 Programmed Death-Ligand 1
  • PD-1 Programmed Death 1
  • CTLA-4 CTLA-4
  • kits comprising: a first container comprising an amphipathic compound and one or more neoantigen(s); and a second container comprising a hydrophobic carrier.
  • the amphipathic compound in the first container comprise 1,2- dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol.
  • DOPC 1,2- dioleoyl-sn-glycero-3-phosphocholine
  • the first container further comprises a T-helper epitope, an adjuvant, or a combination thereof.
  • the T-helper epitope is a peptide comprising the amino acid sequence AQYIKANSKFIGITEL (SEQ ID NO: 1) and the adjuvant is a polyLC polynucleotide.
  • the components of the first container are subject to lyophilization to form a dry cake.
  • the kit may be used in inducing an antibody immune response and/or a cell-mediated immune response to the one or more neoantigen(s) in a subject.
  • the composition is present in a unit dosage container.
  • the unit dosage container is a vial.
  • the vial is an amber sealed glass vial.
  • Fig. 1 shows interferon-gamma (IFN-y) ELISPOT responses of HLA-A2 transgenic mice vaccinated with KRAS mutant G12V antigen (KRAS-G12V) prepared in DPX formulation. Specifically, DPX was formulated with the KRAS mutant antigen G12, along with T-helper epitope A16L T-helper and dldC adjuvant (DPX-KRAS).
  • IFN-y interferon-gamma
  • KRAS-WT wild-type peptide
  • KRAS-G12V mutated peptide
  • Some hurdles include, for example, (i) the absence of technologies to rapidly identify' neoantigens and/or their neoepitopes, (ii) the identification of a vaccine composition that can be produced in a rapid and cost-effective manner for a production scale as small as one patient, e g. for personalized medicine, (iii) the identification of a suitable vaccine composition (e.g. delivery platform) that generates sufficiently strong and specific immune responses against a neoantigen, preferably after a single administration and with low doses of the neoantigen to avoid cross-reactivity, (iv) the ability' of the vaccine composition to effectively deliver long peptide antigens (e.g.
  • a vaccine composition that is suitable to generate an immune response (e.g. a CTL immune response) against multiple peptide neoantigens targeting multiple epitopes across abroad range of epitopes, and (vi) the ability of the vaccine composition to induce potent immune responses to neoantigens or neoepitopes that have not been selected for their proven abi 1 i ty to be strongly immunogenic and are apt to be weakly immunogenic.
  • an immune response e.g. a CTL immune response
  • the ability of the vaccine composition to induce potent immune responses to neoantigens or neoepitopes that have not been selected for their proven abi 1 i ty to be strongly immunogenic and are apt to be weakly immunogenic.
  • the importance of a vaccine composition having the ability to improve the immunogenicity of a neoantigen is significantly greater than in traditional vaccine epitope selection.
  • the KRAS neoantigen vaccine composition may be capable of inducing an effective immune response against multiple epitopes at the same time.
  • KRAS neoantigen vaccine compositions that are capable of inducing potent immune responses, even in respect of weakly immunogenic neoantigen(s). Moreover, the KRAS neoantigen vaccine compositions as disclosed herein should be compatible and amenable to cost-effective, scalable manufacturing capabilities.
  • the KRAS neoantigen compositions of the invention are capable of inducing unusually strong immune responses.
  • the present disclosure relates to a vaccine composition
  • a vaccine composition comprising: one or more neoantigen(s). wherein the one or more neoantigen(s) is associated with KRAS, a pharmaceutically acceptable carrier, and optionally an adjuvant and/or T-helper epitope.
  • KRAS neoantigen
  • a pharmaceutically acceptable carrier e.g., a pharmaceutically acceptable carrier
  • an adjuvant and/or T-helper epitope e.g., a pharmaceutically acceptable carrier
  • a vaccine composition disclosed herein may comprise one or more neoantigen(s).
  • the one or more neoantigen(s) may be associated with KRAS, a pharmaceutically acceptable carrier, and optionally an adjuvant and/or T-helper epitope.
  • a vaccine may include an immune educating composition.
  • Vaccine compositions as disclosed herein may be administered to a subject in a therapeutically effect amount.
  • a "therapeutically effective amount” means an amount of the vaccine or active ingredient (e.g., one or more neoantigens) effective to stimulate, induce, maintain, boost or enhance an immune response in a subject.
  • a therapeutically effective amount of the vaccine is an amount capable of inducing a clinical response in a subject in the treatment of a particular disease or disorder. Determination of a therapeutically effective amount of the vaccine is well within the capability of those skilled in the art, especially in light of the disclosure provided herein.
  • the therapeutically effective amount may vary according to a variety of factors such as the subject' s condition, weight, sex and age.
  • the vaccine compositions as disclosed herein comprise one or more KRAS neoantigens.
  • the KRAS neoantigenic vaccine compositions additionally include one or more neoantigens of proteins other than KRAS.
  • the term "neoantigen” refers to a class of tumor antigens which arise from tumor-specific mutations in an expressed protein.
  • the neoantigen can be derived from any cancer, tumor or cell thereof (e.g., a cancer, tumor or cell thereof comprising a tumor- induced KRAS mutation).
  • the term encompasses both a neoantigenic peptide and a polynucleotide encoding a neoantigenic peptide.
  • neoantigenic peptide refers specifically to the peptide neoantigen
  • the term “neoantigen” more broadly encompasses the polynucleotide that encodes a neoantigenic peptide. Mutations can include, for example, insertions, substitutions, and/or deletions, at one or more locations in the amino acid sequence of the expressed protein. In certain embodiments, the mutation may be produced as a result of proteolytic processing (e.g., proteasome digestion) of the protein to the neoantigenic peptide.
  • a neoantigen peptide comprising a mutation may be produced, for example, as a result of proteasome digestion of a protein described herein (e.g., a KRAS protein comprising a G12V substitution mutation).
  • a neoantigen that is isolated or obtained directly from an originating source e.g. a subject
  • a synthetic or recombinantly generated neoantigen that is identical in sequence to a neoantigen from an originating source
  • a neoantigen which is made from a neoantigen of an originating source or a fragment thereof e.g. a subject
  • the mutations in the expressed protein that create the neoantigen may be patientspecific.
  • patient-specific it is meant that the mutation(s) are unique to an individual subject. However, it is possible that more than one subject will share the same mutation(s). Thus, a “patient-specific” mutation may be shared by a small or large sub-population of subjects.
  • the size of the neoantigenic peptide may be about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22. about 23, about 24, about 25, about 26. about 27. about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120 or greater amino acid residues, and any range derivable therein.
  • the neoantigenic peptide may be greater than 25 amino acids in length. In some embodiments, the neoantigenic peptide may be 5 to 50 amino acids in length, 5 to 40 amino acids in length, 5 to 30 amino acids in length, 5 to 27 amino acids in length, 5 to 26 amino acids in length, 5 to 25 amino acids in length, 5 to 24 amino acids in length, 5 to 23 amino acids in length, 5 to 22 amino acids in length, 5 to 21 amino acids in length, 5 to 20 amino acids in length.
  • the neoantigenic peptide may be 9 to 10 or 9 to 11 amino acids in length.
  • a neoantigenic peptide described herein can comprise modifications such as, but not limited to, terminal-NH2 acylation, e.g.. by alkanoyl (C1-C20) or thioglycolyl acetylation, terminal-carboxyl amidation (e.g., ammonia, methylamine, etc.), glycosylation, side chain oxidation, biotinylation, phosphorylation, addition of a surface active material, e.g. a lipid, or can be chemically modified, e.g., acetylation, etc.
  • bonds in the peptide can be other than peptide bonds, e.g.. covalent bonds, ester or ether bonds, disulfide bonds, hydrogen bonds, ionic bonds, etc.
  • a neoantigenic peptide described herein can comprise amino acid mimetics or unnatural amino acid residues, e.g. D- or L-naphylalanine; D- or L- phenylglycine; D- or L-2-thieneylalanine; D- or L-1,-2, 3-, or 4-pyreneylalanine; D- or L-3 thienylalanine; D- or L-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- or L-(2- pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine; D-(trifluoromethyl)-phenylglycine; D-(trifluoro-methyl)-phenylalanine; D-p-fluorophenylalanine; D- or L-p-biphenyl- phenyla
  • Aromatic rings of a non-natural amino acid include, e.g., thiazolyl, thiophenyl, pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings.
  • Modified peptides that have various amino acid mimetics or unnatural amino acid residues may have increased stability in vivo. Such peptides may also have improved shelf-life or manufacturing properties.
  • the neoantigenic peptide may comprise one or more neoepitopes.
  • the term “epitope” refers to a peptide sequence which can be recognized by the immune system, specifically by antibodies, B cells or T cells.
  • a "neoepitope” (e g., atumor specific neoepitope) is an epitope of a neoantigenic peptide which comprises a tumor-specific mutation as compared to the native amino acid sequence.
  • neoepitopes may be identified by screening neoantigens for anchor residues that have the potential to bind patient HLA. The neoepitopes are normally ranked using algorithms, such as NetMHC, that can predict peptide binding to HLA.
  • T-cell neoepitope is to be understood as meaning a mutated peptide sequence which can be bound by the MHC molecules of class I or II in the form of a peptide-presenting MHC molecule or MHC complex.
  • the T-cell neoepitope should typically be one that is amenable to recognition by T cell receptors so that a cell-mediated immune response can occur.
  • T-cell epitopes presented by MHC class I molecules are ty pically peptides between 8 and 15 amino acids in length, and more often between 9 and 11 amino acids in length.
  • T-cell epitopes presented by MHC class II molecules are typically peptides between 5 and 24 amino acids in length, and more often between 13 and 17 amino acids in length. If the neoantigen is larger than these sizes, it will be processed by the immune system into fragments of a size more suitable for interaction with MHC class I or II molecules. Therefore, T-cell neoepitopes may be part of a larger peptide than those mentioned above.
  • B-cell neoepitope is to be understood as meaning a mutated peptide sequence which can be recognized by B cells and/or by 7 antibodies.
  • B-cell epitopes are typically at least five amino acids, more often at least six amino acids, still more often at least seven or eight amino acids in length, and may be continuous ("linear") or discontinuous ("conformational”); the latter being formed, for example, by the folding of a protein to bring non-contiguous parts of the primary amino acid sequence into physical proximity.
  • Linear B-cell epitopes ty pically vary from 5 to 20 amino acids in length.
  • At least one of the neoepitopes of the neoantigenic peptide is a patient-specific neoepitope.
  • patient-specific neoepitope it is meant that the mutation(s) in the neoepitope are unique to an individual subject. However, it is possible that more than one subject will share the same mutation(s). Thus, a "patient- specific neoepitope" may be shared by a small or large sub-population of subjects.
  • the KRAS neoantigenic vaccine composition comprises at least one, at least two, at least three, at least four, at least five, or any greater number of different neoantigens.
  • the KRAS neoantigenic vaccine composition comprises one, two, three, four or five different neoantigens.
  • different neoantigens it is broadly meant that the neoantigens do not share the exact same sequence.
  • the KRAS neoantigenic vaccine composition may include neoantigens from one or more different proteins (e.g., a different tumor-specific antigen).
  • neoantigenic peptides can comprise a diverse set of peptides that are unique to an individual. These peptides may have different solubility properties which would make them difficult to formulate in conventional types of vaccine formulations, such as aqueous buffer or emulsion type formulations. Additionally, there may be pre-existing tolerance to these peptides in the host from which they were derived. These aspects, among others, may cause the neoantigenic peptides to be weakly immunogenic. Therefore, it is important to deliver them in a vaccine formulation that is capable of generating a robust immune response, as disclosed herein.
  • the vaccine compositions as disclosed herein comprise neoantigens that are weakly immunogenic.
  • the neoantigens may be weakly immunogenic for a variety of reasons, such as lack of heterogeneity in their sequence; small size; insufficient foreignness for recognition by the immune system; decreased susceptibility to antigen processing and presentation, increased degradability or insolubility, and limited neoantigen processing and presentation due to their expression only by tumor cells.
  • neoantigens are more susceptible to these factors than are regular antigens.
  • weakly immunogenic it is meant that in conventional vaccines (e.g. aqueous vaccines, emulsions, etc.), the neoantigens have little or no ability' to induce, maintain and/or boost a neoantigen- specific immune response.
  • a weakly immunogenic neoantigen is one that has little or no ability to induce, maintain and/or boost a neoantigen- specific immune response after a single administration of the neoantigen.
  • a weakly immunogenic neoantigen is one that when formulated in an aqueous vaccine, is unable to sufficiently induce an immune response.
  • a weakly immunogenic neoantigen is one that when formulated in an aqueous vaccine, is unable to sufficiently induce an immune response after a single administration of the vaccine composition.
  • These embodiments are in contrast to when the same neoantigen is formulated in a comparable vaccine composition as disclosed herein (i.e. having the same components, except formulated in a hydrophobic carrier with an amphipathic compound), whereby the neoantigen is now able to sufficiently induce an immune response.
  • "sufficiently induce an immune response” means that the neoantigen is able to induce an immune response to the extent that it can provide a therapeutic effect, e.g. in the treatment of cancer.
  • a weakly immunogenic neoantigen is one that upon exposure to the subject in an aqueous vaccine, induces no immune response or induces an immune response that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20- fold, 30-fold, 40-fold or 50-fold less efficacious as compared to the immune response induced upon exposure to the subject in a vaccine composition as described herein.
  • the immune response is measured after a single administration of the neoantigen.
  • the immune response may be measured, for example, by enzyme-linked immunosorbent spot assay (ELISPOT).
  • a weakly immunogenic neoantigen is one that when administered in an aqueous vaccine is unable to provide a measurable therapeutic benefit to the subject; whereas a measurable therapeutic benefit can be achieved when the neoantigen is administered in a composition as disclosed herein.
  • the measurable therapeutic benefit may, for example, be a reduction in tumor size or an increased cancer survival prognosis.
  • the measurable therapeutic benefit is a reduction in tumor size of at least 25%, 50%, 75%, 80%, 85%, 90%, 95% or 100%.
  • the measurable therapeutic benefit may be detectable with a vaccine of the invention after only a single administration.
  • a weakly immunogenic neoantigen is one that, when administered in an aqueous vaccine at a high dose amount, is less efficacious in generating an immune response than when administered at a low dose amount in a composition of the present invention.
  • the high dose amount (as measured in mice) is at least 100 micrograms, 200 micrograms, 300 micrograms, 400 micrograms, 500 micrograms or more.
  • the low dose amount (as measured in mice) is about 10 micrograms, 20 micrograms, 30 micrograms, 40 micrograms, 50 micrograms, 60 micrograms or 75 micrograms or less. The skilled person will readily appreciate equivalent or appropriate doses in humans.
  • the low dose amount is about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%. about 40%, about 35%, about 30% or about 25% of the high dose amount.
  • the immune response is measured after a single administration of the neoantigen.
  • weakly immunogenic neoantigens may include, for example, purified and synthetic peptide neoantigens derived from cancer-associated antigens.
  • weak antigenicity is a root cause of why the immune system typically fails to control tumor growth.
  • Many cancer antigens stimulate a weak, and thus slow, immune response that provides the opportunity’ and time for tumor cells to develop immune evasion mechanisms and to ultimately gain the upper hand.
  • neoantigens may also exhibit this weak antigenicity 7 .
  • weakly immunogenic neoantigens may represent a particularly suitable type of neoantigen for use in the compositions and methods disclosed herein.
  • the vaccine compositions disclosed herein comprise a tumor- specific neoantigen that is weakly immunogenic.
  • the neoantigen may be selected from mutated somatic proteins of a cancer using selection algorithms such as NetMHC which look for motifs predicted to bind to MHC class I and/ or MHC class II proteins.
  • the neoantigen may comprise or consist of the neoantigens disclosed Rive et al., 2020, Mishto et al., 2019, Bergmann-Leitner et al. Cell Immuol, 1998, and/or Kubuschok et al. Clin Cancer Res, 2016, each incorporated by reference in their entirety for all intended purposes. .
  • the term “neoantigen” also includes a polynucleotide that encodes a neoantigenic peptide.
  • Nucleic acid-based vaccination strategies are known, wherein a vaccine composition that contains a polynucleotide is administered to a subject. The neoantigenic peptide encoded by the polynucleotide is expressed in the subject, such that the neoantigenic peptide is ultimately present in the subject, just as if the vaccine composition itself had contained the neoantigenic peptide.
  • nucleotide encompasses a chain of nucleotides of any length (e.g. 9, 12. 18, 24, 30, 60, 150. 300, 600. 1500 or more nucleotides) or number of strands (e.g. singlestranded or double-stranded).
  • Polynucleotides may be DNA (e.g. genomic DNA or cDNA) or RNA (e.g. mRNA) or combinations thereof. They may be naturally occurring or synthetic (e.g.
  • polynucleotide may contain modifications of one or more nitrogenous bases, pentose sugars or phosphate groups in the nucleotide chain. Such modifications are well-known in the art and may be for the purpose of e.g. improving stability of the polynucleotide.
  • polynucleotides encoding neoantigenic peptides described herein can be synthesized by chemical techniques, for example, the phosphotriester method of Matteucci, et al., J. Am. Chem. Soc. 103 :3185 (1981). Polynucleotides encoding neoantigenic peptides comprising or consisting of an analog can be made simply by substituting the appropriate and desired nucleic acid base(s) for those that encode the native epitope.
  • Polynucleotides encoding neoantigenic peptides described herein can also comprise a ubiquitination signal sequence, and/or a targeting sequence such as an endoplasmic reticulum (ER) signal sequence to facilitate movement of the resulting peptide into the endoplasmic reticulum.
  • a targeting sequence such as an endoplasmic reticulum (ER) signal sequence to facilitate movement of the resulting peptide into the endoplasmic reticulum.
  • ER endoplasmic reticulum
  • the polynucleotide may be delivered in various forms.
  • a naked polynucleotide may be used, either in linear form, or inserted into a plasmid, such as an expression plasmid.
  • a live vector such as a viral or bacterial vector may be used.
  • RNA messenger RNA
  • regulatory sequences relating to the transcription process e.g. a promoter
  • protein expression may be effected in the absence of a promoter.
  • suitable regulatory sequences as the circumstances require.
  • the polynucleotide is present in an expression cassette, in which it is operably linked to regulatory sequences that will permit the polynucleotide to be expressed in the subject to which the composition of the invention is administered.
  • the choice of expression cassette depends on the subject to which the composition is administered as well as the features desired for the expressed polypeptide.
  • an expression cassette typically includes a promoter that is functional in the subject and can be constitutive or inducible; a ribosome binding site; a start codon (ATG) if necessary; the polynucleotide encoding the neoantigenic peptide; a stop codon; and optionally a 3' terminal region (translation and/or transcription terminator). Additional sequences such as a region encoding a signal peptide may be included. The polynucleotide encoding the neoantigenic peptide may be homologous or heterologous to any of the other regulatory sequences in the expression cassette.
  • Sequences to be expressed together with the neoantigenic peptide are typically located adjacent to the polynucleotide encoding the protein to be expressed and placed in proper reading frame.
  • the open reading frame constituted by the polynucleotide encoding the neoantigenic peptide to be expressed solely or together with any other sequence to be expressed is placed under the control of the promoter so that transcription and translation occur in the subject to which the composition is administered.
  • the one or more neoantigens described herein is a neoantigenic peptide or a polynucleotide encoding a neoantigenic peptide associated with Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS).
  • KRAS also know n as, e.g., GTPase KRas, or V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog).
  • KRAS has been shown to modulate cell cycle progression (e.g., cell division), as well as induce apoptosis, growth arrest, and replicative senescence under in response to various environmental triggers, e.g., ultraviolet exposure, ionizing irradiation, cellular stress, and/or heat shock.
  • the KRAS gene encodes a KRAS protein which is a p21 GTPase belonging to the small GTPase superfamily. From a cell signaling standpoint, KRAS protein can function as an on/off switch transmitting extracellular signals of receptor ty rosine kinases (e.g., EGFR) and initiating the cascade of signal transduction. Activated KRAS phosphorylates and activates Raf, which in turn phosphorylates and activates MEK. which downstream can promote cellular survival.
  • the KRAS gene is a member of a class of genes known as oncogenes, which when mutated can cause normal cells to become cancerous.
  • KRAS4A see, e.g. SEQ ID NO: 18
  • KRAS4B see, e.g., SEQ ID NO: 19
  • KRAS4A is the predominant splice variant and expressed in many tissues, hence contributing to its focus in cancer studies, there is significant KRAS4A expression in some tissues (e.g., stomach, intestine, kidney and heart) and cancers (e.g., colon cancer).
  • a single amino acid substitution, or a single nucleotide substitution may be accountable for an activating mutation.
  • Abnormally active KRAS protein may direct cells to proliferate in an aberrant (e.g., uncontrolled) way.
  • the cancer is a glioma cancer.
  • the cancer is a lung cancer.
  • the cancer is ovarian cancer.
  • the cancer is a head and neck cancer.
  • the cancer is breast cancer.
  • the cancer is a prostate cancer.
  • the cancer is a colorectal cancer.
  • the cancer is a bladder cancer.
  • the cancer is a hematologic malignancy, for example, leukemia, a lymphoma, or a myeloma.
  • Non-limiting examples of leukemia are acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), and chronic myeloid leukemia (CML).
  • the lymphoma is Hodgkin's lymphoma or Non-Hodgkin‘s lymphoma.
  • the NonHodgkin's lymphoma is Diffuse Large B-cell Lymphoma (DLBCL).
  • the KRAS polynucleotide described herein is wild type or comprises a mutation (e.g., a substitution, a deletion, or an addition).
  • the KRAS polynucleotide comprises a ribonucleic acid (RNA), RNA derivative, deoxyribonucleic acid (DNA), or DNA derivative.
  • the KRAS polynucleotide comprises or encodes a messenger RNA (mRNA), an antisense RNA, an interfering RNA, a catalytic RNA. or a ribozyme.
  • mRNA messenger RNA
  • a KRAS mRNA described is wild type or comprises a mutation.
  • a KRAS polynucleotide described herein comprises one or more mutations.
  • the KRAS polynucleotide comprises one or more mutations at codons 12 or 13 in exon 1.
  • the KRAS polynucleotide comprises one or more mutations at codons 61. 63. 117, 119. or 146. In some instances, the KRAS polynucleotide comprises one or more mutations at positions corresponding to amino acid residues 12, 13, 18, 19, 20, 22, 24, 26, 36, 59, 61, 63, 64, 68, 110, 116, 117, 119, 146, 147, 158, 164, 176, or a combination thereof of the KRAS polypeptide.
  • KRAS polynucleotide comprises one or more mutations at positions corresponding to amino acid residues selected from G12V, G12D, G12C, G12A, G12S, G12F, G12R, G12L, G12T, G13C, G13D, G13V, GBR, G13H, G13A, V14G, V14I, S17G, A18D, L19F, T20M, T2OR, I21R, Q22K, I24N, I24V, N26K, H27N, D33E, P34L, P34R, T35A, I36L, I36M, T50I, D57N, T58I, A59G, A59S, A59T, A59E, G60V, Q61K, Q61H, Q61L, Q61R.
  • Non-limiting examples of amino acid mutations comprise amino acid substitutions, insertions, and/or deletions. Amino acid substitution means that an amino acid residue is substituted for a replacement amino acid residue at the same position.
  • Inserted amino acid residues may be inserted at any position and may be inserted such that some or all of the inserted amino acid residues are immediately adjacent one another or may be inserted such that none of the inserted amino acid residues is immediately adjacent to another inserted amino acid residue.
  • the KRAS neoantigenic peptide described herein is derived from a substitution mutation, e.g.. a KRAS G12V substitution mutation described herein.
  • the substitution mutation may be selected from G12V, G12D, G12C, G12A, G12S, G12F, G13C, G13D, G13V, A18D, L19F, T2OR, Q22K, I24N, N26K, I36L, I36M, A59G, A59E, Q61K, Q61H, Q61L, Q61R, E63K, Y64D, Y64N, R68S, Pl IOS, K117N, C118S, A146T, A146P, A146V, K147N, T158A, R164Q, K176Q, or a combination thereof of a w ild-type KRAS polypeptide.
  • a wild-type KRAS polypeptide described herein may comprise, for example, the amino acid sequence of any one of SEQ ID NOs: 11, 13, or 15.
  • the KRAS neoantigenic peptide is derived from one or more of a G12V, G12D, and/or a G12C mutation.
  • the neoantigen disclosed herein is derived from a substitution mutation such as, but not limited to, a KRAS G12V, G12D, and/or G12C mutation
  • the nonantigenic peptide can be a peptide comprising 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 amino acid residues of the protein, e.g., KRAS including the substitution, e.g., G12V, G12D, and/or a G12C mutation.
  • the substitution may be positioned at any position along the length of the neoantigenic peptide.
  • Neoantigenic peptides may be similarly derived from tumor specific insertion mutations where the nonantigenic peptide comprises one or more, or all of the inserted residues.
  • a KRAS neoantigenic mutation described herein comprises a G12V mutation, e.g., a KRAS G12V substitution mutation.
  • a KRAS neoantigen comprising a G12V mutation include the amino acid sequence of SEQ ID NOs: 10, 14, and 16.
  • the KRAS neoantigen comprising a G12V mutation comprises at least about 50% or more, about 60% or more, about 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.
  • a KRAS neoantigenic mutation described herein comprising a G12D mutation.
  • Non-limiting examples of a KRAS neoantigen comprising a G12V mutation include the amino acid sequences of SEQ ID NOs: 9 and 12.
  • the KRAS neoantigen comprising a G12D mutation comprises at least about 50% or more, about 60% or more, about 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% ormore, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more sequence identity with SEQ ID NO: 9 and 12.
  • neoantigenic peptides described herein may be derived from fusion polypeptides, a frame shift mutation, in-frame deletions, and splice variants.
  • the tumor specific polypeptides resulting from these mutations can be characterized by the existence of a transition point between a polypeptide sequence encoded by the germline and the tumor specific mutant polypeptide encoded by the tumor specific mutation.
  • the neoantigenic peptide can be a peptide of about 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 amino acid residues that encompasses the transition point between the germline encoded native sequence and the tumor specific polypeptide sequence.
  • the transition point can be positioned anywhere along the length of the neoantigenic peptide. For example, it can be located in the N terminal third of the neoantigenic peptide, the central third of the neoantigenic peptide or t e C terminal third of the neoantigenic peptide.
  • the transition point is located 2-5 residues away from the N terminal end or 2-5 residues away from the C terminal end.
  • a neoantigenic peptide derived from frame shift mutation and splice variants can also be a peptide consisting of tumor specific mutant residues.
  • the neoantigen may be a purified neoantigen, e.g. , from about 25% to 50% pure, from about 50% to about 75% pure, from about 75% to about 85% pure, from about 85% to about 90% pure, from about 90% to about 95% pure, from about 95% to about 98% pure, from about 98% to about 99% pure, or greater than 99% pure.
  • the amount of neoantigen used in a single treatment with a composition as described herein may vary depending on the type of neoantigen and characteristics of the subject (e.g. size, weight, age, sex, etc.).
  • an effective amount of neoantigen to use in a particular application.
  • the term "effective amount” as used herein means an amount effective, at dosages and for periods of time necessary’, to achieve the desired result.
  • the amount of neoantigen used in a single dose of a composition as described herein may be from 0.001 to 5 mg/unit dose of the composition. In certain embodiments, the amount of neoantigen will be about 0.250 mg/unit dose of the composition. In certain embodiments, the amount of neoantigen will be about 1 mg/mL of the composition. [00148] In some embodiments, the amount of neoantigen used in a single dose of a composition as described herein may be about 100 micrograms.
  • the amount of neoantigen used in a single dose of a composition as described herein may be about 50 micrograms.
  • oil-based compositions of the invention are capable of generating comparable immune responses against a neoantigenic peptide at both a low dose amount (e g. 50 micrograms) and a high dose amount (e.g. 100 micrograms), after a single administration.
  • a low dose amount e g. 50 micrograms
  • a high dose amount e.g. 100 micrograms
  • the compositions of the invention are for delivery of a low dose amount of a neoantigen.
  • the term "low dose amount” refers to a lower dose amount of the neoantigen in a composition of the invention that remains capable of providing a comparable immune response to a higher dose amount of the same neoantigen in a composition of the invention and/or in a conventional ty pe of vaccine formulation, such as an aqueous buffer or emulsion type formulation.
  • the term "low dose amount” encompasses any dose amount of the neoantigen that is less than the minimum required dose amount to generate an immune response using an aqueous-based formulation, but is sufficient to induce an immune response using a composition of the invention.
  • the low dose amount is about 10 micrograms, 20 micrograms, 30 micrograms, 40 micrograms, 50 micrograms, 60 micrograms or 75 micrograms or less, as measured in mice. In an embodiment, the low dose amount is about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30% or about 25% of a high dose amount.
  • the high dose amount may be a dose amount typically used in an aqueous-based formulation. In an embodiment, the high dose amount of the neoantigen is at least 100 micrograms, 200 micrograms, 300 micrograms, 400 micrograms, 500 micrograms or more, as measured in mice. The skilled person will readily appreciate equivalent or appropriate doses in humans based on the dosing in mice.
  • amphipathic compound is a compound having both hydrophilic and hydrophobic (lipophilic) parts or characteristics.
  • amphipathic compound may be used interchangeably with “amphiphile” or “amphiphilic”.
  • suitable amphipathic compounds may also include emulsifiers such as those described herein below.
  • emulsifiers that are encompassed herein by the term “amphipathic compound” include, without limitation, polysorbates (e.g. sorbitan monooleate), mannide oleate (ArlacelTM A), lecithin, TweenTM 80, and SpansTM 20, 80, 83 and 85.
  • the amphipathic compound can facilitate the incorporation of vaccine components with hydrophilic affinity into a hydrophobic carrier such as an oil in the absence of water.
  • the vaccine components can include, without limitation, neoantigens and/or adjuvants and/or other ingredients (e.g. T- helper epitopes) that can facilitate the production of an immune response.
  • the hydrophobic portion of an amphipathic compound is typically a large hydrocarbon moiety, such as a long chain of the form CHs(CH2)n, with n > 4.
  • the hydrophilic portion of an amphipathic compound is usually either a charged group or a polar uncharged group.
  • Charged groups include anionic and cationic groups. Examples of anionic charged groups include the following (wherein the hydrophobic part of the molecule is represented by "R"): carboxylates: RCO2 sulfates: RSO4 sulfonates: RSth and phosphates (the charged functionality in phospholipids).
  • Cationic charged groups include e.g. amines: RNH3 + ("R" again representing the hydrophobic part of the molecule).
  • Uncharged polar groups include e g. alcohols with large R groups, such as diacyl glycerol (DAG).
  • Amphipathic compounds may have several hydrophobic parts, several hydrophilic parts, or several of both. Proteins and some block copolymers are examples. Steroids, cholesterol, fatty acids, bile acids, and saponins, are also amphiphiles.
  • amphipathic compounds there are numerous amphipathic compounds which may be used, and the vaccine compositions disclosed herein may contain a single type of amphipathic compound or a mixture of different types of amphipathic compounds.
  • the amphipathic compound is a lipid.
  • lipid has its common meaning in the art in that it is any organic substance or compound that is soluble in nonpolar solvents, but generally insoluble in polar solvents (e.g. w ater).
  • Lipids are a diverse group of compounds including, without limitation, fats, waxes, sterols, fat-soluble vitamins, monoglycerides, diglycerides, triglycerides and phospholipids.
  • Lipids may be membrane- forming lipids.
  • membrane-forming lipids' it is meant that the lipids, alone or together with other lipids and/or stabilizing molecules, are capable of forming a lipid membrane.
  • the lipid membranes may form closed lipid vesicles or any other structure, such as for example lipid sheets.
  • amphiphilic lipids it is meant that the lipids possess both hydrophilic and hydrophobic (lipophilic) properties.
  • suitable lipids may include those with at least one fatty acid chain containing at least 4 carbons, and typically about 4 to 28 carbons in length.
  • the fatty acid chain may contain any number of saturated and/or unsaturated bonds.
  • the lipid may be a natural lipid or a synthetic lipid.
  • Nonlimiting examples of amphiphilic lipids may include phospholipids, sphingolipids, sphingomyelin, cerobrocides, gangliosides, ether lipids, sterols, cardiolipin, cationic lipids and lipids modified with poly (ethylene glycol) and other polymers.
  • Synthetic lipids may include, without limitation, the following fatty acid constituents: lauroyl, myristoyl, palmitoyl, stearoyl, arachidoyl, oleoyl, linoleoyl, erucoyl, or combinations of these fatty acids.
  • the amphipathic compound is a phospholipid or a mixture of phospholipids.
  • a "phospholipid” is a member of a group of lipid compounds that yield on hydrolysis phosphoric acid, an alcohol, fatty acid, and nitrogenous base.
  • Phospholipids that are preferably used in the preparation of the composition of the present disclosure are those with at least one head group selected from the group consisting of phosphoglycerol, phosphoethanolamine, phosphoserine, phosphocholine and phosphoinositol. More preferred are lipids which are about 94-100% phosphatidylcholine. Such lipids are available commercially in the lecithin Phospholipon® 90 G (Phospholipid GmBH, Germany) or lecithin S100 (Lipoid GmBH, Germany).
  • the phospholipid used in the preparation of the composition of the present disclosure is dioleoyl phosphatidylcholine (DOPC), l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), Dioleoyl Phosphatidylethanolamine (DOPE), 1 ,2-dipalmitoyl-sn-glycero-3 -succinate (DGS), or a combination thereof.
  • the phospholipid used in the preparation of the composition of the present disclosure is dioleoyl phosphatidylcholine (DOPC).
  • DOPC dioleoyl phosphatidylcholine
  • a mixture of DOPC and unesterified cholesterol may be used.
  • a mixture of Lipoid S 100 lecithin and unesterified cholesterol may be used.
  • positively charged lipids are used in the composition of the present disclosure.
  • exemplary' cationic lipids suitable for use in the compositions of the present disclosure include but are not limited to, 1.2-dioleoyl-3- trimethylammonium-propane (DOTAP), l-[2-(oleoyloxy)ethyl]-2-oleyl-3-(2- hydroxyethyl)imidazolinium chloride (DOTIM), N-[l-(2,3-dioleyloxy)propyl]-N,N,N- trimethylammonium chloride (DOTMA), dioctadecylamidoglycylspermine-4trifluoroacetic acid (DOGS), dioleyldimethylammonium chloride (DODAC), dimethyldioctadecylammonium bromide (DDAB), l,2-distearoyl-3-dimethylammonium-prop
  • DOTAP 1.2-dioleoyl
  • DODMA Dimethyldioctadecylammonium Bromide Salt
  • EPC Dimethyldioctadecylammonium Bromide Salt
  • EPC Dimethyldioctadecylammonium Bromide Salt
  • EPC l,2-dilauroyl-sn-glycero-3-ethylphosphocholine chloride salt
  • N4-Cholesteryl-Spermine HC1 Salt GL67
  • DORI N-(3-aminopropyl)-N,N-dimethyl-2,3- bis(dodecyloxy)-l-propanammonium bromide
  • GAP-DLRIE 2,3dioleyloxy-N- [2[sperminecarboxaminino]ethyl]-N,N-dimethyl-l -propanaminium trifluroacetate
  • DOSPA 2,3dioleyloxy-N- [2[sperminecarboxaminino
  • cationic lipids include those described in, for example, Audouy and Hoekstra, Mol Membr Biol, Apr-Jun 2001:18(2): 129-43; Shim et al., Asian Journal of Pharmaceutical Sciences 8(2):72-80, April 2013; and Faneca et al (2013) Cationic Liposome-Based Systems for Nucleic Acid Delivery: From the Formulation Development to Therapeutic Applications.
  • DMRIE 1,2- distearoyl-sn-glycero-3-phosphoethanolamine
  • SAINT 2 1.2-dimyristyloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide
  • DSPE 1,2- distearoyl-sn-glycero-3-phosphoethanolamine
  • a mixture of DOPC and unesterified cholesterol may be used.
  • a mixture of Lipoid S 100 lecithin and unesterified cholesterol may be used.
  • the cholesterol may be used in an amount equivalent to about 10% of the weight of phospholipid (e.g. in a DOPC cholesterol ratio of 10: 1 w/w or a S 100 lecitinxholesterol ratio of 10: 1 w/w).
  • the cholesterol is used to stabilize the formation of phospholipid vesicles. If a compound other than cholesterol is used, one skilled in the art can readily determine the amount needed.
  • the lipid vesicle particles comprise a synthetic lipid.
  • the lipid vesicle particles comprise synthetic DOPC.
  • the lipid vesicle particles comprise synthetic DOPC and cholesterol.
  • liposomes that comprise lipids which are about 94-100% phosphatidylcholine may be used.
  • lipids are available commercially in the form of lecithin Phospholipon® 90 G (Phospholipid GmBH, Germany).
  • cationic lipids such as l,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and l-[2- (oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)imidazolinium chloride (DOTIM) may be used.
  • DOTAP l,2-dioleoyl-3-trimethylammonium-propane
  • DOTIM l-[2- (oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)imidazolinium chloride
  • the amphipathic compound comprises a mixture of DOPC and cholesterol that was lyophilized and then reconstituted in mineral oil, mannide oleate in mineral oil (e.g. MontanideTM ISA 51), or MS80 oil.
  • the amphipathic compound comprises a mixture of at least one hydrophobic phase agent, DOPC and cholesterol that was lyophilized and then reconstituted in mineral oil, mannide oleate in mineral oil (e.g. MontanideTM ISA 1), or MS80 oil.
  • compositions disclosed herein may comprise about 120 milligrams of DOPC and about 12 milligrams of cholesterol.
  • Sphingomyelin contains sphingosine, an amino alcohol with a long unsaturated hydrocarbon chain. A fatty acyl side chain is linked to the amino group of sphingosine by an amide bond, to form ceramide. The hydroxyl group of sphingosine is esterified to phosphocholine. Like phosphoglycerides, sphingomyelin is amphipathic.
  • Lecithin which also may be used, is a natural mixture of phospholipids typically derived from chicken eggs or sheep's wool.
  • Phospholipids can be purchased, for example, from Avanti lipids (Alabastar, AL, USA), and lipoid LLC (Newark, NJ, USA).
  • Cholesterol and/or cholesterol derivatives may be used in the composition of the present disclosure.
  • the cholesterol is usually used in an amount equivalent to about 10% of the amount of phospholipid. If a compound other than cholesterol is used to stabilize the composition, one skilled in the art can readily determine the amount needed in the composition.
  • Cholesterol derivatives suitable for use in the present disclosure include cholesterol P-D-glucoside, cholesterol 3-sulfate sodium salt, positively charged cholesterol such as DC-cholesterol and other cholesterol like molecules such as Campesterol, Ergosterol, Betulin, Lupeol, (3-Sitosterol, a, P-Amyrin and bile acids.
  • the lipid vesicle particles comprise DOPC and cholesterol at aDOPC:Cholesterol ratio ofabout 10: 1 (w/w). In some embodiments, the lipid vesicle particles comprise DOPC and cholesterol at a DOPC cholesterol ratio of about 8: 1 (w/w), about 9:1 (w/w), about 11 : 1 (w/w), or about 12: 1 (w/w).
  • the compositions disclosed herein comprise about 66 mg/ml of DOPC and cholesterol. In other embodiments, the compositions disclosed herein comprise about 55 mg/ml, 56 mg/ml, 57 mg/ml, 58 mg/ml, 59 mg/ml, 60 mg/ml, 61 mg/ml, 62 mg/ml, 63 mg/ml, 64 mg/ml, 65 mg/ml, 67 mg/ml, 68 mg/ml, 69 mg/ml, 70 mg/ml, 71 mg/ml. 72 mg/ml, 73 mg/ml, 74 mg/ml, or 75 mg/ml of DOPC and cholesterol.
  • compositions disclosed herein comprise about 60 mg/ml of DOPC and about 6 mg/ml of cholesterol.
  • the amphipathic compound may be substantially evenly dispersed in the hydrophobic carrier, whereby the presence of the amphipathic compound alone is sufficient to facilitate the incorporation of vaccine components with hydrophilic affinity (e.g. a neoantigen) into a hydrophobic carrier.
  • hydrophilic affinity e.g. a neoantigen
  • the amphipathic compound may be closely associated with the neoantigen so as to make the neoantigen miscible in the hydrophobic carrier.
  • close associated it is meant that the amphipathic compound is in such proximity with the neoantigen that the neoantigen is presented in a form that it is miscible in the hydrophobic carrier.
  • the close association may or may not involve physical interaction between the neoantigen and the amphiphile.
  • the hydrophilic part of the amphipathic compound is oriented towards the hydrophilic moieties on the neoantigen.
  • the amphipathic compounds may remain substantially separate from one another or they may form various different types of structures, assemblies or arrays.
  • Exemplary embodiments of the types of structures, assemblies or arrays that the amphipathic compounds may form include, without limitation: single layer sheets, bilayer sheets, multilayer sheets, single layer vesicular structures (e.g. micelles), bilayer vesicular structures (e.g. unilamellar or multilamellar vesicles), or various combinations thereof.
  • single layer it is meant that the amphipathic compounds do not form a bilayer, but rather remain in a layer with the hydrophobic part oriented on one side and the hydrophilic part oriented on the opposition side.
  • bilayer it is meant that the amphipathic compounds form a two-layered sheet, typically with the hydrophobic part of each layer internally oriented toward the center of the bilayer with the hydrophilic part externally oriented.
  • multilayer is meant to encompass any combination of single and bilayer structures. The form adopted may depend upon the specific neoantigen, the specific amphipathic compound, and/or the specific hydrophobic carrier that is used.
  • the structure, assembly or array formed by the amphipathic compound may partially or completely surround the neoantigen.
  • the amphipathic compound may form a closed vesicular structure around the neoantigen.
  • the vesicular structure is a single layer vesicular structure.
  • An example of such a structure is a micelle.
  • a typical micelle in aqueous solution forms an aggregate with the hydrophilic parts in contact with the surrounding aqueous solution, sequestering the hydrophobic parts in the micelle center.
  • an inverse/reverse micelle forms with the hydrophobic parts in contact with the surrounding aqueous solution, sequestering the hydrophilic parts in the micelle center.
  • a spherical reverse micelle can package a neoantigen with hydrophilic affinity within its core.
  • the vesicular structure is a micelle or an inverse/reverse micelle.
  • the size of the micelles or inverse/reverse micelles range from 2 nm (20 A) to 20 nm (200 A) in diameter.
  • the size of the micelles or inverse/reverse micelles is about 2 nm. 3 nm, 4 nm, 5 nm, 6 nm. about 7 nm, about 8 nm, about 9 nm, or about 10 nm in diameter.
  • the size of the micelles or inverse/reverse micelles is about 10 nm in diameter.
  • the vesicular structure is a bilayer vesicular structure, such as for example, a liposome.
  • Liposomes are completely closed lipid bilayer membranes containing an entrapped aqueous volume. Liposomes may be unilamellar vesicles (possessing a single bilayer membrane) or multilamellar vesicles characterized by multimembrane bilayers, each bilayer may or may not be separated from the next by an aqueous layer.
  • a general discussion of liposomes can be found in Gregoriadis 1990; and Frezard 1999.
  • liposomes is intended to encompass all such vesicular structures as described above, including, without limitation, those described in the art as “niosomes”, “transfersomes” and “virosomes”
  • Any liposomes may be used in this invention, including liposomes made from archaebacterial lipids.
  • Any amphipathic lipid with at least one fatty acid chain containing at least 4 carbons, typically about 4 to 28 carbons in length may be used.
  • the fatty acid chain may contain any number of saturated and/or unsaturated bonds.
  • Amphipathic lipids contemplated may be phospholipids, sphingolipids, sphingomyelin, cerobrocides, gangliosides.
  • Particularly useful liposomes use phospholipids and unesterified cholesterol in the liposome formulation.
  • the cholesterol is used to stabilize the liposomes and any other compound that stabilizes liposomes may replace the cholesterol.
  • Other liposome stabilizing compounds are known to those skilled in the art. For example, saturated phospholipids produce liposomes with higher transition temperatures indicating increased stability.
  • Liposome compositions may be obtained, for example, by using natural lipids, synthetic lipids, sphingolipids, ether lipids, sterols, cardiolipin, cationic lipids and lipids modified with poly (ethylene glycol) and other polymers.
  • Synthetic lipids may include the following fatty acid constituents: lauroyl, myristoyl, palmitoyl, stearoyl, arachidoyl, oleoyl, linoleoyl, erucoyl, or combinations of these fatty acids.
  • Liposomes can adsorb to virtually any type of cell and then release an incorporated agent (e.g. neoantigen).
  • the liposome can fuse with the target cell, whereby the contents of the liposome empty into the target cell.
  • a liposome may be endocytosed by cells that are phagocytic.
  • Liposomes have been used in the preparation of compositions comprising a hydrophobic carrier as a vesicle to encapsulate antigens as well as an emulsifier to stabilize the formulation (see e.g. W02002/038175, W02007/041832, W02009/039628, WO2009/146523 and WO2013/049941).
  • Hydrophilic antigens are typically entrapped in the aqueous interior, while hydrophobic antigens can be intercalated in the lipid bilayer or dispersed in the oil phase.
  • pre-manufactured liposomes may be used in the vaccine compositions disclosed herein.
  • one or more of the components of the composition may be encapsulated in, or mixed or suspended with, liposomes in an aqueous phase; lyophilized; and then reconstituted in the hydrophobic carrier.
  • the liposomes may reorganize to form alternate structures in the hydrophobic carrier.
  • bilayer and mutilayer vesicular structures include, without limitation: niosomes, transfersomes, virosomes, multilamellar vesicles (MLV), oligolamellar vesicles (OLV), unilamellar vesicles (UV), small unilamellar vesicles (SUV), medium-sized unilamellar vesicles (MUV), large unilamellar vesicles (LUV), giant unilamellar vesicles (GUV), multivesicular vesicles (MW), single or oligolamellar vesicles made by reverse-phase evaporation method (REV), multilamellar vesicles made by the reverse-phase evaporation method (MLV-REV), stable plurilamellar vesicles (SPLV), frozen and thawed MLV (FATMLV), vesicles prepared by extrusion methods
  • MLV multilam
  • compositions disclosed herein comprise a hydrophobic carrier, preferably a liquid hydrophobic substance.
  • a hydrophobic carrier preferably a liquid hydrophobic substance.
  • These compositions may be referred to herein interchangeably as an "oil-based formulation", an "oil-based vaccine”, an “oil-based depot vaccine”, an “oil- based depot forming vaccine”, a “hydrophobic vaccine”, a “hydrophobic composition” or a “hydrophobic vaccine composition”.
  • the hydrophobic carrier may be an essentially pure hydrophobic substance or a mixture of hydrophobic substances.
  • Hydrophobic substances that are useful in the compositions described herein are those that are pharmaceutically and/or immunologically acceptable.
  • the carrier is typically a liquid but certain hydrophobic substances that are not liquids at atmospheric temperature may be liquefied, for example by warming, and may also be useful.
  • Oil or a mixture of oils is a particularly suitable carrier for use in the compositions disclosed herein.
  • Oils should be pharmaceutically and/or immunologically acceptable.
  • Suitable oils include, for example, mineral oils (especially light or low viscosity’ mineral oil such as Drakeol® 6VR), vegetable oils (e.g. , soybean oil), nut oils (e.g., peanut oil), or mixtures thereof.
  • the hydrophobic carrier is a hydrophobic substance such as vegetable oil, nut oil or mineral oil. Animal fats and artificial hydrophobic polymeric materials, particularly those that are liquid at atmospheric temperature or that can be liquefied relatively easily, may also be used.
  • the hydrophobic carrier may be, or comprise, Incomplete Freund' s Adjuvant (IFA), a mineral oil-based model hydrophobic carrier.
  • IFA Incomplete Freund' s Adjuvant
  • the hydrophobic carrier may be, or comprise, a mannide oleate in mineral oil solution, such as that commercially available as Montanide® ISA 51 (SEPPIC, France). While these carriers are commonly used to prepare water-in-oil emulsions, the present disclosure avoids this ty pe of formulation by use of an amphipathic compound to suspend the components in the absence of substantial quantities of water, as described herein.
  • Immunovaccine Inc. has developed vaccine delivery platforms referred to as VacciMax® and DepoVaxTM (DPX) (see e.g. US Patent Nos. 6,793,923 and 7,824,686; W02002/038175; W02007/041832; W02009/039628; W02009/043165 and
  • DPX is a lipid-in-oil formulation that can be formulated with any antigen, or mixture of antigens. Unlike water-in-oil emulsion-based vaccines, which rely on oil entrapping water droplets containing antigen and adjuvant, DPX based formulations rely on lipids to facilitate the incorporation of antigens and adjuvants directly into the oil, without the need for emulsification.
  • the vaccine compositions disclosed herein may comprise Immunovaccine Inc.'s delivery platform DPX.
  • compositions disclosed herein may further comprise one or more additional components as are known in the art (see e.g. Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985; and The United States Pharmacopoeia: The National Formulary (USP 24 NF19) published in 1999).
  • the vaccine compositions may additionally comprise an adjuvant, a T-helper epitope, an emulsifier and/or an excipient.
  • the vaccine compositions disclosed herein may comprise one or more adjuvants.
  • adjuvants include, without limitation, alum, other compounds of aluminum, Bacillus of Calmette and Guerin (BCG), TiterMaxTM, RibiTM.
  • BCG Bacillus of Calmette and Guerin
  • TiterMaxTM TiterMaxTM
  • RibiTM RibiTM.
  • FCA Freund's Complete Adjuvant
  • CpG ODN CpG-containing oligodeoxynucleotides
  • lipid A mimics or analogs lipopeptides and polyFC polynucleotides.
  • An exemplary CpG ODN is 5 -TCCATGACGTTCCTGACGTT-3' (SEQ ID NO: 3).
  • the skilled person can readily select other appropriate CpG ODNs on the basis of the target species and efficacy.
  • An exemplary lipopeptide includes, without limitation, Pam3Cys-SKKKK (EMC MicrocoUections, Germany; SEQ ID NO: 4) or variants, homologs and analogs thereof.
  • the Pam2 family of lipopeptides has been shown to be an effective alternative to the Pam3 family of lipopeptides.
  • the pharmaceutical or vaccine compositions may comprise a polyI:C polynucleotide as an adjuvant.
  • PolykC polynucleotides are polynucleotide molecules (either RNA or DNA or a combination of DNA and RNA) containing inosinic acid residues (I) and cytidylic acid residues (C), and which induce the production of inflammatory cytokines, such as interferon.
  • the polyPC polynucleotide is double-stranded.
  • they are ty pically composed of one strand consisting entirely of cytosine-containing nucleotides and one strand consisting entirely of inosine-containing nucleotides, although other configurations are possible.
  • each strand may contain both cytosine- containing and inosine- containing nucleotides.
  • either or both strand may additionally contain one or more non-cytosine or non-inosine nucleotides.
  • the polyI:C polynucleotide may be a single- stranded molecule containing inosinic acid residues (I) and cytidylic acid residues (C).
  • the single- stranded polyLC may be a sequence of repeating dldC.
  • the sequence of the single- stranded polyLC may be a 26-mer sequence of (IC)13, i.e. ICICICICICICICICICICIC (SEQ ID NO: 5).
  • ICICICICICICICICICICICICIC SEQ ID NO: 5
  • polyl C can be segmented every 16 residues without an effect on its interferon activating potential (Bobst 1981). Furthermore, the interferon inducing potential of a polyl: C molecule mismatched by introducing a uridine residue every 12 repeating cytidylic acid residues (Hendrix 1993), suggests that a minimal double stranded polyl: C molecule of 12 residues is sufficient to promote interferon production. Others have also suggested that regions as small as 6-12 residues, which correspond to 0.5-1 helical turn of the double stranded polynucleotide, are capable of triggering the induction process (Greene 1978).
  • polyl C polynucleotides are ty pically about 20 or more residues in length (commonly 22, 24, 26, 28 or 30 residues in length). If semi-synthetically made (e.g. using an enzyme), the length of the strand may be 500. 1000 or more residues.
  • PolyLC acts as a mimic of viral genomes and is particularly useful for modulating the immune system in vivo.
  • Synthetic poly I/poly C homopolymers for example have been reported to enhance innate immunity by inducing interferon gamma non- specifically when delivered systemically in vivo by intravenous or intramuscular injection (Krown 1985, Zhu 2007).
  • poly inosinic and cytidylic acid polymers have been described over the y ears (de Clercq 1978, Bobst 1981, de Clercq 1975, Guschlbauer 1977, Fukui 1977, Johnston 1975, US Patent No.
  • polyLC is also useful as an adjuvant in vaccine compositions.
  • the enhancement of innate immunity can lead to an enhanced antigen specific adaptive immunity', possibly through a mechanism that involves, at least in part.
  • NK cells, macrophages and/or dendritic cells Chorigos 1985, Salem 2006, Alexopoulou 2001, Trumpfheller 2008.
  • Evidence for the use of polyLC molecules in this context originates from various vaccine studies for controlling infectious diseases (Houston 1976, Stephen 1977, Ichinohe 2007, Sloat 2008, Agger 2006, Padalko 2004) and the prevention or treatment of cancer by a variety of vaccine modalities (Zhu 2007.
  • polyLC enhances humoral responses as evident from enhanced antibody responses against specific infectious disease antigens.
  • PolyLC is also a potentiator of antigen-specific cellular responses (Zhu 2007, Zaks 2006, Cui 2006, Riedl 2008).
  • TLR toll like receptors
  • TLR3 toll like receptors
  • polyLC molecules may exert their effect, at least in part, by interacting with receptors other than TLRs, such as the RNA helicase retinoic acid induced protein I (RIG-I)/melanoma differentiation associated gene 5 (MDA5) (Alexopoulou 2001, Yoneyama 2004, Gowen 2007, Dong 2008).
  • receptors other than TLRs such as the RNA helicase retinoic acid induced protein I (RIG-I)/melanoma differentiation associated gene 5 (MDA5) (Alexopoulou 2001, Yoneyama 2004, Gowen 2007, Dong 2008).
  • RIG-I RNA helicase retinoic acid induced protein I
  • MDA5 melanoma differentiation associated gene 5
  • a "polyLC”, “polyLC polynucleotide” or “polyLC polynucleotide adjuvant” is a double- or single- stranded polynucleotide molecule (RNA or DNA or a combination of DNA and RNA), each strand of which contains at least 6 contiguous inosinic or cytidylic acid residues, or 6 contiguous residues selected from inosinic acid and cytidylic acid in any order (e.g.
  • PolyLC polynucleotides will typically have a length of about 8, 10, 12, 14, 16, 18, 20, 22, 24, 25, 26, 28, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300. 500, 1000 or more residues.
  • Preferred polyEC polynucleotides may have a minimum length of about 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30 nucleotides and a maximum length of about 1000, 500, 300, 200, 100, 90, 80, 70, 60, 50, 45 or 40 nucleotides.
  • Each strand of a double- stranded polyEC polynucleotide may be a homopolymer of inosinic or cytidylic acid residues, or each strand may be a heteropolymer containing both inosinic and cytidylic acid residues.
  • the polymer may be interrupted by one or more non-inosinic or non-cytidylic acid residues (e.g. uridine), provided there is at least one contiguous region of 6 1, 6 C or 6 VC residues as described above.
  • each strand of a polyEC polynucleotide will contain no more than 1 non- I/C residue per 6 VC residues, more preferably, no more than 1 non-I/C residue per every 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 or 30 VC residues.
  • the inosinic acid or cytidylic acid (or other) residues in the polyEC polynucleotide may be derivatized or modified as is known in the art, provided the ability of the polyEC polynucleotide to promote the production of an inflammatory’ cytokine, such as interferon, is retained.
  • Non-limiting examples of derivatives or modifications include e.g. azido modifications, fluoro modifications, or the use of thioester (or similar) linkages instead of natural phosphodiester linkages to enhance stability 7 in vivo.
  • the polyEC polynucleotide may also be modified to e.g. enhance its resistance to degradation in vivo by e.g. complexing the molecule with positively charged poly -lysine and carboxymethylcellulose, or with a positively charged synthetic peptide.
  • the polyEC polynucleotide adjuvant is a traditional form of polyEC with an approximate molecular weight of 989,486 Daltons, containing a mixture of varying strand lengths of polyl and polyC of several hundred base pairs (Thermo Scientific; USA).
  • the vaccine compositions as disclosed herein may comprise an adjuvant that activates or increases the activity of TLR2.
  • an adjuvant which "activates” or “increases the activity” of a TLR2 includes any adjuvant, in some embodiments a lipid-based adjuvant, which acts as a TLR2 agonist.
  • activating or increasing the activity’ of TLR2 encompasses its activation in any monomeric, homodimeric or heterodimeric form, and particularly includes the activation of TLR2 as a heterodimer with TLR1 or TLR6 (i.e. TLR1/2 or TLR2/6).
  • an adjuvant that activates or increases the activity of TLR2 include lipid-based adjuvants, such as those described in WO2013/049941.
  • the vaccine composition as disclosed herein may comprise a lipid-based adjuvant, such as disclosed for example in WO2013/049941.
  • the lipid-based adjuvant is PAM2Cys-Ser-(Lys)4 (SEQ ID NO: 2) or PAMsCys- Ser-(Lys)4 (SEQ ID NO: 4).
  • the vaccine composition as disclosed herein may comprise a lipid A mimic or analog adjuvant, such as for example those disclosed in International Patent Application No. PCT/CA2015/051309 and the references cited therein.
  • the adjuvant may be JL-265 or JL-266 as disclosed in PCT/CA2015/051309.
  • adjuvants include, without limitation, chemokines, colony stimulating factors, cytokines, 1018 ISS, aluminum salts, Amplivax, AS04, AS 15. ABM2, Adjumer, Algammulin, AS01B, AS02 (SBASA), AS02A.
  • BCG CalcitrioL Chitosan, Cholera toxin, CP-870,893, CpG, polyEC, CyaA, DETOX (Ribi Immunochemicals), Dimethyldioctadecylammonium bromide (DDA), Dibutyl phthalate (DBP), dSLIM, Gamma inulin, GM-CSF, GMDP, Glycerol, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch.
  • compositions herein may comprise one or more pharmaceutically acceptable adjuvants.
  • at least one of the neoantigens may be coupled to at least one of the adjuvants.
  • compositions herein may comprise a polyEC polynucleotide adjuvant, a lipid-based adjuvant, a lipid A mimic or analog, or any combination thereof.
  • compositions may comprise a combination of a polyEC polynucleotide adjuvant and a lipid-based adjuvant, such as described in the adjuvanting system disclosed in United States Provisional Patent Application No. 62/256,875 filed on November 18, 2015.
  • the amount of adjuvant used depends on the type and amount of neoantigen and on the ty pe of adjuvant. One skilled in the art can readily determine the amount of adjuvant needed in a particular application by empirical testing. T-helper epitopes
  • compositions disclosed herein may also comprise at least one T-helper epitope or T-helper antigen.
  • T-helper epitopes are a sequence of amino acids (natural or non-natural amino acids) that have T-helper activity'. T-helper epitopes are recognized by T-helper lymphocytes, which play an important role in establishing and maximizing the capabilities of the immune system, and are involved in activating and directing other immune cells, such as for example cytotoxic T lymphocytes.
  • a T-helper epitope can consist of a continuous or discontinuous epitope. Hence not every amino acid of a T-helper is necessarily part of the epitope. Accordingly, T-helper epitopes, including analogs and segments of T-helper epitopes, are capable of enhancing or stimulating an immune response. Immunodominant T-helper epitopes are broadly reactive in animal and human populations with widely divergent MHC types (Celis 1988, Demotz 1989, Chong 1992). The T-helper domain of the subject peptides may have from about 10 to about 50 amino acids, and more particularly about 10 to about 30 amino acids. When multiple T- helper epitopes are present, then each T-helper epitope acts independently.
  • the T-helper epitope may form part of a neoantigen described herein.
  • the neoantigen may contain an epitope that functions as a T-helper epitope.
  • the T-helper epitope is a separate molecule from the neoantigen.
  • the T-helper epitope may be fused to the neoantigen.
  • T-helper epitope analogs may include substitutions, deletions and insertions of from one to about 10 amino acid residues in the T-helper epitope.
  • T-helper segments are contiguous portions of a T-helper epitope that are sufficient to enhance or stimulate an immune response.
  • An example of T-helper segments is a series of overlapping peptides that are derived from a single longer peptide.
  • compositions as disclosed herein may comprise as a T-helper epitope or antigen, the modified Tetanus toxin peptide A16L (830 to 844; AQYIKANSKFIGITEL (SEQ ID NO: 1), with an alanine residue added to its amino terminus to enhance stability (Slingluff 2001).
  • T-helper epitopes which may be used in the present compositions include, for example, hepatitis B surface antigen helper T cell epitopes, pertussis toxin helper T cell epitopes, measles virus F protein helper T cell epitope. Chlamydia trachomitis major outer membrane protein helper T cell epitope, diphtheria toxin helper T cell epitopes, Plasmodium falciparum circumsporozoite helper T cell epitopes, Schistosoma mansoni triose phosphate isomerase helper T cell epitopes. Escherichia coli TraT helper T cell epitopes and immune-enhancing analogs and segments of any of these T-helper epitopes.
  • the T-helper epitope may be a universal T-helper epitope.
  • a universal T-helper epitope as used herein refers to a peptide or other immunogenic molecule, or a fragment thereof, that binds to a multiplicity of MHC class II molecules in a manner that activates T cell function in a class II (CD4+ T cells)-restricted manner.
  • An example of a universal T-helper epitope is PADRE (pan-DR epitope) comprising the peptide sequence AKXVAAWTLKAAA (SEQ ID NO: 6), wherein X may be cyclohexylalanyl.
  • PADRE specifically has a CD4+ T-helper epitope, that is, it stimulates induction of a PADRE-specific CD4+ T-helper response.
  • Tetanus toxoid has other T-helper epitopes that work in the similar manner as PADRE. Tetanus and diphtheria toxins have universal epitopes for human CD4+ cells (Diethelm-Okita 2000).
  • the T-helper epitope may be a tetanus toxoid peptide such as F21E comprising the peptide sequence FNNFTVSFWLRVPKVS ASHLE (amino acids 947- 967; SEQ ID NO: 7).
  • the T-helper epitope is fused to at least one of the one or more neoantigens in the composition as disclosed herein (e.g. a fusion peptide).
  • the vaccine compositions disclosed herein may comprise one or more emulsifiers.
  • the emulsifier may be a pure emulsifying agent or a mixture of emulsifying agents.
  • the emulsifier(s) should be pharmaceutically and/or immunologically acceptable.
  • an emulsifier may be of particular relevance to preparing compositions that are water-free or substantially free of water.
  • an emulsifier may be used to assist in stabilizing the amphipathic compound, mixture of amphipathic compound and neoantigen, or the mixture of amphipathic compound, neoantigen and other vaccine components (e.g. polyEC and/or lipid-based adjuvant, T-helper epitope, etc.) when the amphipathic compound or mixtures are resuspended into the hydrophobic carrier.
  • the use of an emulsifier may, for example, promote more even distribution of the amphipathic compound or mixture in the hydrophobic carrier.
  • the emulsifier may be amphipathic and therefore, the emulsifier may include a broad range of compounds.
  • the emulsifier may be a surfactant, such as for example, a non-ionic surfactant.
  • emulsifiers which may be used include polysorbates, which are oily liquids derived from polyethylene glycolyated sorbital, and sorbitan esters. Polysorbates may include, for example, sorbitan monooleate.
  • Typical emulsifiers are well-known in the art and include, without limitation, mannide oleate (ArlacelTM A), lecithin, TweenTM 80, SpansTM 20, 80, 83 and 85.
  • the emulsifier for use in the vaccine compositions is mannide oleate.
  • the emulsifier is generally pre-mixed with the hydrophobic carrier.
  • a hydrophobic carrier which already contains an emulsifier may be used.
  • a hydrophobic carrier such MontanideTM ISA 51 already contains the emulsifier mannide oleate.
  • the hydrophobic carrier may be mixed with emulsifier before combining with the amphipathic compound, mixture of amphipathic compound and neoantigen, or the mixture of amphipathic compound, neoantigen and other vaccine components (e.g. polyI:C and/or lipid-based adjuvant, T-helper epitope, etc.).
  • the emulsifier is used in an amount effective to promote even distribution of the amphipathic compound in the hydrophobic carrier and/or to assist in the formation of structures, assemblies or arrays described herein.
  • the volume ratio (v/v) of hydrophobic carrier to emulsifier is in the range of about 5: 1 to about 15: 1, more particularly 10: 1.
  • an “emulsion” refers to a mixture of two or more liquids that are normally immiscible wherein droplets of one liquid are dispersed in the other.
  • ahydrophobic substance e.g., oil
  • an aqueous substance e g. water
  • W/O water-in-oil
  • Water-in- oil emulsion refers to an emulsion of a hydrophobic phase in an aqueous phase.
  • a dispersion of oil droplets in water is an oil-in-water (O/W) emulsion in which the oil (hydrophobic phase) forms a discontinuous phase and the water (aqueous phase) forms a continuous phase.
  • Oil-in-water emulsion or “O/W”, as used herein, refers to an emulsion of a hydrophobic phase in an aqueous phase.
  • a phase or substance that is hydrophobic may also be called lipophilic.
  • a phase or substance that is aqueous may also be called hydrophilic or lipophobic.
  • ionizable aminoglycoside refers to any polyamino sugar having the ability to bind with a negatively charged molecule (e.g., polynucleotide) through electrostatic interactions.
  • the polyamino sugar may be naturally occurring, semi-synthetic, or fully synthetic. Through interaction with the negatively charged molecule such as a polynucleotide the ionizable aminoglycoside may protect the polynucleotide from nuclease attack.
  • composition of the present disclosure contains an ionizable aminoglycoside.
  • Ionizable aminoglycosides useful in the compositions of the present disclosure include, but are not limited to, chitosan, cationic alginate, cationic gelatin, cationic dextran, DEAE-dextran hydrochloride, aminated cellulose, aminated sucrose, aminated trehalose, N-acetyl-D-glucosamine, D-(+)-glucosamine hydrochloride, glycyrrhizic acid ammonium salt, or derivatives thereof.
  • the ionizable aminogly coside is a polymer.
  • the ionizable aminoglycoside is chitosan, chitosan derivative, or a chitosan like molecule.
  • Chitosan derivatives suitable for use in the present disclosure include, but are not limited to N- trimethyl chitosan, mono-N-carboxymethyl chitosan (MCC), N-palmitoyl chitosan (NPCS), EDTA-chitosan, low molecular weight chitosan, galactosylated chitosan, N-dodecylated chitosan, thiolated chitosan or combinations thereof.
  • the chitosan like molecules include, but are not limited to, N-acetyl-D-glucosamine, D-(+)-glucosamine hydrochloride, galactosamine, N-acetylgalactosamine, cellulose acetate, mannosamine, N-acetylneuraminic acid, alginic acid, Trehalose-6,6-dibehenate (TDB) with Dimethyldioctadecylammonium bromide (DDA), heptakis(6-deoxy-6-amino)-(3-cyclodextrin heptahydrochloride, DEAE-dextran hydrochloride, and glycyrrhizic acid ammonium salt. It is contemplated that the chitosan, chitosan derivative, or a chitosan like molecule may also function as a carrier (e.g., a structural carrier).
  • a carrier e.g.,
  • the chitosan or chitosan derivative used in the composition has a molecular weight of about 10 kDa to 200 kDa. In some embodiments, the chitosan or chitosan derivative used in the composition has a molecular weight of about 60 kDa to 150 kDa, about 80 kDa to 150 kDa, about 90 kDa to 110 kDa, about 100 kDa to 120 kDa, about 100 kDa, or about 120 kDa.
  • the chitosan or chitosan derivative used in the composition has a molecular weight of about 80 kDa, about 90 kDa, about 100 kDa, about 110 kDa, about 120 kDa, about 130 kDa, about 140 kDa, or about 150 kDa. In one embodiment, the chitosan or chitosan derivative used in the composition has a molecular weight of about 100 kDa.
  • Degree of deacetylation is another main parameter characterizing chitosan.
  • the degree of deacetylation (DDA, %) is defined as the molar fraction of D-glusoamine units in the copolymers (chitosan) composed of N-acetylglucosamine units and D-glusoamine units (Shigemasa Y et al., Int. J. Biol. Macromol. 1996;18:237-242, which is incorporated hereby by reference in its entirety).
  • the chitosan or chitosan derivative used in the composition has a degree of deacetylation (DDA) of about 15% to 95%.
  • the chitosan or chitosan derivative used in the composition has a degree of deacetylation (DDA) of about 15% to 30%, about 20% to 30%, about 20% to 40%, about 25% to 50%, about 30% to 60%, about 40% to 70%, about 50% to 80%, about 60% to 90%, about 70% to 95%.
  • the chitosan or chitosan derivative used in the composition has a degree of deacetylation (DDA) of about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%. 70%, 75%, 80%, 85%, 90%, or 95%.
  • the chitosan or chitosan derivative has a DDA% of about 75%, of about 90%.
  • the chitosan or chitosan derivative is added in a concentration of about 0.1 mg/mL to about 5 mg/mL. In some embodiments, the chitosan or chitosan derivative is added in a concentration of about 0.25 mg/mL to about 4 mg/mL, about 0.5 mg/mL to about 3 mg/mL, about 0.75 mg/mL to about 2.5 mg/mL, about 1 mg/mL to about 2 mg/mL. In some embodiments, the chitosan or chitosan derivative is added in a concentration of about 0.5 mg/mL to about 3 mg/mL or about 1 mg/mL to about 2 mg/mL.
  • the chitosan or chitosan derivative is added in a concentration of about 0.1 mg/ml, about 0.25 mg/ml, about 0.5 mg/ml, about 0.75 mg/ml, about 1 mg/mL, about 1.25 mg/ml, about 1.5 mg/mL, about 1.75 mg/ml, about 2 mg/mL, about 2.25 mg/ml, about 2.5 mg/ml, about 2.75 mg/ml, about 3 mg/ml, about 3.25 mg/ml. about 3.5 mg/ml, about 3.75 mg/ml. about 4 mg/ml. about 4.25 mg/ml. about 4.5 mg/ml. about 4.75 mg/ml, about 5 mg/ml.
  • the chitosan or chitosan derivative is added in a concentration of about 0.1 mg/mL, about 0.5 mg/mL, about 1.0 mg/mL, about 1.5 mg/mL, about 2 mg/mL, about 2.5 mg/mL, or about 3 mg/mL.
  • the delivery platform includes cell-penetrating peptides, nanoparticulate encapsulation, virus like particles, liposomes, or any combination thereof.
  • the cell-penetrating peptide is TAT peptide, CADY peptide, penetratin, herpes simplex virus VP22, transportan, Antp, or any combination thereof.
  • the vaccine compositions disclosed herein are water-free or substantially free of water, i.e. the vaccine compositions are not emulsions.
  • compositions contain no water at all.
  • compositions may be substantially free of water.
  • substantially free of water is intended to encompass embodiments where the hydrophobic earner may still contain small quantities of water, provided that the water is present in the non-continuous phase of the carrier.
  • individual components of the composition may have small quantities of bound water that may not be completely removed by processes such as lyophilization or evaporation and certain hydrophobic carriers may contain small amounts of water dissolved therein.
  • compositions as disclosed herein that are "substantially free of water” contain, for example, less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% or 0.01% water on a weight/weight basis of the total weight of the carrier component of the composition.
  • the compositions that still contain small quantities of water do not contain a sufficient amount of water such that an emulsion would be formed.
  • water-free vaccine compositions as disclosed herein may be capable of generating significantly higher antibody titers and more potent cell-mediated immune responses with lower doses of one or more of the components, e.g. neoantigen, adjuvant(s), T-helper epitope, etc. This is based on the unique mechanism of action of DPX in forcing active uptake of the vaccine components.
  • kits of the present disclosure contains one or more components of the compositions disclosed herein.
  • the kit can further comprise one or more additional reagents, packaging material, containers for holding the components of the kit, and an instruction set or user manual detailing preferred methods of using the kit components.
  • the containers are vials.
  • the kit contains pre-formulated vaccine in separate containers in a ready-to-use format.
  • the pre-formulated vaccine in each separate container may be the same or different.
  • the kit comprises at least one container comprising an amphipathic compound, a neoantigen and a hydrophobic carrier.
  • the vaccine may be provided with all components, except the carrier, in one container (e.g., as a lyocake) ready for reconstitution in the carrier or as individual components in separate containers for formulation, lyophilization and reconstitution in the carrier.
  • one container e.g., as a lyocake
  • the kit may comprise a first container comprising a neoantigen; and a second container comprising a carrier.
  • the vaccine components in the first container may be in the form of a dry cake that is ready to be re-suspended in the carrier.
  • the vaccine in addition to neoantigen and carrier, may optionally further comprise one or more of a T-helper epitope, an adjuvant, and an emulsifier.
  • a T-helper epitope an adjuvant
  • an emulsifier an emulsifier
  • the T-helper epitope is a peptide comprising the amino acid sequence FNNFTVSFWLRVPKVSASHLE (SEQ ID NO: 7).
  • the T-helper epitope is a peptide comprising the amino acid sequence AQYIKANSKFIGITEL (SEQ ID NO: 1).
  • the adjuvant is a polyI:C polynucleotide.
  • the amphipathic compound is one or more lipids, such as phospholipids.
  • the lipids are l,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC) and cholesterol.
  • the kit may additionally contain an agent that interferes with DNA replication.
  • the agent that interferes with DNA replication may be included in the kit in a separate container, or the agent may be included with other components.
  • the agent that interferes with DNA replication that is included in the kit is an alkylating agent, such as for example, cyclophosphamide.
  • the kit may additionally contain an immune response checkpoint inhibitor.
  • the immune response checkpoint inhibitor may be included in the kit in a separate container, or it may be included with other components.
  • the immune response checkpoint inhibitor may be an inhibitor of Programmed Death-Ligand 1 (PD-L1), Programmed Death 1 (PD-1), CTLA-4, PD-L2, LAG3, TIM3. 41BB, 2B4, A2aR. B7H1, B7H3. B7H4. BTLA, CD2.
  • T cell costimulatoiy IIR
  • LAIR1 inducible T cell costimulatoiy
  • MARCO macrophage receptor with collageneous structure
  • PS phosphatidylserine
  • OX-40 SLAM
  • TIGIT TIGIT
  • VISTA VTCN1
  • the kit as disclosed herein may be used in practicing the methods disclosed herein.
  • the kit is for use in inducing an antibody immune response and/or cell- mediated immune response to the neoantigen in a subject.
  • the kit is for preparing a vaccine composition that is water-free or substantially free of water.
  • compositions disclosed herein may find application in any instance in which it is desired to administer a neoantigen to a subject.
  • the subject may be a vertebrate, such as a fish, bird or mammal, preferably a human.
  • the "immune response” may either be a cell-mediated immune response or an antibody (humoral) immune response.
  • the vaccine compositions disclosed herein may be used for inducing a cell-mediated immune response to the neoantigen.
  • “inducing” or “to induce” means that there is an improved efficacy in eliciting or generating an antibody immune response to the neoantigen.
  • “improved efficacy”, “improving the efficacy” or the like refers to any change or alteration in the immune response of a subject that is capable of rendering the compositions more effective in treating a disease or disorder (e.g., cancer). In some embodiments, this may involve accelerating the appearance of an immune response and/or improving the persistence or strength of an immune response. In some embodiments, this may involve inducing an immune response to the neoantigen but not the wild-type version of the neoantigen.
  • “inducing” or “to induce” refers to the ability’ to generate, elicit, strengthen, or prolong a neoantigenspecific recall response in a subject that has previously been primed by an earlier immunization or other exposure to the neoantigen.
  • a recall immune response is the immune response occurring on the second and/or subsequent exposures to a neoantigen, reestablishing an immune response that was previously produced by a prime immunization or other exposure to the neoantigen (e.g. appearance of the tumor neoantigen).
  • inducing refers to the ability to maintain and/or boost a neoantigen-specific antibody immune response in a subject that has previously been primed by an earlier immunization or other exposure to the neoantigen.
  • maintain and/or boost' it is meant that the previously induced immune response is enhanced, elevated, improved, strengthened, or prolonged to the benefit of the subject.
  • “potentiate” encompasses instances in which the antibody immune response to the antigen is made more effective or an adverse event is avoided, abolished or lessened in strength and/or duration.
  • “potentiating” refers to the ability to reduce the occurrence of an adverse event.
  • the terms "cell-mediated immune response”, “cellular immunity”, “cellular immune response” or “cytotoxic T-lymphocyte (CTL) immune response” refer to an immune response characterized by the activation of macrophages and natural killer cells, the production of neoantigen- specific cytotoxic T lymphocytes and/or the release of various cytokines in response to a neoantigen.
  • Cytotoxic T lymphocytes are a sub-group of T lymphocytes (a type of white blood cell) which are capable of inducing the death of infected somatic or tumor cells; they kill cells that are infected with viruses (or other pathogens), or that are otherwise damaged or dysfunctional.
  • cytotoxic T cells express T cell receptors that can recognize a specific peptide antigen bound to Class I MHC molecules.
  • cytotoxic T cells also express CD8 (i.e. CD8+ T cells), which is attracted to portions of the Class I MHC molecule. This affinity keeps the cytotoxic T cell and the target cell bound closely together during antigen-specific activation.
  • Cellular immunity protects the body by, for example, activating antigen-specific cytotoxic T-lymphocytes (e.g. antigen-specific CD8+ T cells) that are able to lyse body cells displaying epitopes of foreign or mutated antigen on their surface, such as cancer cells displaying tumor-specific neoantigens; activating macrophages and natural killer cells, enabling them to destroy intracellular pathogens; and stimulating cells to secrete a variety of cytokines that influence the function of other cells involved in adaptive immune responses and innate immune responses.
  • antigen-specific cytotoxic T-lymphocytes e.g. antigen-specific CD8+ T cells
  • Cellular immunity is an important component of the adaptive immune response and following recognition of neoantigen by cells through their interaction with neoantigenpresenting cells such as dendritic cells, B lymphocytes and to a lesser extent, macrophages, protect the body by various mechanisms such as: 1. activating antigen- specific cytotoxic T-lymphocytes that are able to induce apoptosis in body cells displaying epitopes of foreign or mutated antigen on their surface, such as cancer cells displaying tumor-specific neoantigens;
  • Cell-mediated immunity is most effective in removing virus-infected cells, but also participates in defending against fungi, protozoans, cancers, and intracellular bacteria. It also plays a major role in transplant rejection.
  • Antigen presenting cells Dendritic cells and B cells (and to a lesser extent macrophages) are equipped with special immunostimulatory receptors that allow for enhanced activation of T cells, and are termed professional antigen presenting cells (APC). These immunostimulatory molecules (also called co- stimulatory molecules) are up-regulated on these cells following infection or vaccination, during the process of antigen presentation to effector cells such as CD4 and CD8 cytotoxic T cells.
  • APC professional antigen presenting cells
  • co- stimulatory molecules such as CD40, CD80, CD86, MHC class I or MHC class II
  • APC such as CD 11c for dendritic cells
  • Cytotoxic T cells (also known as Tc, killer T cell, or cytotoxic T-lymphocyte (CTL)) are a sub-group of T cells which induce the death of cells that are infected with viruses (and other pathogens), or expressing tumor antigens or neoantigens.
  • CTL cytotoxic T-lymphocyte
  • CTLs directly attack other cells carrying certain foreign or abnormal molecules on their surface.
  • the ability of such cellular cytotoxicity can be detected using in vitro cytolytic assays (chromium release assay).
  • induction of adaptive cellular immunity can be demonstrated by the presence of such CTLs, wherein neoantigen loaded target cells are lysed by CTLs following vaccination or infection.
  • Naive cytotoxic T cells are activated when their T cell receptor (TCR) strongly interacts with a peptide-bound MHC class I molecule. This affinity depends on the type and orientation of the antigen/MHC complex, and is what keeps the CTL and infected cell bound together. Once activated the CTL undergoes a process called clonal expansion in which it gains functionality, and divides rapidly, to produce an army of "armed"-effector cells.
  • TCR T cell receptor
  • Activated CTL will then travel throughout the body in search of cells bearing that unique MHC Class I + peptide. This could be used to identify such CTLs in vitro by using peptide- MHC Class I tetramers in flow cytometric assays.
  • effector CTL When exposed to these infected or dysfunctional somatic cells, effector CTL release perforin and granulysin: cytotoxins which form pores in the target cell's plasma membrane, allowing ions and water to flow into the infected cell, and causing it to burst or lyse.
  • ELISA enzyme linked immunosorbant assay
  • ELISPOT enzyme linked immunosorbent spot assay
  • CTLs are also capable of producing important cytokines such as IFN-y
  • quantitative measurement of IFN-gramma IFN-y-producing CD8 cells can be achieved by ELISPOT and by flowcytometric measurement of intracellular IFN-y in these cells.
  • CD4+ "helper" T cells CD4+ lymphocytes, or helper T cells, are immune response mediators, and play an important role in establishing and maximizing the capabilities of the adaptive immune response. These cells have no cytotoxic or phagocytic activity; and cannot kill infected cells or clear pathogens, but, in essence "manage" the immune response, by directing other cells to perform these tasks.
  • Two types of effector CD4+ T helper cell responses can be induced by a professional APC, designated Thl and Th2, each designed to eliminate different types of pathogens.
  • Helper T cells express T cell receptors (TCR) that recognize antigen bound to Class II MHC molecules.
  • TCR T cell receptors
  • the activation of a naive helper T cell causes it to release cytokines, which influences the activity of many cell ty pes, including the APC that activated it.
  • Helper T cells require a much milder activation stimulus than cytotoxic T cells.
  • Helper T cells can provide extra signals that "help" activate cytotoxic cells.
  • Two types of effector CD4+ T helper cell responses can be induced by a professional APC, designated Thl and Th2, each designed to eliminate different types of pathogens. The two Th cell populations differ in the pattern of the effector proteins (cytokines) produced.
  • Thl cells assist the cell-mediated immune response by activation of macrophages and cytotoxic T cells; whereas Th2 cells promote the humoral immune response by stimulation of B cells for conversion into plasma cells and by formation of antibodies.
  • a response regulated by Thl cells may induce lgG2a and lgG2b in mouse (IgGl and lgG3 in humans) and favor a cell mediated immune response to a neoantigen. If the IgG response to an antigen is regulated by Th2 type cells, it may predominantly enhance the production of IgGl in mouse (lgG2 in humans).
  • the measure of cytokines associated with Thl or Th2 responses will give a measure of successful vaccination. This can be achieved by specific ELISA designed for Thl -cytokines such as IFN-y, IL-2, IL- 12, TNF-a and others, or Th2- cytokines such as IL-4. IL-5, IL10 among others.
  • cytokines released from regional lymph nodes gives a good indication of successful immunization.
  • APC immune effector cells
  • CD4 and CD8 T cells several cytokines are released by lymph node cells.
  • lymph node cells By culturing these LNC in vitro in the presence of neoantigen, a neoantigen-specific immune response can be detected by measuring release if certain important cytokines such as IFN-y, IL-2, IL-12, TNF-a and GM-CSF. This could be done by ELISA using culture supernatants and recombinant cy tokines as standards.
  • Successful immunization may be determined in a number of ways known to the skilled person including, but not limited to, hemagglutination inhibition (HAFI) and serum neutralization inhibition assays to detect functional antibodies; challenge studies, in which vaccinated subjects are challenged with the associated pathogen to determine the efficacy of the vaccination; and the use of fluorescence activated cell sorting (FACS) to determine the population of cells that express a specific cell surface marker, e.g. in the identification of activated or memory lymphocytes.
  • FACS fluorescence activated cell sorting
  • a skilled person may also determine if immunization with a composition as disclosed herein elicited an antibody and/or cell mediated immune response using other known methods. See, for example, Coligan et al., ed. Current Protocols in Immunology, Wiley Interscience, 2007.
  • the composition disclosed herein is capable of generating an enhanced cell-mediated immune response that is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold or at least 10-fold greater than when the neoantigen is formulated in an aqueous-based vaccine formulation.
  • aqueous-based vaccine it is meant a vaccine that comprises identical components as the oilbased formulations described herein, with the exception that the hydrophobic carrier is replaced with an aqueous carrier and the aqueous-based vaccine does not comprise an amphipathic compound.
  • the composition disclosed herein is capable of generating the enhanced cell-mediated immune response with only a single administration of the composition.
  • the compositions disclosed herein are for delivery' of the neoantigen by single administration.
  • the composition disclosed herein is capable of generating the enhanced cell-mediated immune response by a low dose amount of the neoantigen, wherein the low dose amount is about 50% of the dose amount in the aqueous-based vaccine formulation.
  • the vaccine compositions disclosed herein may be used for inducing an antibody immune response to the neoantigen.
  • an "antibody immune response” or “humoral immune response” (used interchangeably herein), as opposed to cell-mediated immunity, is mediated by secreted antibodies which are produced in the cells of the B lymphocyte lineage (B cells). Such secreted antibodies bind to antigens, such as for example those on the surfaces of foreign substances, pathogens (e.g. viruses, bacteria, etc.) and/or cancer cells, and flag them for destruction.
  • B cells B lymphocyte lineage
  • “humoral immune response” refers to antibody production and may also include, in addition or alternatively, the accessory processes that accompany it. such as for example the generation and/or activation of T-helper 2 (Th2) or T-helper 17 (Thl7) cells, cytokine production, isotype switching, affinity maturation and memory cell activation.
  • “Humoral immune response” may also include the effector functions of an antibody, such as for example toxin neutralization, classical complement activation, and promotion of phagocytosis and pathogen elimination.
  • the humoral immune response is often aided by CD4+ Th2 cells and therefore the activation or generation of this cell type may also be indicative of a humoral immune response.
  • the term “humoral immune response” is used interchangeably herein with “antibody response” or “antibody immune response”.
  • an "antibody” is a protein comprising one or more polypeptides substantially or partially encoded by immunoglobulin genes or fragments of immunoglobulin genes.
  • the recognized immunoglobulin genes include the K, X, a, y, 5, 8 and p constant region genes, as well as myriad immunoglobulin variable region genes.
  • Light chains are classified as either K or X.
  • Heavy’ chains are classified as y. p, a, 5. or 8, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
  • a typical immunoglobulin (antibody) structural unit comprises a protein containing four polypeptides.
  • Each antibody structural unit is composed of two identical pairs of polypeptide chains, each having one "light” and one "heavy” chain.
  • the N-terminus of each chain defines a variable region primarily responsible for antigen recognition.
  • Antibody structural units e.g. of the IgA and IgM classes
  • Antibodies are the antigen-specific glycoprotein products of a subset of white blood cells called B lymphocytes (B cells). Engagement of neoantigen with antibody expressed on the surface of B cells can induce an antibody response comprising stimulation of B cells to become activated, to undergo mitosis and to terminally differentiate into plasma cells, which are specialized for synthesis and secretion of antigen-specific antibody.
  • B cells are the sole producers of antibodies during an immune response and are thus a key element to effective humoral immunity. In addition to producing large amounts of antibodies, B cells also act as antigen-presenting cells and can present neoantigenic peptide to T cells, such as T helper CD4 or cytotoxic CD8+ T cells, thus propagating the immune response. B cells, as well as T cells, are part of the adaptive immune response. During an active immune response, induced for example by either vaccination or natural infection, antigenspecific B cells are activated and clonally expand. During expansion, B cells evolve to have higher affinity for the epitope. Proliferation of B cells can be induced indirectly by activated T-helper cells, and also directly through stimulation of receptors, such as the TLRs.
  • Antigen presenting cells such as dendritic cells and B cells
  • the adjuvant stimulates the cells to become activated and the neoantigen provides the blueprint for the target.
  • Different types of adjuvants may provide different stimulation signals to cells.
  • polyEC a TLR3 agonist
  • Adjuvants such as Pam3Cys, Pam2Cys and FSL- 1 are especially adept at activating and initiating proliferation of B cells, which is expected to facilitate the production of an antibody response (Moyle 2008; So 2012).
  • a humoral immune response is one of the common mechanisms for effective infectious disease vaccines (e.g. to protect against viral or bacterial invaders). However, a humoral immune response can also be useful for combating cancer. Whereas a cancer vaccine is typically designed to produce a cell-mediated immune response that can recognize and destroy cancer cells, B cell mediated responses may target cancer cells through other mechanisms which may in some instances cooperate with a cytotoxic T cell for maximum benefit. Examples of B cell mediated (e.g. humoral immune response mediated) anti-tumor responses include, without limitation: 1) Antibodies produced by B cells that bind to surface antigens (e.g. neoantigens) found on tumor cells or other cells that influence tumorigenesis.
  • surface antigens e.g. neoantigens
  • Such antibodies can, for example, induce killing of target cells through antibody-dependent cell-mediated cytotoxicity (ADCC) or complement fixation, potentially resulting in the release of additional antigens that can be recognized by the immune system; 2) Antibodies that bind to receptors on tumor cells to block their stimulation and in effect neutralize their effects; 3) Antibodies that bind to factors released by or associated with a tumor or tumor- associated cells to modulate a signaling or cellular pathway that supports cancer; and 4) Antibodies that bind to intracellular targets and mediate anti-tumor activity through a currently unknown mechanism.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • One method of evaluating an antibody response is to measure the titers of antibodies reactive with a particular antigen. This may be performed using a variety of methods known in the art such as enzy me-linked immunosorbent assay (ELISA) of antibody-containing substances obtained from animals.
  • ELISA enzy me-linked immunosorbent assay
  • the titers of serum antibodies which bind to a particular neoantigen may be determined in a subject both before and after exposure to the neoantigen. A statistically significant increase in the titer of neoantigen-specific antibodies following exposure to the neoantigen would indicate the subject had mounted an antibodyresponse to the neoantigen.
  • immunological assays e.g. radioimmunoassay (RIA)
  • immunoprecipitation assays e.g., Western blot
  • protein blot e.g., Western blot
  • neutralization assays e.g., neutralization of viral infectivity in an in vitro or in vivo assay.
  • the vaccine compositions disclosed herein may be useful for treating or preventing diseases and/or disorders ameliorated by a cell-mediated immune response or a humoral immune response.
  • the vaccines may find application in any instance in which it is desired to administer a neoantigen to a subject to induce a cell-mediated immune response or a humoral immune response.
  • the vaccines may find application for the delivery of a personalized vaccine.
  • the present disclosure relates to a method comprising administering the composition as described herein to a subject in need thereof.
  • the method is for inducing an antibody immune response and/or cell-mediated immune response to a neoantigen in said subject.
  • the method is for the treatment and/or prevention of cancer.
  • Treating” or “treatment of, or “preventing” or “prevention of, as used herein, refers to an approach for obtaining beneficial or desired results.
  • Beneficial or desired results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilization of the state of disease, prevention of development of disease, prevention of spread of disease, delay or slowing of disease progression (e.g. suppression), delay or slowing of disease onset, conferring protective immunity against a disease-causing agent and amelioration or palliation of the disease state.
  • Treating” or “preventing” can also mean prolonging survival of a patient beyond that expected in the absence of treatment and can also mean inhibiting the progression of disease temporarily or preventing the occurrence of disease, such as by preventing infection in a subject. "Treating” or “preventing” may also refer to a reduction in the size of a tumor mass, reduction in tumor aggressiveness, etc.
  • the methods and compositions disclosed herein may be for use in treating and/or preventing cancer in a subject in need thereof.
  • the subject may have cancer or may be at risk of developing cancer.
  • cancer refers to cells that exhibit abnormal growth, characterized by a significant loss of control of cell proliferation or cells that have been immortalized.
  • cancer or “tumor” includes metastatic as well as non-metastatic cancer or tumors.
  • a cancer may be diagnosed using criteria generally accepted in the art, including the presence of a malignant tumor.
  • cancers that may be capable of being treated and/or prevented by the use or administration of a composition as disclosed herein include carcinoma, adenocarcinoma, lymphoma, leukemia, sarcoma, blastoma, myeloma, and germ cell tumors.
  • particularly suitable embodiments may include glioblastoma, multiple myeloma, ovarian cancer, breast cancer, fallopian tube cancer, prostate cancer or peritoneal cancer.
  • the cancer may be caused by a pathogen, such as a virus.
  • Viruses linked to the development of cancer are known to the skilled person and include, but are not limited to, human papillomaviruses (HPV), John Cunningham virus (JCV), Human herpes virus 8, Epstein Barr Virus (EBV), Merkel cell polyomavirus, Hepatitis C Virus and Human T cell leukaemia virus- 1
  • HPV human papillomaviruses
  • JCV John Cunningham virus
  • EBV Epstein Barr Virus
  • Merkel cell polyomavirus Hepatitis C Virus
  • Hepatitis C Virus and Human T cell leukaemia virus- 1
  • the cancer is one that expresses one or more tumor- specific neoantigens.
  • the cancer is breast cancer, ovarian cancer, prostate cancer, fallopian tube cancer, peritoneal cancer, glioblastoma or diffuse large B cell lymphoma.
  • the methods and compositions disclosed herein may be useful for either the treatment or prophylaxis of cancer; for example, a reduction of the severity of cancer (e.g. size of the tumor, aggressiveness and/or invasiveness, malignancy, etc.) or the prevention of cancer recurrences.
  • the method for treating and/or preventing cancer first comprises identifying one or more neoantigens or neoepitopes in the patients' tumor cells.
  • neoantigens can be identified using whole genome/exome sequencing.
  • whole genome/exome sequencing may be used to identify mutated neoantigens that are uniquely present in a tumor of an individual patient.
  • the collection of identified neoantigens can be analyzed to select (e.g. based on algorithms) a specific, optimized subset of neoantigens and/or neoepitopes for use as a personalized cancer vaccine.
  • neoantigenic peptides may be produced by’ any method known the art and then may be formulated into a vaccine composition or kit as described herein and administered to a subject.
  • the vaccine composition upon administration to a subject, induces a tumor-specific immune response in the treatment of cancer.
  • the immune response specifically targets the tumor cells without a significant effect on normal cells of the body which do not express the neoantigen.
  • the composition may comprise at least one patient- specific neoepitope such that the tumorspecific immune response is patient- specific for the subject or a subset of subjects, i.e. a personalized immunotherapy.
  • the vaccine composition as disclosed herein may be administered by any suitable route.
  • the route of administration is subcutaneous injection.
  • the methods disclosed herein may also comprise administering an agent that interferes with DNA replication.
  • an agent that interferes with DNA replication is administered when the methods disclosed herein are used in the treatment or prevention of cancer.
  • the expression "interferes with DNA replication” is intended to encompass any action that prevents, inhibits or delays the biological process of copying (i.e., replicating) the DNA of a cell.
  • the skilled person will appreciate that there exist various mechanisms for preventing, inhibiting or delaying DNA replication, such as for example DNA cross-linking, methylation of DNA, base substitution, etc.
  • the methods according to the invention encompass the use of any agent that interferes with DNA replication by any means known in the art.
  • the agent that interferes with DNA replication is a drug.
  • the agent that interferes with DNA replication is one which, when used at doses that are non-chemotherapeutic, is capable of selectively affecting DNA replication in cells of the immune system, with the intent of modulating the immune system to enhance vaccine responses.
  • non-chemotherapeutic it is meant that the dose of the agent is a dose lower than that which would be used to directly and selectively destroy malignant or cancerous cells and tissues.
  • an agent that interferes with DNA replication include agents that interfere with DNA replication to cause programmed cell death, with the ability to selectively target rapidly dividing cells of the immune system.
  • the purpose of such agents is to modulate cells of the immune system to enhance vaccine responses.
  • Such agents are typically used at doses that are not expected to be chemotherapeutic and are considered acceptable for use in humans.
  • the purpose of selectively targeting immune cells may be to reduce the number of immune suppressive cells, and/or deplete useful immune cells involved in mediating the immune response for the purposes of inducing rapid proliferation upon removal of the drug targeting DNA replication.
  • Interference with DNA replication leading to cell death may be caused by numerous mechanisms, including but not limited to, the formation of DNA cross-linking (e.g. by alkylating agents, platinum compounds, etc ), methylation of DNA (i.e. by methylating agents), base substitution (i.e. by nucleoside analogs).
  • DNA cross-linking e.g. by alkylating agents, platinum compounds, etc
  • methylation of DNA i.e. by methylating agents
  • base substitution i.e. by nucleoside analogs.
  • Exemplary agents and their mechanisms are described in Cancer Chemotherapy and Biotherapy: Principles and Practice (Cabner B.A., 5th edition, Lippincott Williams & Wilkins. PA, USA, 2011).
  • the agent that interferes with DNA replication is an alkylating agent.
  • Alkylating agents include, but are not limited to, cyclophosphamide, temozolomide, ifosfamide, mafosfamide, melphalan, busulfan, bendamustine, uramustine, carmustine or bis- chloroethylnitrosourea (BCNU), chlorambucil, mitomycin C. and their derivatives, active metabolites or metabolite intermediates.
  • a suitable derivative may be, for example and without limitation, palifosfamide (e g. a derivative of ifosfamide).
  • the agent that interferes with DNA replication is a platinum compound.
  • Platinum compounds include, but are not limited to, carboplatin, cisplatin, oxaliplatin and their derivatives.
  • the agent that interferes with DNA replication is a methylating agent. Methylating agents include, but are not limited to, temzolomide, procarbazine and dacarbazine, and their derivatives.
  • the agent that interferes with DNA replication is a nucleoside analog.
  • nucleoside analogs include gemcitabine, 5- fluorouracil, cytosine arabinoside (Ara-C) and their derivatives.
  • any drug that inhibits DNA replication indirectly by inhibiting enzymes critical to DNA replication such as topoisomerase I, topoisomerase II or DNA polymerase, may also be used.
  • Such drugs include, for example and without limitation, doxorubicin, daunorubicin, mitoxantrone, etoposide, teniposide, topotecan, camptothecin, irinotecan, acyclovir and ganciclovir.
  • agents that interfere with DNA replication include, without limitation, those listed below in Table 1. As the skilled person will appreciate, these are examples of agents that may be used. Additional agents include, for example, any drug or compound that interferes with DNA replication by a similar mechanism and/or that has a similar functional group.
  • the agent that interferes with DNA replication is a nitrogen mustard alkylating agent, or any intermediary or active metabolite thereof.
  • Nitrogen mustards are non-specific DNA alkylating agents. Nitrogen mustards form cyclic aminium ions (aziridinium rings) by intramolecular displacement of the chloride by the amine nitrogen. This azidirium group is then capable of alkylating DNA by attacking the N-7 nucleophilic center on the guanine base. Upon displacement of the second chlorine, a second alkylation step occurs that results in the formation of interstrand cross-links (ICLs). These lesions are highly cytotoxic since they block fundamental metabolic processes such as DNA replication and transcription.
  • ICLs interstrand cross-links
  • the methods of the invention encompass the use of any such non-specific nitrogen mustard DNA alkylating agents.
  • Particularly suitable nitrogen mustard alkylating agents may include for example, and without limitation, cyclophosphamide, palifosfamide, bendamustine, and ifosfamide.
  • Ifosfamide is a nitrogen mustard alkylating agent.
  • the IUPAC name for ifosfamide is N-3-bis(2-chloroethyl)-l,3,2-oxazaphosphinan-2-amide-2-oxide. Ifosfamide commonly known as Ifex®.
  • the chemical structure of ifosfamide is:
  • Palifosfamide is an active metabolite of ifosfamide that is covalently linked to the amino acid lysine for stability. Palifosfamide irreversibly alkydates and cross-links DNA through GC base pairs, resulting in irreparable 7-atom inter-strand cross-links; inhibition of DNA replication and/or cell death. Palifosfamide is also known as Zymafos®.
  • Bendamustine is another nitrogen mustard alkylating agent.
  • the IUPAC name for Bendamustine is 4-[5-[Bis(2-chloroethyl)amino]-l-methylbenzimidazol-2-yl]butanoic acid, and it is commonly referred to as Treakisym®, Ribomustin®, Levact® and Treanda®.
  • the chemical structure of bendamustine is:
  • Also encompassed by the methods of the invention is the use of intermediary and/or active metabolites of DNA alkylating agents, and particularly intermediary and/or active metabolites of the nitrogen mustard DNA alkylating agents described herein.
  • Such metabolites include, without limitation, aldophosphamide, 4-hydroxycyclophosphamide, 4- hydroxyifosfamide, chloracetaldehyde and phosphamide mustard.
  • the agent that interferes with DNA replication may be any suitable pharmaceutically acceptable salt, ester, tautomer, stereoisomer, racemic mixture, solvate, hydrate or prodrug of the alkylating agents, platinum compounds, methylating agents, or nucleoside analogs described herein.
  • the agent that interferes with DNA replication for use in the methods of the invention is cyclophosphamide.
  • Cyclophosphamide N,N-bis(2- chloroethyl)-l,3,2-oxazaphosphinan-2-amine 2-oxide
  • cytophosphane is a nitrogen mustard alkylating agent.
  • the chemical structure of cyclophosphamide is:
  • Cyclophosphamide is also known and referred to under the trade-marks Endoxan®, Cytoxan®, Neosar®, Procytox® and Revimmune®.
  • Other nitrogen mustard alkylating agents in the same class as cyclophosphamide include, without limitation, palifosfamide, bendamustine and ifosfamide.
  • Cyclophosphamide is a prodrug which is typically administered via intravenous infusion, but also can be administered parenterally and orally (de Jonge 2005) with little difference in bioavailability (Juma 1979).
  • CPA is converted to its active metabolites, 4- hydroxy-CPA and aldophosphamide, by oxidation by P450 enzymes in the liver (Emmenegger 2007, Emmenegger 2011).
  • the active metabolites of CPA are lipid soluble and enter cells through passive diffusion. Intracellular 4-OH-CPA spontaneously decomposes into phosphoramide mustard which is the ultimate active metabolite.
  • Phosphoramide mustard catalyzes intra- and interstrand DNA cross-links as well as DNA- protein cross-links that inhibit DNA replication leading to cell death (de Jonge 2005). Phosphoramide mustard is eliminated by enzymatic conversion to carboxyphoshphamide by cytoplasmic aldehyde dehydrogenase (ALDH) (Emmenegger 2007, Emmenegger 2011).
  • ADH cytoplasmic aldehyde dehydrogenase
  • low dose CPA has been appreciated for its immune modulatory and anti-angiogenic effects.
  • high dose CPA low doses of CPA, A pically 100-300 mg/m 2 , lack widespread cytotoxic activity but do appear to enhance immune- mediated tumor elimination by selectively modulating cells of the immune system and also by reducing angiogenesis within the tumor microenvironment.
  • the mechanisms of action and uses of low dose CPA are further described, for example, in WO2014/153636.
  • the methods disclosed herein comprise administering an agent that interferes with DNA replication.
  • the agent that interferes with DNA replication is typically administered in an amount sufficient to provide an immune-modulating effect.
  • the expression “immune-modulating effect” refers to the ability of the agent that interferes with DNA replication to alter (modulate) one or more aspects of the immune system and/or cells of the immune system.
  • the "amount sufficient to provide an immune-modulating effect” is an amount of the agent that is capable of selectively affecting DNA replication in cells the immune system.
  • the amount of agent may be an amount sufficient to selectively target rapidly dividing cells of the immune system to cause programmed cell death.
  • the "amount sufficient to provide an immune-modulating effect” may interchangeably be referred to herein as a "low dose" amount.
  • the expression "low dose” typically refers to a dose of cyclophosphamide that is less than or equal to 300 mg/m 2 , such as for example 25-300 mg/m 2 and more particularly 100-300 mg/m 2 .
  • the low 7 dose amount of cyclophosphamide is 10, 25, 50, 75 or 100 mg BID (two times daily).
  • the low dose amount of cyclophosphamide is 50 mg BID.
  • the "low dose” amounts of other agents that interfere with DNA replication, as encompassed herein, would be known to those skilled in the art, or could be determined by routine skill.
  • the methods disclosed herein comprise a cycle of low dose metronomic cyclophosphamide.
  • “metronomic” is meant to refer to a frequent administration of a lower-than-normal dose amount of the agent that interferes with DNA replication (e g. cyclophosphamide).
  • the term "normal dose amount” may refer, for example and without limitation, to either: (i) the established maximum tolerated dose (MTD) or standard dose via a traditional dosing schedule, or (ii) in instances where a low dose single bolus amount has been established for a particular agent that interferes with DNA replication, than to that low dose amount.
  • the same, lower, or higher cumulative dose over a certain time period as would be administered via a traditional dosing schedule may ultimately be administered.
  • this is achieved by extending the time frame during which the dosing is conducted and/or increasing the frequency of administrations, while decreasing the amount administered as compared to the normal dose amount.
  • a metronomic regimen may comprise administering the same amount over a period of several days by administering frequent low doses.
  • metronomic treatment with the agent that interferes with DNA replication is intended to encompass a daily low dose administration of the agent over a certain period of time, such as for example a period of 2, 3, 4, 5, 6 or 7, or more, consecutive days.
  • the agent that interferes with DNA replication may be provided at frequent regular intervals or varying intervals.
  • a dose of the agent that interferes with DNA replication may be administered even' 1, 2, 3, 4, 6, 8, 12 or 24 hours.
  • a dose of the agent that interferes with DNA replication may be administered once every' 2, 3, or 4 days.
  • a dose of the agent that interferes w ith DNA replication may be administered two times daily.
  • metronomic treatment may occur in a cyclic fashion, alternating between on and off periods of administration.
  • Particularly suitable are intervals where the agent that interferes with DNA replication is administered to the subject daily on alternating weekly intervals. For instance, a one-week period of administration of the agent that interferes with DNA replication is followed by a one week suspension of treatment, and the cycle repeats.
  • the methods disclosed herein comprise administering the agent that interferes with DNA replication to the subject daily for a period of 7 consecutive days, beginning every second week.
  • the administration of the agent that interferes wdth DNA replication begins about 7 days prior to the first administration of the depot-forming vaccine.
  • the agent that interferes with DNA replication may be administered at a dose of 50 mg BID (two times daily) on each day of administration.
  • the agent that interferes with DNA replication may be administered as a priming agent during the intermittent period between each administration of the depot-forming vaccine and/or non-depot-forming vaccine.
  • the frequency and duration of the administration of the agent that interferes with DNA replication may be adjusted as desired for any given subject within the parameters described above.
  • Factors that may be taken into account include, e.g. : the nature of the one or more neoantigens in the vaccine; the type of disease or disorder; the age, physical condition, body weight, sex and diet of the subject; and other factors.
  • the agent that interferes with DNA replication may be administered by any suitable delivery means and any suitable route of administration.
  • the agent that interferes with DNA replication is administered orally, such as in the form of a pill, tablet or capsule.
  • the agent is administered by injection (e.g., intravenous).
  • the agent is cyclophosphamide, and it is administered orally.
  • the agent that interferes with DNA replication is cyclophosphamide.
  • the methods disclosed herein may also comprise administering an immune response checkpoint inhibitor.
  • an “immune response checkpoint inhibitor” refers to any compound or molecule that totally or partially reduces, inhibits, interferes with or modulates one or more checkpoint proteins.
  • Checkpoint proteins regulate T-cell activation or function. Numerous checkpoint proteins are known, such as for example CTLA-4 and its ligands CD80 and CD86; and PD-1 and its ligands PD-L1 and PD-L2.
  • Checkpoint proteins are responsible for costimulatory or inhibitory interactions of T-cell responses.
  • Checkpoint proteins regulate and maintain self-tolerance and the duration and amplitude of physiological immune responses.
  • the term “immune response checkpoint inhibitor” may be used interchangeably with "checkpoint inhibitor”.
  • the immune response checkpoint inhibitor is an inhibitor of Programmed Death-Ligand 1 (PD-L1, also known as B7-H1, CD274), Programmed Death 1 (PD-1, CD279), CTLA-4 (CD154), PD-L2 (B7-DC, CD273), LAG3 (CD223), TIM3 (HAVCR2, CD366).
  • PD-L1 Programmed Death-Ligand 1
  • PD-1 Programmed Death 1
  • CD279 CTLA-4
  • B7-L2 B7-DC, CD273
  • LAG3 CD223)
  • TIM3 HVCR2, CD366
  • 41BB CD137
  • 2B4, A2aR B7H1.
  • the immune response checkpoint inhibitor is an inhibitor of PD-L1, PD-1, CTLA-4 or any combination thereof.
  • the immune response checkpoint inhibitor is an inhibitor of PD-L1 or PD-1.
  • the inhibitor of PD-L1 or PD-1 may be an anti-PD-1 or anti-PD-Ll antibody, such as for example and without limitation, those disclosed in WO 2015/103602.
  • the anti-PD-1 antibody or anti-PD-Ll antibody may be selected from: nivolumab, pembrolizumab.
  • the anti-PD-1 antibody may be RMP1-4 or J43 (BioXCell) or a human or humanized counterpart thereof.
  • the immune response checkpoint inhibitor is an inhibitor of CTLA-4.
  • the inhibitor of CTLA-4 may be an antibody, such as for example and without limitation, ipilimumab (Bristol-Myers Squibb) or BN 13 (BioXCell).
  • the anti-CTLA-4 antibody may be UC10-4F10-11, 9D9 or 9H10 (BioXCell) or a human or humanized counterpart thereof.
  • the one or more immune response checkpoint inhibitors may be administered by any suitable route.
  • the route of administration of the one or more immune response checkpoint inhibitors is parenteral, mucosal, oral, sublingual, transdermal, topical, inhalation, intranasal, aerosol, intraperitoneal, intratumoral, intraocular, intratracheal, intrarectal, intragastric, vaginal, by gene gun, dermal patch or in eye drop or mouthwash form.
  • the immune response checkpoint inhibitor may be administered by subcutaneous injection.
  • the frequency and duration of the administration of the immune response checkpoint inhibitor may be adjusted as desired for any given subject. Factors that may be taken into account include, e.g. : the nature and type of the specific checkpoint inhibitor; the nature of the one or more neoantigens in the vaccine; the type of disease or disorder; the age, physical condition, body weight, sex and diet of the subject; and other factors.
  • the one or more immune response checkpoint inhibitors may be administered before, after or concurrently with the depot-forming vaccine and/or nondepot-forming vaccine.
  • the immune response checkpoint inhibitor may be administered at a time subsequent to the first administration with the depot-forming vaccine.
  • the immune response checkpoint inhibitor may be administered at a time before or after the first administration of the non-depot-forming vaccine.
  • administration of the immune response checkpoint inhibitor may begin on the same day as the first administration of the depot-forming vaccine and may be administered at a desired schedule thereafter.
  • the desired schedule may be administration of the immune response checkpoint inhibitor every 1, 2, 3, 4, 6, 8, 12 or 18 hours; every 1. 2, 3, 4, 5 or 6 days; or every 1, 2, 3 or 4 weeks.
  • the desired schedule may be once every 3 days.
  • the vaccine compositions may be prepared by known methods in the art having regard to the present disclosure. Exemplary 7 embodiments for preparing the vaccine compositions disclosed herein are described below, without limitation.
  • neoantigen is used generally to describe how a neoantigen (e.g., one or more KRAS neoantigens) may be formulated in the vaccine compositions of the present disclosure.
  • neoantigen encompasses both the singular form “neoantigen” and the plural “neoantigens”. It is not necessary that all neoantigens be introduced into the vaccine composition in the same way.
  • the neoantigen and optionally other vaccine components are reconstituted in a suitable solvent together with an amphipathic compound.
  • the vaccine components are then dried to form a dry cake, and the dry cake is resuspended in a hydrophobic carrier.
  • the step of drying may be performed by various means known in the art, such as by lyophilization, freeze- drying, spray freeze-drying, or spray drying, rotary evaporation, evaporation under pressure, etc. Low 7 heat drying that does not compromise the integrity 7 of the components can also be used.
  • Heat can also be used to assist in resuspending the neoantigen/amphipathic compound mixture.
  • dry cake ', “lyocake”’, “dried preparation”, “dried lipid/neoantigen preparation” or “dried preparation comprising lipids and neoantigens”, used interchangeably, do not necessarily mean that the preparation is completely dry.
  • a small component of volatile and/or non-volatile material will remain in the dried preparation. In an embodiment, the non-volatile material will remain.
  • dry cake “lyocake”.
  • the dried preparation is completely free of water.
  • the dried preparation may contain a residual moisture content based on the limitations of the drying process (e.g. lyophilization). This residual moisture content will typically be less than 2%, less than 1%, less than 0.5%, less than 0.25%, less than 0. 1%, less than 0.05% or less byweight of the dried preparation. This residual moisture content will not be more than 5% by weight of the dried preparation as this would result in a product that is not clear.
  • the drying is performed by lyophilization.
  • lyophilization As used herein, “lyophilization”, “lyophilized” and “freeze-dry ing” are used interchangeably. As is well known in the art. lyophilization works by freezing the material and then reducing the surrounding pressure to allow the volatile solvent (e.g. water) in the material to sublime directly from the solid phase to the gas phase.
  • volatile solvent e.g. water
  • any conventional freeze-drying procedure may be used to carry 7 out the drying step of the methods disclosed herein.
  • the lyophilization is performed by sequential steps of loading, freezing, evacuation and drying (e.g. primary drying and secondary drying).
  • the lyophilization is performed according to the protocol set forth here. Briefly , the mixture is frozen to a temperature of about -50°C. Evacuation is then performed by reducing the pressure to about 100 micron (mTorr). The mixture is then dried. A primary drying is performed for about 55 hours by increasing the temperature to about -40°C under the reduced pressure. Then, a secondary drying is performed for about 20 minutes by further increasing the temperature to about 35°C under the reduced pressure.
  • Freezing It is important to cool the material below its triple point, i.e., the lowest temperature at which the solid and liquid phases of the material can coexist. This ensures that sublimation rather than melting will occur in the following steps.
  • the methods disclosed herein for preparing a dried lipid/neoantigen preparation may further comprise a step of confirming that the sized lipid vesicle particles have retained a mean particle size of ⁇ 120 nm and PDI of ⁇ 0.1.
  • a mean particle size of ⁇ 120 nm and PDI of ⁇ 0.1 there are several techniques, instruments and services that are available to measure the mean particle size and PDI of lipid vesicle particles, such as for example and without limitation TEM, SEM, AFM, FTIR, XPS, XRD, MALDI-TOF-MS, NMR and DLS.
  • the present invention relates to a method for preparing a pharmaceutical composition.
  • the pharmaceutical composition is prepared by first preparing a dried lipid/KRAS neoantigen preparation according to the methods disclosed herein, and then solubilizing the dried preparation in a hydrophobic carrier.
  • the dried lipid/KRAS neoantigen preparation is restored to a liquid state by dissolving the dried constituents in a hydrophobic carrier.
  • the hydrophobic carrier may be added by any means that will dissolve the dried constituents (e.g. the lipid and KRAS neoantigen) in the hydrophobic carrier.
  • the dried lipid/KRAS neoantigen preparation may be solubilized in the hydrophobic carrier by mixing of the two together.
  • solubilizing involves adding the hydrophobic carrier to the dried lipid/KRAS neoantigen preparation, allowing it to sit for 1-30 minutes, and then gently shaking or mixing the mixture for 1-15 minutes. This process can be repeated until the dried constituents are dissolved in the hydrophobic carrier (e.g. a clear solution is obtained).
  • solubilizing involves adding the hydrophobic carrier to the dried lipid/KRAS neoantigen preparation, allowing it to sit for 5 minutes, and then gently shaking or mixing for 1 minute. This process can be repeated until the dried constituents are dissolved in the hydrophobic carrier (e.g. a clear solution is obtained).
  • the step of solubilizing the dried lipid/KRAS neoantigen in a hydrophobic carrier results in a composition in which the dried constituents are fully dissolved in the hydrophobic carrier.
  • the dried constituents may not be completely dissolved in the hydrophobic carrier, but they are dissolved to a sufficient degree to reproducibly provide a clear solution.
  • a KRAS neoantigen stock may be prepared with a single solubilized KRAS neoantigen.
  • the KRAS neoantigen stock may be prepared by combining individual stock preparations of different solubilized KRAS neoantigens. These individual stock preparations may each comprise one or more different KRAS neoantigens or additional therapeutic agent(s).
  • each individual stock preparation comprises a single KRAS neoantigen, all of which are then combined to form, in whole or in part, to form the KRAS neoantigen stock.
  • a KRAS neoantigen stock may be prepared by combining dry KRAS neoantigens, adding a solvent and mixing the KRAS neoantigens in the solvent.
  • the KRAS neoantigen stock may be prepared by combining one or more dry powder KRAS neoantigens with one or more solubilized KRAS neoantigens.
  • a KRAS neoantigen stock is prepared by sequentially adding individual stock preparations, each comprising one or more different KRAS neoantigens, into a compatible solvent with mixing.
  • '‘compatible’ 7 it is meant that the solvent will not cause the solubilized KRAS neoantigens to come out of solution.
  • KRAS neoantigen stock comprising one or more solubilized KRAS neoantigens can be prepared.
  • the above procedures are exemplary, without limitation.
  • the mixing of the KRAS neoantigen stock and the lipid mixture may be performed by any suitable means.
  • the mixing is by shaking and/or mixing at 300 RPM for about 1 hour.
  • the mixing is performed using a Silverson AX60 high speed mixer (e.g. at 3000 rpm for a period of 15-45 minutes).
  • the "suitable solvent” is one that is suitable for solubilizing the neoantigen, adjuvants and/or amphipathic compound, and can be determined by the skilled person.
  • sodium phosphate buffer (0.2M, pH 6.0) or sodium phosphate buffer (0.1M, pH 7.0) may be used.
  • acetate buffer (0.1M, pH 9.5) may be used.
  • sodium carbonate buffer (100 mM, pH 10.5 ⁇ 0.5) may be used.
  • apolar protic solvent such as an alcohol (e.g.
  • tert-butanol, n-butanol, isopropanol, n-propanol, ethanol or methanol), water, acetate buffer, formic acid or chloroform may be used.
  • the same solvent can be used to solubilize each of the amphipathic compound, neoantigen and adjuvants, and the solubilized components are then mixed.
  • the neoantigen, adjuvants and amphipathic compound may be mixed prior to solubilization, and then solubilized together.
  • only one or more of the amphipathic compound, neoantigen or adjuvants are solubilized, and the non- solubilized component(s) are added.
  • Exemplary solvents that may be used for solubilizing the neoantigen include, for example and without limitation, zwitterionic solvents.
  • zwitterionic solvents include HEPES (4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid), MOPS (3-(N- Morpholino) propanesulfonic acid) and MES (2-(N-morpholino)ethanesulfonic acid).
  • exemplary solvents for solubilizing the neoantigen are aqueous salt solutions.
  • Salts provide useful properties in solubilizing the neoantigens, and it has also been recognized that certain salts provide stability 7 to the dried lipid/neoantigen preparation.
  • Non-limiting examples of such solvents include sodium acetate, sodium phosphate, sodium carbonate, sodium bicarbonate, potassium acetate, potassium phosphate, potassium carbonate, potassium bicarbonate, EDTA (Ethylenedi aminetetraacetic acid) and Tris-HCl.
  • the solvent is aqueous sodium acetate. It has been observed in the course of the present invention that sodium acetate imparts favorable properties to the dried lipid/neoantigen preparation for subsequent solubilization in the hydrophobic carrier. This is observed over a broad pH range (e.g. 6.0-10.5). For dissolution of multiple different neoantigens, a molarity 7 in the range of 50-200 mM may be preferred.
  • the sodium acetate may be 25-250 mM sodium acetate having a pH in the range of 6.0-10.5.
  • the solvent is 50 mM sodium acetate having a pH of 6.0 ⁇ 1.0.
  • the solvent is 100 mM sodium acetate having a pH of 9.5 ⁇ 1.0.
  • the solvent is 100 mM sodium acetate having a pH of 9.5 ⁇ 0.5.
  • the solvent is aqueous sodium phosphate.
  • the sodium phosphate may be 25-250 mM sodium phosphate having a pH in the range of 6.0- 8.0.
  • the solvent is 50 mM sodium phosphate having a pH of 7.0 ⁇ 1 .0.
  • the solvent is 100 mM sodium phosphate having a pH of 6.0 ⁇ 1.0.
  • the solvent is 50 mM sodium phosphate having a pH of 7.0.
  • the solvent is 100 mM sodium phosphate having a pH of 6.0.
  • neoantigen(s) and/or therapeutic agent(s) it may be advantageous to initially solubilize the neoantigen(s) and/or therapeutic agent(s) in a mild/weak acidic solvent (e.g. for basic neoantigen(s) and/or therapeutic agent(s)) or a mild/weak basic solvent (e.g. for acidic neoantigen(s) and/or therapeutic agent(s)).
  • exemplary acidic solvents that may be used include, without limitation, hydrochloric acid, acetic acid.
  • Exemplary basic solvents that may be used include, without limitation, sodium hydroxide, sodium bicarbonate, sodium acetate and sodium carbonate.
  • an exemplary solvent may be dimethyl sulfoxide (DMSO).
  • the disclosed methods allow multiple different therapeutic agents to be formulated in a single pharmaceutical composition.
  • the KRAS neoantigens and/or therapeutic agents may have different characteristics relating to isoelectric point, solubility (e.g. co-solubility), stability and/or immunogenicity, and do not need to be pre-selected based on these characteristics for compatibility in the disclosed method.
  • the KRAS neoantigens and/or therapeutic agents are peptide antigens (e.g., neoantigens).
  • the neoantigens do not need to be subjected to extensive peptide selection steps to determine suitability for use in the disclosed methods or to determine compatibility with other peptide antigens (e.g. cosolubility). More particularly, in an embodiment, the neoantigens do not need to be pre-selected based on any characteristic relating to isoelectric point, solubility, stability and/or immunogenicity 7 .
  • isoelectric point has its ordinary meaning in the art in that it refers to the pH at which a particular molecule (e.g. neoantigen and/or therapeutic agent) carries no net electrical charge.
  • the isoelectric point (pl) value can affect the solubility 7 of a molecule at a given pH.
  • amino acids that make up polypeptides/proteins may be positive, negative, neutral or polar in nature, and as a whole give a polypeptide/protein its overall charge.
  • polypeptides/proteins carry a net positive charge, whereas above their pl they carry' a net negative charge.
  • pl is often an important consideration in the formulation of pharmaceutical compositions, such as peptide-based formulations.
  • a number of algorithms for estimating isoelectric points have been developed, most of which use the Henderson-Hasselbalch equation with different pK values.
  • each of the solubilized neoantigens and/or therapeutic agents is at a concentration of between about 0. 1 mg/mL and 10 mg/mL in the sized lipid parti cl e/KRAS neoantigen mixture.
  • each of the solubilized neoantigens/therapeutic agents is at a concentration of at least about 0.5 mg/mL, 0.6 mg/mL, 0.7 mg/mL, 0.8 mg/mL, 0.9 mg/mL, 1.0 mg/mL, 1.1 mg/mL, 1.2 mg/mL, 1.3 mg/mL, 1.4 mg/mL, 1.5 mg/mL, 1.6 mg/mL, 1.7 mg/mL, 1.8 mg/mL, 1.9 mg/mL or 2.0 mg/mL.
  • the neoantigens are peptide antigens.
  • the methods involve the use of five or more different neoantigens, each of the different solubilized first and second neoantigens is at a concentration of about 0.5 mg/mL or 1.0 mg/mL.
  • the neoantigens are peptide antigens.
  • the neoantigen and adjuvants are reconstituted together or separately in sodium phosphate buffer with S 100 lipids and cholesterol (Lipoid, Germany). These vaccine components are then lyophilized to form a dry cake. Just prior to injection, the dry cake is resuspended in IS A51 VGoil (SEPPIC, France) to prepare a water-free oil-based vaccine composition.
  • the neoantigen and adjuvants are reconstituted together or separately in acetate buffer (0. IM, pH 9.5) with DOPC and cholesterol (Lipoid, Germany). These vaccine components are then lyophilized to form a ry cake. Just prior to injection, the dry cake is resuspended in IS A51 VG oil (SEPPIC, France) to prepare a water-free oil-based vaccine composition.
  • a conjugated neoantigen/T-helper epitope is reconstituted in 0.2% PEG-H2O with lipids DOPC and cholesterol (Lipoid, Germany).
  • the polyLC and lipid-based adjuvants are reconstituted in water, and then added to the neoantigen-lipid mixture.
  • These vaccine components are then lyophilized to form a dr ’ cake.
  • the dry cake is resuspended in IS A51 VG oil (SEPPIC, France) to prepare a water-free vaccine composition.
  • lipid vesicle particle may be used interchangeably with “lipid vesicle”.
  • a lipid vesicle particle refers to a complex or structure having an internal environment separated from the external environment by a continuous layer of enveloping lipids.
  • the expression “layer of enveloping lipids” can mean a single layer lipid membrane (e.g. as found on a micelle or reverse micelle), a bilayer lipid membrane (e.g. as found on a liposome) or any multilayer membrane formed from single and/or bilayer lipid membranes.
  • the layer of enveloping lipids is typically a single layer, bilayer or multilayer throughout its circumference, but it is contemplated that other conformations may be possible such that the layer has different configurations over its circumference.
  • the lipid vesicle particle may contain, within its internal environment, other vesicle structures (/. e. it may be multivesicular).
  • lipid vesicle particle encompasses many different types of structures, including without limitation micelles, reverse micelles, unilamellar liposomes, multilamellar liposomes and multivesicular liposomes.
  • the mean particle size of the liposomes is >80 nm.
  • the lipid vesicle particles have a mean particle size of 120 nm and a PDI of ⁇ 0.1.
  • the mean particle size of the liposomes used in the methods disclosed herein is in the range of 80 nm to 120 nm, with a PDI of ⁇ 0.1.
  • mean refers to the arithmetic mean of the particle size of the lipid vesicle particles in a given population. It is a synonym for average.
  • lipid vesicle particles may not be perfectly spherical, and therefore the “particle size” of a given vesicle particle may not be an exact measure of its diameter. Rather, the particle size may be defined by other means known in the art, including for example: the diameter of the sphere of equal area or the largest perpendicular distance between parallel tangents touching opposite sides of the particle (Feref s statistical diameter).
  • lipid vesicle particles there are several techniques, instruments and services that are available to measure the mean particle size of lipid vesicle particles, such as electron microscopy (transmission, TEM, or scanning, SEM), atomic force microscopy (AFM), Fourier-transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), powder X-ray diffraction (XRD), matrixassisted laser desorption/ionization time-of-flight mass spectrometry’ (MALDI- TOF-MS), nuclear magnetic resonance (NMR) and dynamic light scattering (DLS).
  • DLS is a well-established technique for measuring the particle size in the submicron size range, with available technology to measure particle sizes of less than 1 nm (LS Instruments, CH; Malvern Instruments, UK).
  • the mean particle size of ⁇ 120 nm is measured by DLS using a Malvern Zetasizer series instrument, such as for example the Zetasizer Nano S, Zetasizer APS, Zetasizer pV or Zetasizer AT machines (Malvern Instruments, UK).
  • the mean particle size of ⁇ 120 nm is measured by DLS using a Malvern Zetasizer Nano S machine.
  • Exemplary conditions and system settings may include:
  • the lipid vesicle particles have a mean particle size of less than or equal to 120 nanometers (i.e. 120 nm) and a PDI of less than or equal to 0.1 (z.e., 0.1). In an embodiment, the lipid vesicle particles have a mean particle size of ⁇ 115 nm, more particularly still ⁇ 110 nm and more particularly still ⁇ 100 nm. In an embodiment, the mean particle size of the lipid vesicle particles is between 50 nm and 120 nm. In an embodiment, the mean particle size of the lipid vesicle particles is between 80 nm and 120 nm.
  • the mean particle size of the lipid vesicle particles is between about 80 nm and about 115 nm, about 85 nm and about 115 nm, about 90 nm and about 115 nm, about 95 nm and about 115 nm, about 100 nm and about 115 nm or about 105 nm and about 115 nm.
  • the mean particle size of the lipid vesicle particles is about 80 nm, about 81 nm, about 82 nm, about 83 nm, about 84 nm, about 85 nm, about 86 nm, about 87 nm, about 88 nm, about 89 nm, about 90 nm, about 91 nm, about 92 nm, about 93 nm, about 94 nm, about 95 nm, about 96 nm, about 97 nm, about 98 nm, about 99 nm, about 100 nm, about 101 nm, about 102 nm, about 103 nm, about 104 nm, about 105 nm.
  • the mean particle size is 120 nm.
  • the PDI of the sized lipid vesicle particles is ⁇ 0.1. In an embodiment, the PDI of ⁇ 0.1 is measured by any instrument and/or machine suitable for measuring the PDI of lipid vesicle particles.
  • PDI size distribution is determined by DLS (Malvern Instruments, UK).
  • the PDI of ⁇ 0. 1 is measured by DLS using a Malvern Zetasizer series instrument, such as for example the Zetasizer Nano S, Zetasizer APS, Zetasizer pV or Zetasizer AT machines (Malvern Instruments, UK).
  • the PDI of ⁇ 0. 1 is measured by DLS using a Malvern Zetasizer Nano S machine. Exemplary conditions and system settings are described above in respect of determining mean particle size.
  • lipid vesicle particles have a mean particle size of 120 nm and a PDI of ⁇ 0. 1 means that it is possible that some lipid vesicle particles in a given population will have a particle size that is greater than 120 nm. This is acceptable so long as the mean particle size remains 120 nm and the PDI remains ⁇ 0. 1.
  • lipid vesicle particles of the present disclosure may be synonymous with lipid nanoparticles.
  • lipid nanoparticle refers to any nano-sized particle (i.e. having a diameter of between 1 nanometer and 1000 nanometers) formed by a lipid membrane.
  • size threshold for a nanoparticle material is limited to between 1 nanometer and 100 nanometers. This latter definition excludes lipid vesicle sizes that are encompassed by the present disclosure (e.g. lipid vesicle particles >100 nm in size), and to this extent is inconsistent with the term “lipid vesicle particles” as used in the present disclosure.
  • the lipid vesicle particles may take on various different shapes, and the shape may change at any given time (e.g. upon dry ing, sizing, and/or mixing procedures).
  • lipid vesicle particles are spherical or substantially spherical structures.
  • substantially spherical it is meant that the lipid vesicles are close to spherical, but may not be a perfect sphere.
  • Other shapes of the lipid vesicle particles include, without limitation, oval, oblong, square, rectangular, triangular, cuboid, crescent, diamond, cylinder or hemisphere shapes. Any regular or irregular shape may be formed.
  • a single lipid vesicle particle may comprise different shapes if it is multivesicular.
  • the outer vesicle shape may be oblong or rectangular while an inner vesicle may be spherical.
  • the lipid vesicle particles are formed from single layer lipid membranes, bilayer lipid membranes and/or multilayer lipid membranes.
  • the lipid membranes are predominantly comprised of and formed by lipids, but may also comprise additional components.
  • the lipid membrane may include stabilizing molecules to aid in maintaining the size and/or shape of the lipid vesicle particle. Any stabilizing molecule known in the art may be used so long as it does not negatively affect the ability of the lipid vesicle particles to be used in the disclosed methods.
  • lipid has its common meaning in the art in that it is any organic substance or compound that is soluble in nonpolar solvents, but generally insoluble in polar solvents (e.g. water). Lipids are a diverse group of compounds including, without limitation, fats, waxes, sterols, fat-soluble vitamins, monoglycerides, diglycerides, triglycerides and phospholipids. For the lipid vesicle particles herein, any lipid may be used so long as it is a membrane-forming lipid.
  • membrane-forming lipid it is meant that the lipid, alone or together with other lipids and/or stabilizing molecules, is capable of forming the lipid membrane of the lipid vesicle particle.
  • the lipid vesicle particles may comprise a single type of lipid or two or more different types of lipids.
  • the lipid or lipids of the lipid vesicle particle are amphiphilic lipids, meaning that they possess both hydrophilic and hydrophobic (lipophilic) properties.
  • any lipid as defined above may be used, particularly suitable lipids may include those with at least one fatty acid chain containing at least 4 carbons, and typically about 4 to 28 carbons.
  • the fatty acid chain may contain any number of saturated and/or unsaturated bonds.
  • the lipid may be a natural lipid or a synthetic lipid.
  • Non-limiting examples of lipids may include phospholipids, sphingolipids, sphingomyelin, cerobrocides, gangliosides, ether lipids, sterols, cardiolipin, cationic lipids and lipids modified with poly (ethylene glycol) and other polymers.
  • Synthetic lipids may include, without limitation, the following fatty acid constituents: lauroyl, myristoyl, palmitoyl, stearoyl, arachidoyl, oleoyl, linoleoyl, erucoyl, or combinations of these Patty acids.
  • the lipid is a phospholipid or a mixture of phospholipids.
  • a “phospholipid” is a member of a group of lipid compounds that yield on hydrolysis phosphoric acid, an alcohol, fatty acid, and nitrogenous base.
  • Phospholipids that may be used include for example, and without limitation, those with at least one head group selected from the group consisting of phosphoglycerol, phosphoethanolamine, phosphoserine, phosphocholine (e.g. DOPC; l,2Dioleoyl-sn-glycero-3- phosphocholine) and phosphoinositol.
  • the phospholipid may be phosphatidylcholine or a mixture of lipids comprising phosphatidylcholine.
  • the lipid may be DOPC (Lipoid GmbH, Germany) or Lipoid S100 lecithin.
  • a mixture of DOPC and unesterified cholesterol may be used.
  • a mixture of Lipoid SI 00 lecithin and unesterified cholesterol may be used.
  • the lipid vesicle particles comprise a synthetic lipid.
  • the lipid vesicle particles comprise synthetic DOPC.
  • the lipid vesicle particles comprise synthetic DOPC and cholesterol.
  • the cholesterol may be used in any amount sufficient to stabilize the lipids in the lipid membrane.
  • the cholesterol may be used in an amount equivalent to about 10% of the weight of phospholipid (e.g. in a DOPC:cholesterol ratio of 10: 1 w/w).
  • the cholesterol may stabilize the formation of phospholipid vesicle particles. If a compound other than cholesterol is used, one skilled in the art can readily determine the amount needed.
  • compositions disclosed herein comprise about 120 mg/ml of DOPC and about 12 mg/ml of cholesterol.
  • Sphingomyelin contains sphingosine, an amino alcohol with a long unsaturated hydrocarbon chain. A fatty acyl side chain is linked to the amino group of sphingosine by an amide bond, to form ceramide. The hydroxyl group of sphingosine is esterified to phosphocholine. Like phosphoglycerides, sphingomyelin is amphipathic.
  • Lecithin which also may be used, is a natural mixture of phospholipids typically derived from chicken eggs, sheep's wool, soybean and other vegetable sources.
  • Phospholipids can be purchased, for example, from Av anti lipids (Alabastar, AL, USA). Lipoid LLC (Newark, NJ, USA) and Lipoid GmbH (Germany), among various other suppliers.
  • the lipid vesicle particles are closed vesicular structures. They are typically spherical in shape, but other shapes and conformations may be formed and are not excluded. Exemplary embodiments of lipid vesicle particles include, without limitation, single layer vesicular structures (e.g. micelles) and bilayer vesicular structures (e.g. unilamellar or multilamellar vesicles), or various combinations thereof.
  • single layer vesicular structures e.g. micelles
  • bilayer vesicular structures e.g. unilamellar or multilamellar vesicles
  • PDI poly dispersity index
  • PDI is a measure of the size distribution of the lipid vesicle particles in a mixture.
  • the PDI can be calculated by determining the mean particle size of the lipid vesicle particles and the standard deviation from that size.
  • DLS dynamic light scattering
  • the PDI would be 0.0.
  • a PDI of ⁇ 0. 1 is required. Any mixture of lipid vesicle particles with a PDI >0. 1 is considered “poly disperse” and is not uniform in size.
  • PDI size distribution is determined by DLS (Malvern Instruments, UK).
  • the PDI of ⁇ 0. 1 is measured by DLS using a Malvern Zetasizer series instrument, such as for example the Zetasizer Nano S, Zetasizer APS, Zetasizer pV or Zetasizer AT machines (Malvern Instruments, UK).
  • the PDI of ⁇ 0.1 is measured by DLS using a Malvern Zetasizer Nano S machine. Exemplary conditions and system setings are described above in respect of determining mean particle size.
  • lipid vesicle particles have a mean particle size of 120 nm and a PDI of ⁇ 0. 1 means that it is possible that some lipid vesicle particles in a given population will have a particle size that is greater than 120 nm. This is acceptable so long as the mean particle size remains 120 nm and the PDI remains ⁇ 0.1.
  • single layer it is meant that the lipids do not form a bilayer, but rather remain in a layer with the hydrophobic part oriented on one side and the hydrophilic part oriented on the opposite side.
  • bilayer it is meant that the lipids form a two-layered sheet, typically with the hydrophobic part of each layer internally oriented toward the center of the bilayer with the hydrophilic part externally oriented.
  • multilayer is meant to encompass any combination of single and bilayer structures. The form adopted may depend upon the specific lipid that is used. Also, the forms used in the methods herein will depend on the size constraints of the disclosed method, i. e. a mean particle size of 120 nm and a PDI of ⁇ 0. 1.
  • the lipid vesicle particle is a bilayer vesicular structure, such as for example, a liposome.
  • Liposomes are completely closed lipid bilayer membranes. Liposomes may be unilamellar vesicles (possessing a single bilayer membrane), multilamellar vesicles (characterized by multimembrane bilayers whereby each bilayer may or may not be separated from the next by an aqueous layer) or multivesicular vesicles (possessing one or more vesicles within a vesicle).
  • a general discussion of liposomes can be found in Gregoriadis 1990; and Frezard 1999.
  • T-helper epitope may be added at any stage in the formulation process. For instance, one or more such additional components may be combined with the neoantigen, adjuvants and/or amphipathic compound either before or after solubilization, or added to the solubilized mixture. In another embodiment, the additional components may instead be added to or combined with the dried mixture of neoantigen, adjuvants and amphipathic compound, or combined with the hydrophobic carrier either before or after resuspension of the dry mixture of neoantigen, adjuvants and amphipathic compound in the hydrophobic carrier. In an embodiment, the T-helper epitope is added to the vaccine composition in the same way as the neoantigen. In an embodiment, the neoantigen and T-helper epitope are a fused peptide.
  • an emulsifier in the hydrophobic carrier to assist in stabilizing the vaccine components of the dry cake when they are resuspended in the hydrophobic carrier.
  • the emulsifier is provided in an amount sufficient to resuspend the dry mixture of neoantigen, adjuvants and amphipathic compound in the hydrophobic carrier and maintain the neoantigen, adjuvants and amphipathic compound in suspension in the hydrophobic carrier.
  • the emulsifier may be present at about 5% to about 15% weight/weight or weight/volume of the hydrophobic carrier.
  • Stabilizers such as sugars, anti-oxidants, or preservatives that maintain the biological activity or improve chemical stability 7 to prolong the shelf life of any of the vaccine components, may be added to such compositions.
  • the present invention relates to a method for preparing a dried preparation comprising lipids and neoantigens, said method comprising the steps of: (a) providing a lipid vesicle particle preparation comprising lipid vesicle particles and the at least one solubilized KRAS neoantigen and optionally additional solubilized therapeutic agent(s) (e.g., other neoantigens, small molecule, etc.); (b) sizing the lipid vesicle particle preparation to form a sized lipid vesicle particle preparation comprising sized lipid vesicle particles and at least one solubilized KRAS neoantigen and optional therapeutic agent, the sized lipid vesicle particles having a mean particle size of 120 nm and a poly dispersity index (PDI) of ⁇ 0.1; and (c) drying the lipid vesicle particle preparation to form a dried preparation comprising lipids and
  • PDI poly dis
  • the present invention relates to a method for preparing a dried preparation comprising lipids and neoantigens, said method comprising the steps of: (a) providing a lipid vesicle particle preparation comprising lipid vesicle particles and at least one solubilized first KRAS neoantigen and/or solubilized first therapeutic agent(s) (e.g., other neoantigens, small molecule, etc.); (b) sizing the lipid vesicle particle preparation to form a sized lipid vesicle particle preparation comprising sized lipid vesicle particles and the at least one solubilized first KRAS neoantigen and/or solubilized first therapeutic agent(s)), the sized lipid vesicle particles having a mean particle size of 120 nm and a poly dispersity index (PDI) of ⁇ 0.1; (c) mixing the sized lipid vesicle particle
  • PDI poly dispers
  • a first KRAS neoantigen is any one or more KRAS neoantigens which are used in the preparation of the nonsized lipid vesicle particle preparation (i.e. incorporated in the methods before the step of sizing the non-sized lipid vesicle preparation).
  • a second KRAS neoantigen is any one or more KRAS neoantigens that are used in the methods herein after preparation of the sized lipid vesicle particle preparation (i.e. incorporated in the methods after the step of sizing the nonsized lipid vesicle preparation).
  • the first KRAS neoantigen and the second KRAS neoantigen are different KRAS neoantigens, meaning that if a certain KRAS neoantigen is used as a first KRAS neoantigen, it is not used again as a second KRAS neoantigen in the preparation of the same composition.
  • the second KRAS neoantigens are of a different t pe than the first KRAS neoantigen (e.g.
  • first KRAS neoantigenic peptides in combination with one or more polynucleotides encoding KRAS neoantigenic peptides as second KRAS neoantigens, etc.).
  • first and second KRAS neoantigens are all of the same type (e.g. all peptide antigens or all polynucleotides encoding polypeptides etc.).
  • the first and second KRAS neoantigens may include some KRAS neoantigens of the same t pe and some KRAS neoantigens of different types, so long as none of the second KRAS neoantigens are identical to a first KRAS neoantigens.
  • therapeutic agent' is any molecule, substance or compound that is capable of providing a therapeutic activity, response or effect in the treatment or prevention of a disease, disorder or condition, including diagnostic and prophylactic agents (e.g., non-KRAS neoantigens, small molecule, etc.).
  • the term “therapeutic agent'’ does not include or encompass a T-helper epitope or an adjuvant, which are separately described in the present specification and are different components that may or may not be included in the methods, dried preparations, compositions, uses, and kits disclosed herein.
  • the methods disclosed herein are for formulating multiple different KRAS neoantigens in a single composition. In an embodiment, the methods disclosed herein are for formulating 2, 3, 4. 5, 6, 7. 8, 9, 10 or more different KRAS neoantigens in a single composition. In an embodiment, the methods disclosed herein are for formulating 2 to 10 different KRAS neoantigens in a single composition. In an embodiment, the methods disclosed herein are for formulating 2, 3, 4, or 5 different KRAS neoantigens in a single composition. In a particular embodiment, the methods disclosed herein are for formulating five different KRAS neoantigens in a single composition. In a particular embodiment, the methods disclosed herein are for formulating three different KRAS neoantigens in a single composition.
  • Step (a) of the disclosed methods is directed to providing a lipid vesicle particle preparation comprising lipid vesicle particles and at least one first KRAS neoantigen.
  • the lipid vesicle particles of the lipid vesicle particle preparation of step (a) may be any of the lipid vesicle particles as described herein.
  • the lipid vesicle particles may have undergone, or have been subjected to, processing steps that impart some level or degree of sizing, such as for example to provide a mean particle size and/or PDI outside the defined criteria of step (b), i.e., a mean particle size of a certain value >120 nm and/or a PDI of a certain value >0.1.
  • lipid vesicle particle preparations containing such lipid vesicle particles are encompassed by step (a) of the methods disclosed herein.
  • the lipid vesicle particles of the lipid vesicle particle preparation of step (a) are not sized. By this, it is meant that prior to step (b) of the methods herein, the lipid vesicle particles have not undergone, nor have they been subjected to, any processing steps to size the lipid vesicle particles.
  • the lipid vesicle particles of the lipid vesicle particle preparation of step (a) are of any size and of any distribution of size.
  • the lipid vesicle particles of the lipid vesicle particle preparation of step (a) are of a size and size distribution as would naturally result by preparing the lipid vesicle particles as described herein.
  • non-sized lipid vesicle particles refers to any embodiment of the lipid vesicle particles prior to sizing step (b).
  • non-sized lipid vesicle particles encompasses both of the embodiments described above whereby the lipid vesicle particles are not sized or the lipid vesicle particles have been subjected to processing steps that impart some level or degree of sizing.
  • the non-sized lipid vesicle particles may be of any size and of any distribution of size.
  • the preparation of step (a) will be referred to herein as a “non-sized lipid vesicle particle preparation”.
  • non-sized lipid vesicle particle preparation encompasses both embodiments whereby the lipid vesicle particles contained therein are not sized or the lipid vesicle particles contained therein have been subjected to processing steps that impart some level or degree of sizing.
  • the lipid vesicle particles of the “non-sized lipid vesicle preparation” may be of any size and of any distribution of size.
  • the non-sized lipid vesicle particles may be of any size within the range of 2 nm to 5 pm, or larger.
  • the mixture may comprise lipid vesicle particles of any number of different sizes within the range of 2 nm to 5 pm, or larger (i.e., any distribution of size).
  • the mean particle size of the non-sized lipid vesicle particles may be of any size within the range of 2 nm to 5 pm, or larger.
  • the non-sized lipid vesicle particles have a mean particle size of any size within the range of 2 nm to 5 pm, or larger. In an embodiment, the non-sized lipid vesicle particles have a mean particle size of > 120 nm. In an embodiment, the nonsized lipid vesicle particles have a mean particle size within the range 3 pm to 5 pm. In an embodiment, the non-sized lipid vesicle particles have a PDI of >0.1.
  • the mean particle size and PDI of the nonsized lipid vesicle particles may be determined, it is not necessary in the practice of the methods disclosed herein to determine, control or monitor the size and PDI of the non-sized lipid vesicle particles.
  • the non-sized lipid vesicle particles may be of any size and of any distribution of size.
  • Procedures for preparing lipid vesicle particles are well known in the art.
  • standard procedures for preparing lipid vesicle particles of any size may be employed.
  • conventional liposome forming processes may be used, such as the hydration of solvent-solubilized lipids.
  • Exemplary’ methods of preparing liposomes are discussed, for example, in Gregoriadis 1990; and Frezard 1999.
  • lipids in dry’ powder form may be added to a solution containing one or more solubilized first KRAS neoantigen.
  • the non-sized lipid vesicle particles are formed in the presence of the one or more first KRAS neoantigen to provide the non-sized lipid vesicle particle preparation.
  • lipids in dry powder form may be combined with one or more dry first KRAS neoantigens, and the dry combination may be solubilized together in an appropriate solvent. These embodiments may be performed with shaking and/or mixing (e.g., at 300 RPM for about 1 hour).
  • lipids may first be dissolved and mixed in an organic solvent. In embodiments where different types of lipid are used, this step will allow 7 a homogenous mixture of the lipids to be formed. In an embodiment, these steps may be carried out in chloroform, chloroform: methanol mixtures, tertiary butanol or cyclohexane. In an embodiment, the lipids are prepared at 10-20mg lipid/mL organic solvent; however, higher or lower concentrations may also be used. After mixing, the organic solvent is removed (e.g., by evaporation) to yield a lipid film.
  • the lipid film may then be frozen and lyophilized to yield a dry 7 lipid film.
  • the dry lipid film may then be hydrated with an aqueous solution containing one or more of the solubilized first KRAS neoantigen to provide the nonsized lipid vesicle particle preparation.
  • the step of hydration may be performed with shaking and/or mixing (e.g., at 300 RPM for about 1 hour).
  • an aqueous solution of lipids may be combined with a solution containing one or more solubilized first KRAS neoantigens.
  • one or more dry 7 first KRAS neoantigens may be added to, and solubilized in, the aqueous solution of lipids to provide a non-sized lipid vesicle preparation.
  • These embodiments may be performed with shaking and/or mixing (e.g., at 300 RPM for about 1 hour).
  • the above procedures are exemplary' methods for providing a non-sized lipid vesicle particle preparation comprising non-sized lipid vesicles and one or more first KRAS neoantigens.
  • the skilled person will recognize that other protocols may be used, and that the non-sized lipid vesicle preparation may be prepared using any acceptable combination of the above protocols and/or other protocols known in the art.
  • the non-sized lipid vesicle particle preparation at least some of one or more of the first KRAS neoantigens is encapsulated in the non-sized lipid vesicle particles. In an embodiment, all or a majority of one or more of the first KRAS neoantigens is encapsulated in the non-sized lipid vesicle particles.
  • each of the first KRAS neoantigens used is encapsulated in the non-sized lipid vesicle particles. In an embodiment, all or a majority of each of the first KRAS neoantigens used is encapsulated in the non-sized lipid vesicle particles.
  • the non-sized lipid vesicle particle preparation is mixed to disintegrate the lipids.
  • This step may be performed, for example and without limitation, by mixing at 3000 rpm for a period of 15-45 minutes or by mixing with glass beads on a shaker.
  • this mixing step is performed during the preparation of the non-sized lipid vesicle particles in the presence of the one or more first KRAS neoantigens (e.g. in the protocols described above).
  • this mixing step is performed after the non-sized lipid vesicle particle preparation is prepared, just prior to sizing.
  • this mixing step is performed both during the preparation of the non-sized lipid vesicle particles in the presence of the one or more first KRAS neoantigens and immediately prior to sizing.
  • the mixing is performed using a Silverson AX60 high speed mixer.
  • the pH is maintained at 9.5 ⁇ 1.0. In an embodiment, just prior to the step of sizing the non-sized lipid vesicle particle preparation, the pH is adjusted to 10.0 ⁇ 0.5. Depending on the lipids, first KRAS neoantigens and/or solvents that are used, it may be appropriate to make adjustments to these exemplary' pH values.
  • step (a) of the disclosed methods comprises (al) providing a KRAS neoantigen stock comprising the at least one solubilized first KRAS neoantigen, and optionally further comprising a solubilized adjuvant; and (a2) mixing the KRAS neoantigen stock with a lipid mixture to form the non-sized lipid vesicle preparation.
  • the “lipid mixture'’ may be a mixture of a single type of lipid (e.g. DOPC only) or it may be a mixture of any two or more different types of lipids (e.g. DOPC and cholesterol).
  • the lipid mixture may be provided as a dry powder mixture, a dry lipid film mixture or a mixture in solution.
  • the non-sized lipid vesicle particle preparation comprises non-sized lipid vesicle particles and at least one solubilized KRAS neoantigens and/or solubilized therapeutic agent(s).
  • the at least one solubilized KRAS neoantigen is a single KRAS neoantigen.
  • the at least one solubilized KRAS neoantigen is 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different solubilized KRAS neoantigens.
  • the at least one solubilized KRAS neoantigen is 2 to 10 different KRAS neoantigens. In an embodiment, the at least one solubilized KRAS neoantigen is 2, 3, 4 or 5 different first KRAS neoantigens. In a particular embodiment, the at least one solubilized KRAS neoantigen is five different solubilized KRAS neoantigens. In a particular embodiment, the at least one solubilized KRAS neoantigen is four different solubilized KRAS neoantigens. In a particular embodiment, the at least one solubilized KRAS neoantigen is three different solubilized KRAS neoantigens.
  • the at least one solubilized KRAS neoantigen and/or solubilized first therapeutic agent(s) is soluble at alkaline pH (i.e. pH > 7) during the membrane size extrusion procedures as described herein.
  • the at least one solubilized KRAS neoantigen and/or solubilized therapeutic agent(s) is soluble at alkaline pH during high pressure membrane extrusion with a 0.2 pm membrane, 0. 1 pm membrane and/or 0.08 pm membrane, such as when the extrusion is performed at 1000-5000 psi back pressure, or more particularly at about 5000 psi.
  • one or more of the solubilized KRAS neoantigen(s) are initially solubilized in a mild/weak basic solvent. In an embodiment, one or more of the solubilized KRAS neoantigen(s) are initially solubilized 50-250 mM sodium hydroxide. In an embodiment, the solvent is 200 mM sodium hydroxide.
  • solubilized KRAS neoantigen(s) may be solubilized in any of the solvents described herein. Based on the present disclosure, the skilled person could also identify other solvents that may be used that solubilize it similar characteristics to those described herein.
  • the lipids may be combined with the solubilized KRAS neoantigen(s) in the same or different solvents as are used for solubilizing one or more of the solubilized KRAS neoantigen(s).
  • the non-sized lipid vesicle particle preparation is prepared and provided in a sodium acetate or sodium phosphate solution.
  • the non-sized lipid vesicle particle preparation is prepared and provided in 25-250 mM sodium acetate having a pH in the range of 6.0-10.5 or 25-250 mM sodium phosphate having a pH in the range of 6.0-8.0.
  • the non-sized lipid vesicle particle preparation is prepared and provided in 50 mM sodium acetate having a pH of 6.0 ⁇ 1.0, 100 mM sodium acetate having a pH of 9.5 + 1.0, 50 mM sodium phosphate having a pH of 7.0 + 1.0 or 100 mM sodium phosphate having a pH of 6.0 ⁇ 1.0.
  • the non-sized lipid vesicle particle preparation is prepared and provided in 100 mM sodium acetate having a pH of 9.5 ⁇ 1.0.
  • the pH of the mixture is adjusted to 10 ⁇ 1.0. In an embodiment, the pH is adjusted to 10 ⁇ 0.5.
  • any other optional components may also be solubilized in the solvents described herein to prepare the nonsized lipid vesicle particle preparation.
  • T-helper epitopes and/or adjuvants may be added at any stage of preparing the solubilized KRAS neoantigen(s) or combining the solubilized KRAS neoantigen(s) with the lipids for the non-sized lipid vesicle particles.
  • the adjuvant and T— helper epitope may be added at any stage and in any order, independent of one another.
  • embodiments of the methods disclosed herein that involve the use of T-helper epitopes and/or adjuvants are those in which the KRAS neoantigen and/or therapeutic agent comprises at least one peptide antigen or a polynucleotide encoding an antigen.
  • one or more T-helper epitopes and/or adjuvants is encapsulated in the non-sized lipid vesicle particles.
  • the Thelper epitope comprises or consists of the modified Tetanus toxin peptide A16L (830 to 844; AQYIKANSKFIGITEL; SEQ ID NO: 1).
  • the adjuvant is a polyEC nucleotide adjuvant.
  • an adjuvant is added during the preparation of the non-sized lipid vesicle particle preparation such that the preparation comprises an adjuvant.
  • the adjuvant may be provided together with the KRAS neoantigen(s) stock.
  • the adjuvant Prior to being added to the KRAS neoantigen(s) stock, the adjuvant may be pre-solubilized in a solvent.
  • the solvent is water or any other solvent described herein.
  • the adjuvant is added to the KRAS neoantigen(s) in a dry form and mixed.
  • the adjuvant is a polyI:C nucleotide adjuvant.
  • Step (b) of the disclosed methods involves sizing the non-sized lipid vesicle particle preparation to form a sized lipid vesicle particle preparation comprising sized lipid vesicle particles and said at least one solubilized KRAS neoantigen(s).
  • the methods disclosed herein require sizing of the non-sized lipid vesicle particles to a mean particle size of 120 nm and a poly dispersity index (PDI) of ⁇ 0.1.
  • PDI poly dispersity index
  • the mean particle size of ⁇ 120 is measured by any instrument and/or machine suitable for measuring the mean particle size of lipid vesicle particles, such as by the methods above.
  • the mean particle size is determined by DLS (Malvern Instruments, UK).
  • the mean particle size of the sized lipid vesicle particles is between about 105 nm and about 115 nm, such as for example when the lipid vesicle particles are formed from DOPC/cholesterol (10: 1 w:w).
  • the non-sized lipid vesicle particle preparation may be sized by high pressure homogenization (microfluidizers), sonication or membrane based extrusion.
  • the sizing of the non-sized lipid vesicle particle preparation is performed using membrane based extrusion to obtain the sized lipid vesicle particles having a mean particle size of 120 nm and a PDI of ⁇ 0.1.
  • membrane based extrusion include passing the non-sized lipid vesicle particle preparation through a 0.2 m polycarbonate membrane and then through a 0.1 pm polycarbonate membrane, and then optionally through a 0.08 pm polycarbonate membrane.
  • Exemplary, nonlimiting protocols may include: (i) passing the non-sized lipid vesicle particle preparation 20-40 times through a 0.2 pm polycarbonate membrane, and then 10-20 times through a 0.1 m polycarbonate membrane; or (ii) passing the non-sized lipid vesicle particle preparation 20-40 times through a 0.2 pm polycarbonate membrane, then 10-20 times through a 0.1 pm polycarbonate membrane, and then 10-20 times through a 0.08 pm polycarbonate membrane.
  • the skilled would be well aware of different membranes and different protocols which may be used to attain the required mean particle size of 120 nm and PDI of ⁇ 0. 1.
  • the sizing of the non-sized lipid vesicle particle preparation is performed using high pressure homogenization to obtain sized lipid vesicle particles having a mean particle size of >120 nm and a PDI of >0.1.
  • the lipid vesicle particles obtained via high pressure homogenization can then be further sized down using membrane based extrusion.
  • Membrane based extrusion can include, for example, passing the sized lipid vesicle particle preparation 5-20 times through a 0. 1 pm polycarbonate membrane or, alternatively, 5-20 times through a 0.08 pm polycarbonate membrane, thereby attaining a mean particle size of 120 nm and PDI of ⁇ 0.1.
  • the sizing may be performed by passing a non-sized lipid vesicle particle preparation 25 times through a 0.2 pm polycarbonate membrane, and then 10 times through a 0.1 pm polycarbonate membrane. In another particular embodiment, the sizing may be performed by passing a non-sized lipid vesicle particle preparation 25 times through a 0.2 pm polycarbonate membrane, then 10 times through a 0.1 pm polycarbonate membrane, and then 15 times through a 0.08 pm polycarbonate membrane.
  • the membrane extrusion is typically performed under high back pressure.
  • the membrane extrusion is performed at 1000 to 5000 psi back pressure. Under these conditions, during the size extrusion process a back pressure of above 5000 psi may signal an issue with the solubility of one or more of the first KRAS neoantigen(s) and/or therapeutic agent(s).
  • Step (c) of the disclosed methods involves mixing the sized lipid vesicle particle preparation with at least one second neoantigen and/or second therapeutic agent to form a mixture.
  • the second KRAS neoantigen and/or second therapeutic agent may be any of the KRAS neoantigen and/or therapeutic agents as described herein.
  • the second KRAS neoantigen and/or second therapeutic agent is one that is not compatible with size extrusion procedures (e.g. precipitates under high pressure extrusion).
  • the second KRAS neoantigen and/or second therapeutic agent is one that tends to be stable (e.g. soluble) in acidic or slightly acidic pH and/or unstable (e.g. insoluble) in alkaline or slightly alkaline pH.
  • the second KRAS neoantigen(s) and/or second therapeutic agent(s) are short, positively charged hydrophobic peptides, such as for example peptides of 5-40 amino acids in length, more particularly 5-20 amino acids in length, and more particularly still 5-10 amino acids in length.
  • the at least one second KRAS neoantigen and/or second therapeutic agent is a single second KRAS neoantigen and/or second therapeutic agent.
  • the at least one second KRAS neoantigen and/or second therapeutic agent is 2, 3, 4, 5, 6, 7, 8, 9 or 10 different second KRAS neoantigens and/or second therapeutic agents.
  • the at least one second KRAS neoantigen and/or second therapeutic agent is 2, 3, 4 or 5 different second KRAS neoantigens and/or second therapeutic agents.
  • KRAS neoantigen and/or therapeutic agent is one or more neoantigens as described herein.
  • the one or more second KRAS neoantigens and/or second therapeutic agents are either solubilized in a solvent prior to mixing with the sized lipid vesicle particle preparation or the one or more second KRAS neoantigens and/or second therapeutic agents are solubilized upon being mixed with the sized lipid vesicle particle preparation.
  • the second KRAS neoantigens and/or second therapeutic agents may be added as a dry powder to a solution containing the sized lipid vesicle particle preparation or both the sized lipid vesicle particle preparation and dry second KRAS neoantigens and/or second therapeutic agents may be mixed together in a fresh solvent.
  • the individual second KRAS neoantigens and/or second therapeutic agents may be solubilized together in the same solvent or separate from each other in different solvents.
  • some of the agents may be solubilized together and others may be solubilized individually.
  • each of the second KRAS neoantigens and/or second therapeutic agents are solubilized separately as KRAS neoantigens and/or therapeutic agent stocks, and added sequentially to the sized lipid vesicle particle preparation.
  • the solvent for solubilizing the second KRAS neoantigen and/or second therapeutic agent may be one or more of the same solvents described herein for solubilizing the first KRAS neoantigen and/or first therapeutic agent. Based on the present disclosure, the skilled person could also identify other solvents that may be used that exhibit similar characteristics to those described herein.
  • the one or more the second KRAS neoantigens and/or second therapeutic agents are solubilized in a mild acid.
  • the mild acid may for example be mild acetic acid.
  • the one or more the second KRAS neoantigens and/or second therapeutic agents are solubilized in a 0. 1-0.5% (w/w) acetic acid solution, more particularly a 0.25% (w/w) acetic acid solution.
  • the second KRAS neoantigens and/or second therapeutic agents Similar to the first the second KRAS neoantigens and/or second therapeutic agents, as used herein “solubilized” with respect to the second KRAS neoantigens and/or second therapeutic agents means that the second KRAS neoantigens and/or second therapeutic agents are dissolved in a solvent. In an embodiment, this may be determined visually by the naked eye by observing a clear solution. A hazy solution is indicative of insolubility and is not desired for the methods disclosed herein as it may be problematic to forming a clear composition when the dried lipid/neoantigen preparation is subsequently solubilized in the hydrophobic carrier.
  • step (c) of the disclosed methods other optional components (e.g, T-helper epitope and/or adjuvant) may also be mixed with the sized lipid vesicle particle preparation.
  • other optional components e.g, T-helper epitope and/or adjuvant
  • T-helper epitopes and/or adjuvants may be added at any stage of preparing the solubilized KRAS neoantigens and/or therapeutic agents or mixing the KRAS neoantigens and/or therapeutic agents with the sized lipid vesicle particle preparation.
  • the adjuvant and T-helper epitope may be added at any stage and in any order, independent of one another.
  • embodiments of the methods disclosed herein that involve the use of T-helper epitopes and/or adjuvants are those in w hich the KRAS neoantigen and/or therapeutic agent comprises at least one peptide antigen or a polynucleotide encoding an antigen.
  • step (c) further comprises mixing a T-helper epitope with the sized lipid vesicle particle preparation.
  • the Thelper epitope comprises or consists of the modified Tetanus toxin peptide A16L (830 to 844; AQYIKANSKFIGITEL: SEQ ID NO: 1).
  • the adjuvant is a polyI:C nucleotide adjuvant.
  • the T-helper epitope may be prepared as an individual stock, solubilized in a suitable solvent.
  • the solvent is a mild acid such as, for example, mild acetic acid (e.g. 0.25% w/w).
  • the T-helper epitope may then be mixed with the sized lipid vesicle particle preparation before, after or concurrently with the one or more second KRAS neoantigens and/or therapeutic agents.
  • the T-helper epitope may be provided together in the same solution as the KRAS neoantigen and/or therapeutic agent stock comprising the second KRAS neoantigens and/or therapeutic agents.
  • the T-helper epitope Prior to being added to the KRAS neoantigen and/or therapeutic agent stock, the T-helper epitope may be pre-solubilized in a solvent, such as for example a mild acid (e.g. 0.25% w/w acetic acid).
  • a mild acid e.g. 0.25% w/w acetic acid
  • the T-helper epitope may be added to the KRAS neoantigens and/or therapeutic stock in a dry form and mixed.
  • the actual mixing of the sized lipid vesicle particle preparation and the one or more second KRAS neoantigens and/or therapeutic agents (and any other optional components) maybe performed under any suitable conditions for obtaining a generally homogenous mixture. However, the mixing should not be performed under aggressive conditions that might cause the sized lipid vesicle particles and/or KRAS neoantigens and/or therapeutic agents to precipitate out of solution.
  • the mixing may be performed with gentle shaking or stirring at 100-500 RPM for a period of 2-60 minutes.
  • the mixing may be performed by shaking/stirring/vortexing at 300 RPM for a period of about 3 minutes.
  • the mixing may be performed by shaking/stirring at 300 RPM for a period of about 15 minutes.
  • the mixture formed in step (c) may hereinafter be referred to as a “sized lipid vesicle particle/neoantigen mixture”.
  • step (c) or step (d) the sized lipid vesicle particle/KRAS neoantigen mixture is dried to form a dried lipid/neoantigen preparation as discussed in more detail in the section above.
  • the present invention relates to a method for preparing a pharmaceutical composition.
  • the pharmaceutical composition is prepared by first preparing a dried lipid/neoantigen preparation according to the methods disclosed herein, and then solubilizing the dried preparation in a hydrophobic carrier.
  • the present invention relates to a method for preparing a dried preparation comprising lipids and a KRAS neoantigen, said method comprising the steps of: (a) providing lipid vesicle particles having a mean particle size of 120 nm and a poly dispersity index (PDI) of ⁇ 0.1; (b) mixing the lipid vesicle particles with at least one solubilized KRAS neoantigen to form a mixture; and (c) dr ing the mixture formed in step (b) to form a dried preparation comprising lipids and a KRAS neoantigen and/or therapeutic agent.
  • the method steps are to be performed in this specific order, but the method may comprise additional steps, such as for example those described herein, without limitation.
  • PDI poly dispersity index
  • the mean particle size of ⁇ 120 is measured by any instrument and/or machine suitable for measuring the mean particle size of lipid vesicle particles. Mean is described in more detail above.
  • the lipid vesicle particles have a mean particle size of less than or equal to 120 nanometers (i.e. 120 nm) and a polydispersity index (PDI) of 0.1.
  • the lipid vesicle particles have a mean particle size of ⁇ 115 nm, more particularly still ⁇ 110 nm and more particularly still ⁇ 100 nm.
  • the mean particle size of the lipid vesicle particles is between 50 nm and 120 nm.
  • the mean particle size of the lipid vesicle particles is between 80 nm and 120 nm.
  • the mean particle size of the lipid vesicle particles is between about 80 nm and about 115 nm, about 85 nm and about 115 nm, about 90 nm and about 115 nm, about 95 nm and about 115 nm, about 100 nm and about 115 nm or about 105 nm and about 115 nm.
  • the mean particle size of the lipid vesicle particles is about 80 nm, about 81 nm, about 82 nm, about 83 nm, about 84 nm, about 85 nm, about 86 nm, about 87 nm, about 88 nm, about 89 nm, about 90 nm, about 91 nm, about 92 nm, about 93 nm, about 94 nm, about 95 nm.
  • the mean particle size is 120 nm.
  • the mean particle size of the lipid vesicle particles is between about 105 nm and about 115 nm, such as for example when the lipid vesicle particles are formed from DOPC/cholesterol (10: 1 w:w).
  • the mean particle size of the lipid vesicle particles is between about 90 nm and about 100 nm, such as for example when the lipid vesicle particles are formed from DOPC alone.
  • the requirement that the lipid vesicle particles have a mean particle size of 120 nm and a PDI of ⁇ 0. 1 means that it is possible that some lipid vesicle particles in a given population will have a particle size that is greater than 120 nm. This is acceptable so long as the mean particle size remains 120 nm and the PDI remains ⁇ 0.1.
  • Lipid vesicle particles as encompassed herein, having a mean particle size of 120 nm and a PDI of ⁇ 0.1 may be prepared and provided by any suitable means.
  • the lipid vesicle particles are prepared in a manner in which their size is controlled in order to achieve the mean particle size of 120 nm and a PDI of ⁇ 0.1.
  • lipid vesicle particles are subjected to one or more sizing steps or protocols to achieve the mean particle size of 120 nm and a PDI of ⁇ 0.1.
  • the lipid vesicle particles may be prepared and provided by any combination of controlling their size during manufacture, performing sizing steps and/or any other means available in the art.
  • the lipid vesicle particles must be subj ected to one or more active steps of sizing in order to achieve the mean particle size of 120 nm and a PDI of ⁇ 0.1.
  • the sizing is performed by filter-extrusion whereby lipid vesicle particles are passed through a filter membrane or a series of filter membranes (e.g. polycarbonate membranes) of appropriate pore size.
  • sized lipid vesicle particles refers to lipid vesicle particles that have been prepared by a means in which their size is controlled to attain a mean particle size of 120 nm and a PDI of ⁇ 0.1 and/or they are sized to meet the criteria of having a mean particle size of 120 nm and a PDI of ⁇ 0. 1 .
  • the skilled person will be well aware of techniques available for providing lipid vesicle particles having a mean particle size of 120 nm and a PDI of ⁇ 0.1.
  • non-sized lipid vesicle particles or a “non-sized lipid vesicle particle preparation” means that the lipid vesicle particles have not be subject to procedures that limit their size to meet the defined size criteria, and/or they do not have a mean particle size of 120 nm and a PDI of ⁇ 0.1.
  • the sized lipid vesicle particles may be prepared from a lipid precursor that naturally forms lipid vesicle particles of the required size.
  • the sized lipid vesicle particles may be prepared using Presome® (Nippon Fine Chemical, Japan).
  • Presome® is a dry' powder precursor made up of different lipid combinations.
  • Presome® is supplied ready to be wetted in a suitable buffer to prepare liposomes.
  • the liposomes formed from Presome® have an average particle size of about 93 nm, and sizing procedures (e.g. membrane extrusion, high pressure homogenization, etc.) can be used to achieve the required mean particle size of 120 nm and PDI of ⁇ 0.1.
  • Presome® may for example be wetted in sodium acetate, pH 9.0 ⁇ 0.5 to form liposomes.
  • the Presome® bulk dry powder may be made from DOPC/cholesterol (10:1 (w/w)) or DOPC alone.
  • lipid vesicle particles of any size may be employed.
  • conventional liposome forming processes may be used, such as the hydration of solvent-solubilized lipids. Exemplary methods of preparing liposomes are discussed, for example, in Gregoriadis 1990; and Frezard 1999.
  • the non-sized lipid vesicle particle preparation is subjected to a sizing procedure to obtain lipid vesicle particles having a mean particle size of 120 nm and a PDI of ⁇ 0.1.
  • the nonsized lipid vesicle particle preparation may be sized by high pressure homogenization (microfluidizers), sonication or membrane based extrusion.
  • the sized lipid vesicle particles may be prepared by adding the lipids to a suitable solvent (e.g. sodium phosphate, 50 mM, pH 7.0), shaking and/or stirring the lipid mixture (e.g. at 300 RPM for about 1 hour) and using membrane based extrusion to obtain the sized lipid vesicle particles.
  • a suitable solvent e.g. sodium phosphate, 50 mM, pH 7.0
  • shaking and/or stirring the lipid mixture e.g. at 300 RPM for about 1 hour
  • Exemplary’, non-limiting embodiments of membrane based extrusion include: (i) passing a non-sized lipid vesicle particle preparation 20-40 times through a 0.2 pm polycarbonate membrane, and then 10-20 times through a 0.1 pm polycarbonate membrane; or (ii) passing a non-sized lipid vesicle particle preparation 2040 times through a 0.2 pm polycarbonate membrane, then 10-20 times through a 0. 1 pm polycarbonate membrane, and then 10-20 times through a 0.08 pm polycarbonate membrane.
  • the sizing may be performed by passing a non-sized lipid vesicle particle preparation 25 times through a 0.2 pm polycarbonate membrane, and then 10 times through a 0. 1 pm polycarbonate membrane. In another particular embodiment, the sizing may be performed by passing a non-sized lipid vesicle particle preparation 25 times through a 0.2 pm polycarbonate membrane, then 10 times through a 0.1 pm polycarbonate membrane, and then 15 times through a 0.08 pm polycarbonate membrane.
  • the sized lipid vesicle particles may be provided in a suitable solvent for mixing with the solubilized KRAS neoantigen and/or therapeutic agent.
  • the solvent may be the same solvent that is used for solubilizing the KRAS neoantigen and/or therapeutic agent, or a different compatible solvent.
  • compatible it is meant that the solvent will not cause the solubilized KRAS neoantigen and/or therapeutic agent to come out of solution during the mixing of step (b).
  • the sized lipid vesicle particles may be provided in a dehydrated form and resuspended by adding the solution containing the solubilized KRAS neoantigen and/or therapeutic agent.
  • step (b) the sized lipid vesicle particles are mixed with at least one solubilized KRAS neoantigen and/or therapeutic agent to form a mixture.
  • the methods disclosed herein are for formulating a single type of KRAS neoantigen in a composition. In another embodiment, the methods disclosed herein are for formulating a mixture of multiple different KRAS neoantigens and/or therapeutic agents in a single composition. In an embodiment, the methods disclosed herein are for formulating a mixture of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more different KRAS neoantigens and/or therapeutic agents in a single composition. In an embodiment, the methods disclosed herein are for formulating a mixture of 10-20 different KRAS neoantigens and/or therapeutic agents in a single composition.
  • any other optional components may also be solubilized or mixed in the solvents described herein.
  • the sized lipid vesicle particles may be provided in the solvents described herein.
  • T-helper epitopes and/or adjuvants may be added at any stage of preparing the solubilized KRAS neoantigens and/or therapeutic agents or mixing the KRAS neoantigens and/or therapeutic agents with the sized lipid vesicle particles.
  • the adjuvant and T-helper epitope may be added at any stage and in any order, independent of one another.
  • embodiments of the methods disclosed herein that involve the use of T-helper epitopes and/or adjuvants are those in which the KRAS neoantigen and/or therapeutic agent comprises at least one peptide neoantigen or a polynucleotide encoding a neoantigen.
  • the Thelper epitope comprises or consists of the modified Tetanus toxin peptide A16L (830 to 844; AQYIKANSKFIGITEL; SEQ ID NO: 1).
  • the adjuvant is a polyLC nucleotide adjuvant.
  • step (b) may be performed using the same or different solvents as are used for preparing the sized lipid vesicle particles and/or for solubilizing the KRAS neoantigens and/or therapeutic agents, T-helper epitopes and/or adjuvants.
  • the mixing of step (b) is performed in a sodium acetate or sodium phosphate solution.
  • step (b) the mixing of step (b) is performed in 25-250 mM sodium acetate having a pH in the range of 6.0-10.5 or 25-250 mM sodium phosphate having a pH in the range of 6.0-8.0.
  • step (b) the mixing of step (b) is performed in 50 mM sodium acetate having a pH of 6.0 ⁇ 1.0, 100 mM sodium acetate having a pH of 9.5 ⁇ 1.0, 50 mM sodium phosphate having a pH of 7.0 ⁇ 1.0 or 100 mM sodium phosphate having a pH of 6.0 ⁇ 1.0.
  • step (b) the mixing of step (b) is performed in 50 mM sodium phosphate having a pH of 7.0, 100 mM sodium phosphate having a pH of 6.0, 50 mM sodium acetate having a pH of 6.0, or 100 mM sodium acetate having a pH of 9.5, 50 mM sodium carbonate having a pH of 10.5, or 100 mM sodium carbonate having a pH of 10.5.
  • the pH of the mixture is adjusted to 10 ⁇ 1.0.
  • the pH is increased to 10 ⁇ 1.0 when it was sodium acetate that was used as the solvent.
  • the actual mixing of the sized lipid vesicle particles and the at least one solubilized KRAS neoantigen and/or therapeutic agent may be performed under any suitable conditions for obtaining a generally homogenous mixture of the sized lipid vesicle particles and the KRAS neoantigen and/or therapeutic agent.
  • the mixing should not be performed under aggressive conditions that might cause the sized lipid vesicle particles, KRAS neoantigen, and/or therapeutic agents to precipitate out of solution.
  • the mixing may be performed with gentle shaking or stirring at 100-500 RPM for a period of 2-60 minutes.
  • the mixing may be performed by shaking/stirring/vortexing at 300 RPM for a period of about 3 minutes.
  • the mixing may be performed by shaking/stirring at 300 RPM for a period of about 15 minutes.
  • the KRAS neoantigens and/or therapeutic agents are only to be mixed with sized lipid vesicle particles (i.e. lipid vesicle particles having a mean particle size of 120 nm and a PDI of ⁇ 0. 1). None of the KRAS neoantigens and/or therapeutic agents are to be added to the lipid vesicle particles prior to sizing, such as for example during formation of the lipid vesicle particles.
  • the provided sized lipid vesicle particles contain no KRAS neoantigens or therapeutic agents. As described herein, this feature of the disclosed methods is relevant to obtaining a clear pharmaceutical composition.
  • step (c) the mixture of sized lipid vesicle particles and at least one solubilized KRAS neoantigen and/or therapeutic agent is dried to form a dried lipid/ KRAS neoantigen preparation.
  • Methods of drying are discussed in more detail above.
  • the present invention relates to a method for preparing a composition comprising one or more lipids, a negatively charged molecule (e.g., KRAS neoantigens that are polynucleotides encoding a KRAS neoantigenic peptide), a carrier comprising a continuous phase of a hydrophobic substance, and an ionizable aminoglycoside.
  • a negatively charged molecule e.g., KRAS neoantigens that are polynucleotides encoding a KRAS neoantigenic peptide
  • a carrier comprising a continuous phase of a hydrophobic substance, and an ionizable aminoglycoside.
  • nucleic acid molecules e.g., polynucleotides, oligonucleotides, mixed polymers, peptide nucleic acid, and the like
  • peptides e.g., polyaminoacids, polypeptides, proteins and the like
  • nucleotides e.g., pharmaceutical and biological compositions, that have negatively charged groups.
  • the present invention relates to a metho for preparing a composition
  • a composition comprising one or more lipids, a negatively charged molecule, a carrier comprising a continuous phase of a hydrophobic substance, and an ionizable aminoglycoside.
  • the method can comprise one or more of the following steps: (a) dissolving the one or more lipids in one or more organic solvents and optionally an aqueous solvent to create a lipid solution, (b) adding the negatively charged molecule to the lipid solution formed in step (a) and mixing; (c) adding the ionizable aminoglycoside to the mixture formed in step (b) and mixing; (d) optionally, adding additional amount of the organic solvent(s) or aqueous solvent to the mixture formed in step (c) thereby the overall Wt/Wt or V/V percentage ratio of organic: aqueous solvent or aqueous: organic solvent in the mixture is between 20-50%; (e) drying the mixture formed in step (c) or (d) to generate a dried preparation; and (I) dissolving the dried preparation in the carrier comprising a continuous phase of a hydrophobic substance, thereby generating said composition.
  • the negatively charged molecule may be present in the composition of the present disclosure at about 0.01-2 mg/mL. In some embodiments, the negatively charged molecule (e.g., polynucleotide) may be present in the composition of the present disclosure at about 0.01 mg/mL, 0.02 mg/mL, 0.03 mg/mL, 0.04 mg/mL 0.05 mg/mL, 0.06 mg/mL, 0.07 mg/mL, 0.08 mg/mL, 0.09 mg/mL, 0.1 mg/mL, 0.11 mg/mL, 0.12 mg/mL, 0.13 mg/mL, 0.14 mg/mL 0.15 mg/mL, 0.16 mg/mL, 0.17 mg/mL, 0.18 mg/mL, 0.19 mg/mL, 0.2 mg/ml, 0.25 mg/mL, 0.3 mg/mL, 0.35 mg/mL, 0.4 mg/mL, 0.45 mg/
  • the negatively charged molecule (e.g., polynucleotide) may be present in the composition of the present disclosure at about 0.01-0.1 mg/mL, 0.05-0.2 mg/mL, 0. 1-0.3 mg/mL, 0.2-0.4 mg/mL, 0.3-0.6 mg/mL, 0.4-0.8 mg/mL, 0.5- 1 mg/mL, 0.8-1.2 mg/rnL, 1-1.5 mg/rnL, or 1-2 mg/mL. In one embodiment, the negatively charged molecule (e.g., polynucleotide) may be present in the composition of the present disclosure at about 0. 1 mg/mL.
  • the composition disclosed herein comprise a single type of negatively charged molecule in a composition.
  • the composition disclosed herein comprise a mixture of multiple different negatively charged molecules in a single composition.
  • the composition disclosed herein comprise a mixture of 2, 3. 4, 5, 6. 7, 8, 9. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21. 22. 23. 24. 25. 26. 27. 28. 29, 30 or more different negatively charged molecules in a single composition.
  • a lipid component or mixture of lipid components such as a phospholipid (e.g., DOPC) and cholesterol
  • a lipid component or mixture of lipid components may be solubilized in one or more organic solvent, such as tert-butanol, ethanol, methanol, chloroform, or a mixture of chloroform and methanol, tert-butanol, a mixture of tert-butanol and ethanol, a mixture of tert-butanol and chloroform, or mixture of tert-butanol and water followed by filtering (e.g., a PTFE 0.2 pm filter) and drying, e.g., by rotary evaporation, freeze-drying to remove the solvents.
  • organic solvent such as tert-butanol, ethanol, methanol, chloroform, or a mixture of chloroform and methanol, tert-butanol, a mixture of tert-butanol and
  • the organic solvent(s) is present in an amount sufficient to prevent the one or more lipids from forming lipid vesicle particles in the lipid solution.
  • the organic solvent(s) and the aqueous solvent are present in a percentage ratio of organic: aqueous solvent or aqueous: organic solvent ratio between 20- 50%.
  • the organic solvent(s) and the aqueous solvent are present in a percentage ratio of organic: aqueous solvent or aqueous: organic solvent ratio of about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50%.
  • the organic solvent is tert-butanol.
  • the lipid solution comprises about 20%-50% tert-butanol.
  • the lipid solution comprises about 20% tert-butanol, about 25% tert-butanol, about 30% tert-butanol, about 35% tert-butanol, about 40% tert-butanol, about 45% tert-butanol, or about 50% tertbutanol.
  • the lipid solution comprises about 30% tert-butanol.
  • Hydration of the resulting lipid mixture may be effected by e.g., injecting the lipid mixture into an aqueous solution or sonicating the lipid mixture and an aqueous solution.
  • the lipid components form single bilayers (unilamellar) or multiple bilayers (multilamellar) surrounding a volume of the aqueous solution with which the lipid components are hydrated.
  • the lipid vesicle particles are then dehydrated, such as by freeze-dr ing or lyophilization, spray freeze-drying, spray drying, or rotary evaporation, and subsequently reconstituted with an aqueous solution.
  • the lipid vesicle particles are combined with the carrier comprising a continuous hydrophobic phase.
  • the hydrophobic phase is essentially water-free. This can be done in a variety of ways.
  • the carrier is essentially water-free, and is composed solely of a hydrophobic substance or a mixture of hydrophobic substances (e.g., use of a 100% mineral oil earner)
  • lipid vesicle particles may simply be mixed with the hydrophobic substance, or if there are multiple hydrophobic substances, mixed with any one or a combination of them.
  • An exemplary preparation method is further described below. In this method, any negatively charged molecules (e.g., polynucleotide) presented in an aqueous solution (e.g., sterile RNase free water) is added to a suitable buffer solution.
  • lipid vesicle particles in sterile RNase free water e.g., particle size ⁇ 120 nm, poly dispersity index (PDI) ⁇ 0. 1
  • PDI poly dispersity index
  • formulation method A is prepared with different polymers and transfection agents by adding the respective stock solutions to the polynucleotide loaded lipid vesicle particles obtained from the above step, mixed well gently by e.g., hand or vortexing for 30 seconds.
  • the final formulation (with and without different polymers and transfection agents) is then dehydrated by freeze-drying or lyophilization or spray-drying; and subsequently reconstituted with an aqueous solution or with a hydrophobic substance or a mixture of hydrophobic substances (e.g., use of a 100% mineral oil carrier) prior to administration.
  • the carrier comprising a continuous phase of a hydrophobic substance contains a discontinuous aqueous phase
  • the carrier will ty pically take the form of an emulsion of the aqueous phase in the hydrophobic phase, such as a water-in-oil emulsion.
  • Such compositions may contain an emulsifier to stabilize the emulsion and to promote an even distribution of the lipid vesicle particles (e.g., liposomes).
  • emulsifiers may be useful even if water-free carrier is used, for the purpose of promoting an even distribution of the lipid vesicle particles (e.g., liposomes) in the carrier.
  • Typical emulsifiers include mannide oleate (ArlacelTM A), lecithin, TweenTM 80, and SpansTM 20, 80, 83 and 85.
  • the weight to volume ratio (w/v) of hydrophobic substance to emulsifier is in the range of about 5: 1 to about 15: 1 with a ratio of about 10: 1 being preferred.
  • the lipid vesicle particles may be added to the finished emulsion, or they may be present in either the aqueous phase or the hydrophobic phase prior to emulsification.
  • polynucleotide includes the polynucleotide in naked form including, for example, in an mRNA or a plasmid such as an expression plasmid, or in a live vector such as a bacteria or virus.
  • lipid or lipid-mixture is dissolved in 20-50% or 100% tert-butanol or ethanol by, e.g., simple vortexing or by shaking at 150-300 RPM in an incubator shaker at room temperature or at 37°C until dissolved.
  • any negatively charged molecules e.g., polynucleotide
  • aqueous solution e.g., sterile RNase free water
  • suitable buffer solution ed with another positively charged molecule/adjuvant with known cryoprotectants
  • the overall Wt/Wt or V/V percentage ratio of organic: aqueous solvent mixture aqueous:organic solvent mixture in the final formulation prior to dehydration is maintained between 20-50%.
  • the one or more lipids when dissolved in the organic solvent(s) are presented in clear solution form.
  • the aqueous solution(s) containing the negatively charged molecule (e.g., polynucleotide) and/or ionizable aminoglycoside the one or more lipids form lipid vesicle particles.
  • the formed lipid vesicle particles are then dehydrated, such as by freeze-dry ing or lyophilization or spray-drying; and subsequently reconstituted with an aqueous solution or with a hydrophobic substance or a mixture of hydrophobic substances (e.g., use of a 100% mineral oil carrier) prior to administration.
  • this method has several advantages over the conventional lipid nanoparticles method including increased solubility and encapsulation of hydrophobic molecules, decreased freeze-drying time, and better oil reconstitution characteristics for hydrophobic or complex compounds.
  • More than one polynucleotide may be incorporated into the composition.
  • two or more polynucleotides encoding different proteins may be incorporated into the composition, or a polynucleotide encoding a protein may be present as well as a polynucleotide encoding an antisense RNA or interfering RNA. Proteins may be expressed as the fusion product of two different polynucleotides. More than one polynucleotide may be under the control of the same regulatory elements, e.g., two or more polynucleotides under transcriptional control of a single promoter.
  • the polynucleotide is present in the aqueous solution used to hydrate the components that are used to form the lipid bilayers of the lipid vesicle particles (e.g., liposomes).
  • the polynucleotide will be encapsulated in the lipid vesicle particles , present in its aqueous interior. If the resulting liposomes are not washed or dried, such that there is residual aqueous solution present that is ultimately mixed with the carrier comprising a continuous phase of a hydrophobic substance, it is possible that additional polynucleotide may be present outside the lipid vesicle particles in the final product.
  • the polynucleotide may be mixed directly with the lipid vesicle particles or with the components used to form the lipid bilayers of the lipid vesicle particles, prior to hydration with the aqueous solution.
  • the polynucleotide may instead be mixed with the carrier comprising a continuous phase of a hydrophobic substance, before, during, or after the carrier is combined with the lipid vesicle particles.
  • the carrier is an emulsion
  • the polynucleotide may be mixed with either or both of the aqueous phase or hydrophobic phase prior to emulsification.
  • the polynucleotide may be mixed with the carrier after emulsification.
  • the technique of combining the polynucleotide with the earner may be used together with encapsulation of the polynucleotide in the lipid vesicle particles as described above, such that polynucleotide is present both within the lipid vesicle particles and in the carrier comprising a continuous phase of a hydrophobic substance.
  • the composition may comprise about 0.1 to 5 mg polynucleotide per ml of the composition and about I mg to 300 mg lipid vesicle particles per ml of the composition.
  • the particle size of the lipid vesicle particles prepared according to the methods described herein prior to dehydration is about 100-5000 nm (0.1 to 5 microns).
  • the particle size of the lipid vesicle particles may vary depending upon the ratio of organic: aqueous or aqueous: organic solvent composition in the final formulation prior to dehydration and also on the choice of organic solvent.
  • the particle size of the lipid vesicle particles prepared according to the methods described herein prior to dehydration is about 100-200 nm.
  • the mean particle size of the lipid vesicle particles prepared according to the methods described herein prior to dehydration is about 100-1000 nm (0. 1 to 1 microns). In some embodiments, the mean particle size of the lipid vesicle particles prepared according to the methods described herein prior to dehydration is about 100 nm, about 115 nm, about 120 nm, about 125 nm, about 130 nm, about 135 nm, about 140 nm, about 145 nm, about 150 nm, about 160 nm, about 170 nm.
  • the lipid vesicle particles prepared according to the methods described herein after reconstitution in a earner comprising a continuous phase of a hydrophobic substance form reverse micelles.
  • the particle size of the lipid vesicle particles prepared according to the methods described herein after reconstitution in a carrier comprising a continuous phase of a hydrophobic substance is about 1-50 nm. In some embodiments, the particle size of the lipid vesicle particles prepared according to the methods described herein after reconstitution in a carrier comprising a continuous phase of a hydrophobic substance is about 1-10 nm, 2-8 nm, 4-9 nm, 5-10 nm, 6-12 nm, 7-15 nm, 8-20 nm, 10-30 nm, 15-40 nm, 20-45 nm, or 30-50 nm.
  • the mean particle size of the lipid vesicle particles prepared according to the methods described herein after reconstitution in a carrier comprising a continuous phase of a hydrophobic substance is about 1-20 nm. In some embodiments, the mean particle size of the lipid vesicle particles prepared according to the methods described herein after reconstitution in a carrier comprising a continuous phase of a hydrophobic substance is about 1 nm, about 2 nm, about 3 nm, about 4 nm, about 5 nm, about 6 nm, about 7 nm, about 8 nm, about 9 nm, about 10 nm, about 11 nm, about 12 nm, about 13 nm, about 14 nm, about 15 nm, about 16 nm, about 17 nm, about 18 nm, about 19 nm, or about 20 nm.
  • PDI of a lipid vesicle particle prepared according to the methods described herein prior to dehydration is between about 0.1 to about 0.7. In some embodiments, PDI of a lipid vesicle particle prepared according to the methods described herein prior to dehydration is about 0. 1 to about 0.4, about 0.2 to about 0.5, about 0.3 to about 0.6, about 0.4 to about 0.7, or about 0.5 to 0.7. In some embodiments, PDI of a lipid vesicle particle described herein is about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, or about 0.7.
  • the adjuvant(s) and/or additional component(s) can be incorporated in the composition together with the polynucleotide at the same processing step, or separately, at a different processing step.
  • the polynucleotide and the adjuvant(s) and/or additional component(s) may both be present in the aqueous solution used to hydrate the lipid bilayer-forming components, such that both the polynucleotide and adjuvant(s) and/or additional component(s) become encapsulated in the lipid vesicle particles.
  • the polynucleotide may be encapsulated in the lipid vesicle particles, and the adjuvant(s) and/or additional component(s) mixed with the carrier comprising a continuous phase of a hydrophobic substance. It will be appreciated that many such combinations are possible.
  • the polynucleotide and the adjuvant(s) and/or additional component(s) may be in the form of a complex, in which they are in intimate contact at least prior to incorporation into the composition.
  • Complexing may but need not necessarily involve a chemical linkage, such as covalent bonding.
  • compositions as described herein may be formulated in a form that is suitable for any administration routes, such as oral, nasal, aerosol, rectal or parenteral administration.
  • Parenteral administration includes intravenous, intraperitoneal, intradermal, subcutaneous, intramuscular, transepithelial, intrapulmonary, intrathecal, and topical modes of administration.
  • the compositions may be formulated for systemic or localized distribution in the body of the subject.
  • the preferred routes are intramuscular, subcutaneous and intradermal to achieve a depot effect. In practice, a depot effect is achieved when the therapeutic agent remains at the site of injection for more than about one hour.
  • the injection site may be anywhere close to, or directly into a lymph node, for example. Alternatively, the injection site may be directly into a spleen, a tumor or other diseased tissue.
  • the volume that may be injected is within the professional judgment of the clinician. The volume depends on the injecting device used and the site of injection. When the injection is intramuscularly or subcutaneous, the injection volume may be about 1 mL. When needleless injection is used, the volume may be as low as 0.01 mL. The volume may be increased by injecting multiple sites. Suitable injection volumes may be about 0.01 mL, about 0.02 mL, about 0.05 mL, about 0.
  • formulations of the present disclosure may create stable depot at the site of delivery’ that protects active ingredient (e.g., a polynucleotide) from degradation by nucleases or by renal or hepatic clearance for an extended period of time (e.g., up to one week or two weeks).
  • active ingredient e.g., a polynucleotide
  • formulations of the present disclosure may also provide controlled and prolonged exposure of active ingredients (e.g., a polynucleotide) to the cells at the delivery' site allowing a direct delivery of treatment into target and limits drug distribution specifically to its target site, thus avoiding systemic effects.
  • the term ‘‘about” means reasonably close.
  • “about” can mean within an acceptable standard deviation and/or an acceptable error range for the particular value as determined by one of ordinary' skill in the art, which will depend on how the particular value is measured. Further, when whole numbers are represented, about can refer to decimal values on either side of the whole number.
  • the term “about” encompasses all of the exemplary values between the one particular value at one end of the range and the other particular value at the other end of the range, as well as reasonably close values beyond each end.
  • the term “about” is used to represent a deviation of ⁇ 2.0 nm, so long as it would not cause the mean particle size to exceed 120 nm. Also, the term “about” is meant to encompass any decimal number of the indicated mean particle size.
  • KRAS peptide comprising the single amino acid substitution G12V, KRAS-G12V5-14 neoantigen (KLVVVGAVGV; SEQ ID NO: 10), was synthesized by Genscript.
  • a formulation table describing preparation of the DPX-KRAS peptide formulation used in the present Example is displayed in Table 2.
  • KRAS peptide stock solution KRAS G12Vs-i4 (SEQ ID NO: 10) was prepared separately in sterile water.
  • the KRAS peptide was added to 250 mM sodium acetate, pH 9.5 and gently mixed by hand.
  • the pH of the formulation was adjusted to 7.0-7.5 with 0.1M sodium hydroxide/0.25% acetic acid and the DNA based polyEC polynucleotide adjuvant (dldC) was then added.
  • the final formulation volume was then filled to 1.0 mL by adding sterile water, mixed well by vortexing for 5 seconds. The vial was then partially stoppered and freeze-dried.
  • the freeze-dried cake was then reconstituted with 0.45 mL of Montanide ISA 51 VG oil diluent to obtain final concentrations of DOPC/Chol 132 mg/mL, KRAS peptide 1 mg/mL, dldC adjuvant 0.4 mg/mL, T-helper peptide A16L 0.5 mg/mL and sodium acetate 0.1 M. 50 pL of this formulation was injected subcutaneously (s.c.) into HLA- A2 transgenic mice.
  • Lymph node cells were stimulated in an interferon gamma (IFN-y) ELISPOT plate (BD Biosciences) with syngeneic dendritic cells unloaded (background) or loaded with an irrelevant peptide (SEQ ID NO: 8), WT-KRAS5-14 (SEQ ID NO: 11). or KRAS-G12V 5 -i4 neoantigen (SEQ ID NO: 10).
  • IFN-y interferon gamma
  • ELISPOT plate BD Biosciences
  • Splenocytes were stimulated in an interferon gamma (IFN-y) ELISPOT plate (BD Biosciences) with an irrelevant peptide (SEQ ID NO: 8), WT-KRAS5-14 (SEQ ID NO: 11), or KRAS-GI2V5-14 neoantigen (SEQ ID NO: 10) or remained unstimulated (background).
  • IFN-y interferon gamma
  • SEQ ID NO: 8 irrelevant peptide
  • WT-KRAS5-14 SEQ ID NO: 11
  • KRAS-GI2V5-14 neoantigen SEQ ID NO: 10
  • SEQ ID NO: 1 Modified Tetanus toxin peptide A16L (amino acids 830 to 844), t- helper epitope, amino acid sequence
  • SEQ ID NO: 3 CpG oligodeoxynucleotide (ODN). nucleic acid sequence
  • AKXVAAWTLKAAA wherein X stands for Cha is (L-cyclohexylalanine)
  • SEQ ID NO: 7 F21E tetanus toxoid peptide (amino acids 947 to 967), t-helper epitope, amino acid sequence

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  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

La présente demande concerne des compositions vaccinales comprenant un ou plusieurs néoantigènes, le ou les néoantigènes étant associés à KRAS, un véhicule pharmaceutiquement acceptable et, éventuellement, un adjuvant et/ou un épitope T-auxiliaire, ainsi que des procédés d'utilisation de telles compositions dans le traitement du cancer.
PCT/US2023/036862 2022-11-07 2023-11-06 Compositions vaccinales comprenant un néo-antigène de kras WO2024102332A1 (fr)

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US202263423335P 2022-11-07 2022-11-07
US63/423,335 2022-11-07

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