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WO2025081013A1 - Compositions de protéine d'ouverture de jonction et de nanoparticules et leurs utilisations - Google Patents

Compositions de protéine d'ouverture de jonction et de nanoparticules et leurs utilisations Download PDF

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WO2025081013A1
WO2025081013A1 PCT/US2024/051011 US2024051011W WO2025081013A1 WO 2025081013 A1 WO2025081013 A1 WO 2025081013A1 US 2024051011 W US2024051011 W US 2024051011W WO 2025081013 A1 WO2025081013 A1 WO 2025081013A1
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composition
cancer
protein
lipid
subject
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Adrian SIMPSON
Amit P. Khandhar
Steven James Reed
Jiho Kim
Gaurav GULATI
Darrick Carter
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HDT Bio Corp
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HDT Bio Corp
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5115—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars

Definitions

  • DSG2 Desmoglein 2 positive solid tumors are difficult to treat for many reasons.
  • the tumor cell tight junctions have the effect of reducing the efficacy of chemotherapeutic agents due to a lack of physical infiltration of the agent into the tumor. Accordingly, there is a great unmet need for compositions that yield therapeutically desired effects in the treatment of cancer.
  • compositions wherein the compositions comprise: (a) a nanoparticle comprising a bioactive agent; and (b) a junction opener protein conjugated to the nanoparticle, wherein the junction opener protein comprises: (i) a shaft domain; (ii) a knob domain; (iii) a multimerization domain; and (iv) a conjugatable moiety.
  • compositions comprising: a nanoparticle comprising a lipid carrier, wherein the lipid carrier comprises a hydrophobic core, wherein the hydrophobic core comprises: (i) a lipid for inclusion in a hydrophilic surface of the nanoparticle, wherein the lipid comprises a cationic lipid, a PEGylated lipid, a phospholipid or a combination thereof; (ii) a hydrophilic surfactant; and (hi) a hydrophobic surfactant; and (iv) a junction opener protein, wherein the junction opener protein in conjugated to the nanoparticle.
  • kits for treating a cancer in a subject comprise: (a) administering to a subject a first composition comprising a junction opener protein, wherein the junction opener protein comprises: (i) a shaft domain; (ii) a knob domain; and (iii) a multimerization domain; and (b) administering to the subject a second composition comprising nanoparticles comprising squalene, squalane, dehydroisosqualene, or a combination thereof.
  • nucleic acids comprising a nucleotide sequence encoding a junction opener protein and a nucleotide sequence encoding a self-replicating nucleic acid, wherein the junction opener protein comprises: (a) a shaft domain; (b) a knob domain; and (c) a multimerization domain.
  • FIGURES 1A-1W show schematic representations of nanoparticle (NP) carriers.
  • FIG. 1A shows an oil-in-water emulsion.
  • FIG. IB shows a nanostructured lipid carrier (NLC).
  • FIG. 1C shows a solid inorganic nanoparticles dispersed within a membrane of the nanoparticle.
  • FIG. ID shows an oil-in-water emulsion with bioactive agents.
  • FIG. IE shows a nanoparticle containing an inorganic particles and bioactive agents within a membrane of the nanoparticle.
  • FIG. IF shows a nanoparticle comprising solid inorganic nanoparticles, and bioactive agents bound or conjugated to inside of the membrane of the nanoparticle.
  • FIG. 1G shows a nanoparticle comprising solid inorganic nanoparticles, and bioactive agents bound or conjugated to the outside of the membrane.
  • FIG. 1H shows a nanoparticle comprising bioactive agents bound or conjugated to inside of the membrane of the nanoparticle.
  • FIG. II shows a nanoparticle comprising bioactive agents bound or conjugated to the outside of the membrane.
  • FIG. 1J and IQ show a nanoparticle having a pegylated lipid, a liquid oil core, and inorganic nanoparticles.
  • FIG. IK and 1R show an oil-in-water emulsion with a nanoparticle having a pegylated lipid, a liquid oil core, and bioactive agents within the membrane of the nanoparticle.
  • FIG. 1G shows a nanoparticle comprising solid inorganic nanoparticles, and bioactive agents bound or conjugated to the outside of the membrane.
  • FIG. 1H shows a nanoparticle comprising bioactive agents bound or conjugated to inside of
  • IL and IS show a nanoparticle comprising a pegylated lipid, a liquid oil core, and inorganic nanoparticles and bioactive agents within the membrane of the nanoparticle.
  • FIG. IM and IT show a nanoparticle comprising a pegylated lipid membrane, a liquid oil core, inorganic nanoparticles within the membrane of the nanoparticle, and bioactive agents bound or conjugated to the inside of the membrane.
  • FIG. IN and 1U show a nanoparticle comprising a pegylated lipid, a liquid oil core, inorganic nanoparticles within the membrane of the nanoparticle, and bioactive agents bound or conjugated to the outside of the membrane.
  • FIG. IM and IT show a nanoparticle comprising a pegylated lipid membrane, a liquid oil core, inorganic nanoparticles within the membrane of the nanoparticle, and bioactive agents bound or conjugated to the outside of the membrane.
  • IO and IV show a nanoparticle comprising a pegylated lipid, a liquid oil core, and bioactive agents bound or conjugated to the inside of the membrane.
  • FIG. IP and 1W show a nanoparticle comprising a pegylated lipid, a liquid oil core, and bioactive agents bound or conjugated to the outside of the membrane. Schematics are not to scale.
  • FIGURE 2 illustrates a junction opener protein conjugated (JOC) to a nanoparticle (NP).
  • FIGURES 3A-3C show the results of an in vivo assay for BT-474 tumor bearing NSG mice treated with junction opener protein conjugated to nanoparticles.
  • FIG. 3A shows a graph of tumor volume following tumor inoculation in NP-047-JOC treated mice (•). Mice were also treated with a squalene containing NP-47-JOC formulation (1 st arrow) and a miglyol containing NP-141-JOC (2 nd arrow). Untreated mice ( ⁇ ) were used as anegative control. Tumor size of each mice was measured by day and plotted related to tumor volumes in NP-JOC untreated mice.
  • FIG. 3B is a graph replotting the data from FIG. 3A as the fold-change in tumor size following NP-047- JOC treatment. Treated mice ( ⁇ ) showed reduction in tumor volumes when compared to untreated controls (•). Y-axis: tumor volume fold-change. X-axis: days post treatment.
  • FIG. 3C is a graph replotting the data from FIG. 3A as the fold-change in tumor size following NP-141- JOC treatment. Treated mice (•) showed no reduction in tumor volumes for all mice when compared to untreated mice ( ⁇ ).
  • FIGURES 4A-4E show the results of an in vitro assay for nanoparticle-junction opener compositions in human DSG2 + BT-474 tumor spheroids.
  • FIG. 4A shows images of tumor spheroid disruption by NP and NP-JOC formulations as indicated.
  • FIGs. 4B and 4C show graphs of cell viability after treatment with squalene emulsions at concentrations of 0.25 pg/mL and 0.0625 pg/mL, respectively.
  • An analysis of FIG. 4B indicates that JOC conjugation showed about 5-55-fold higher killing of tumor cell relative to other tested formulations.
  • FIG. 4A shows images of tumor spheroid disruption by NP and NP-JOC formulations as indicated.
  • FIGs. 4B and 4C show graphs of cell viability after treatment with squalene emulsions at concentrations of 0.25 pg/mL and 0.0625 pg/mL, respectively.
  • FIGs. 4C indicates thatNP-047-JOC showed higher killing of tumor cell as compared to all other tested formulations.
  • Y-axis Number of viable cells.
  • X-axis Conditions.
  • FIGs. 4D and 4E show the ratio of viable cells to the concentration of JOC following treatment with squalene emulsions at concentrations of 0.25 pg/mL and 0.0625 pg/mL. respectively. Because formulations were normalized using squalene rather than JOC concentration this comparison allows for the comparison of NP-JOC formulations to NP controls.
  • Y-axis Number of viable cells.
  • X-axis Conditions.
  • FIGURES 5A-5B show results of an in vitro assay performed in human DSG2 + BT- 474 tumor spheroids treated with JOC, NP, and NP-JOC formulations and with transduced with NucLight red [mKate2],
  • FIG. 5A shows images of tumor spheroid disruption by NP and NP- JOC formulations. Reduction in fluorescence indicates cell death.
  • FIG. 5B shows images of tumor spheroid disruption by JOC protein alone. JOC alone showed minimal tumor disruption and no significant cell death.
  • FIGURES 6A-6B show results of an in vivo assay performed in HER2/DSG2 transgenic mice treated with nanoparticle-junction opener compositions.
  • FIG. 6A shows a graph of ELISA assay for JOC and other formulations in mouse serum.
  • Y-axis JOC in serum (nanograms/milliliter, ng/mL);
  • X-axis Time (minutes).
  • FIG. 6B shows a graph of NP-JOC halflives for each formulation evaluated.
  • Y-axis Half Life (minutes); X-axis: Condition.
  • DSG2 Desmoglein 2
  • DSG2 is a membrane glycoprotein of epithelial cells and promotes formation of tight junctions between cells.
  • Many epithelial cancers are known to highly upregulate the production of DSG2 resulting in formation of a network of cellular "staples" reminiscent of poorly organized junctions that make tumors difficult to permeate by cells and treatments.
  • junction opener proteins (2) nanoparticles, complexes, and aggregates; (3) bioactive agents; (4) lyophilized compositions; (5) pharmaceutical compositions; (6) dosing; (7) administration; (8) safety and efficacy; (9) therapeutic applications; and (10) kits.
  • references to “A and/or B,” when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
  • the term “about” or “approximately” means a range of up to ⁇ 20 % of a given value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” is implicit and in this context means within an acceptable error range for the particular value.
  • an effective amount or “therapeutically effective amount” refers to an amount that is sufficient to achieve or at least partially achieve the desired effect.
  • compositions comprising tumor suppressant and/or tumor killing properties.
  • the compositions comprise junction opener proteins.
  • the compositions comprise nanoparticles.
  • the compositions comprise lipid carriers.
  • the compositions comprise bioactive agents.
  • the compositions and methods described herein comprise junction opener proteins and/or nucleic acids encoding the junction opener proteins.
  • junction opener proteins target tumor tight junctions.
  • the junction opener proteins open the tumor tight junction.
  • the tumor tight junction comprises a desmoglein-2 (DSG2) protein.
  • DSG2 desmoglein-2
  • the junction opener proteins provided herein can bind to DSG2 activating the mitogen-activated protein kinase (MAPK) pathway leading to matrix metalloproteinase (MMP) activation causing transient DSG2 cleavage which is utilized by the adenovirus for cellular entry.
  • MAPK mitogen-activated protein kinase
  • MMP matrix metalloproteinase
  • the junction opener proteins increase therapeutic efficacy of cancer therapeutics due to deeper penetration in the tumor as a result of DSG2 cleavage. Additionally, the junction opener proteins also lead to significant tumor accumulation, which, in some embodiments, can be exploited as a tumor target by nanoparticles encapsulating chemotherapeutics.
  • the junction opener proteins are capable of binding DSG2 proteins.
  • the junction opener proteins comprise an adenovirus fiber shaft domain, a knob domain and a multimerization domain.
  • the junction opener proteins comprise one or more conjugatable moi eties.
  • the nucleic acids encoding the junction opener proteins comprise a DNA, a RNA or a combination thereof.
  • compositions provided herein are engineered to secrete the junction opener protein. In some embodiments, the compositions provided herein are engineered to secrete the junction opener protein on the surface of a nanoparticle.
  • Adenovirus Fiber Polypeptides are engineered to secrete the junction opener protein on the surface of a nanoparticle.
  • the junction opener proteins provided herein comprise one or more adenovirus fiber polypeptides.
  • An adenovirus virion is an icosahedron characterized by a fiber located at the base of each of the 12 vertices of the capsid. The fiber on the virion is a homotrimer comprising three individual fiber polypeptides.
  • Each adenovirus fiber polypeptide includes an N-terminal tail, which interacts with the penton base protein of the capsid and contains the signals necessary fortransport of the protein to the cell nucleus; a shaft, which contains a number of repeating motifs around 15 amino acid residues long (referred to as the shaft domain); and a C -terminal knob domain that contains the detenninants for receptor binding. All adenoviruses attach to their receptors through the knob structure on the end of the fiber.
  • the fiber polypeptides spontaneously assemble into homotrimers (referred to as fibers) that are located on the outside of the adenovirus virion at the base of each of the twelve vertices of the capsid.
  • the polypeptides described herein comprise one or more shaft domains.
  • the polypeptides further comprise a knob domain.
  • the knob domain is a sequence that opens a tumor tight junction.
  • the polypeptides do not include a tail domain from an adenovirus fiber polypeptide.
  • the shaft domain and the knob domain are independently derived from any adenovirus serotype that uses DSG2 as an epithelial cell receptor for viral binding.
  • the serotypes comprise Ad3, Ad7. Adi 1, Adi 4, and Adl4a.
  • DSG2 comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to an amino acid sequence of SEQ ID NO: 68 or SEQ ID NO: 69
  • polypeptides described herein comprise one or more domains.
  • the polypeptides comprise a shaft domain, a knob domain, a multimerization domain, or any combination thereof.
  • the polypeptides comprise at least two identical domains.
  • the polypeptides comprise at least two non-identical domains.
  • the at least two non-identical domains comprise amino acid sequences that are derived from different adenoviruses.
  • polypeptides described herein comprise one or more functional variants of the domains provided herein.
  • the functional variant comprises one or more mutations (substitutions, additions, deletions, chimeras, etc.), wherein the functional variant retain or improve binding affinity to DSG2, retain or improve formation of multimers (such as dimers) via the multimerization domain, or combinations thereof.
  • nucleic acids encoding a junction opener protein comprise a DNA, a RNA or a combination thereof.
  • the nucleic acids encoding the junction opener protein can be prepared and isolated using standard molecular biological techniques.
  • the nucleic acids further comprise additional nucleotide sequence, wherein the additional nucleotide sequence function to promote expression and/or purification of the encoded protein, including but not limited to polyA sequences, modified Kozak sequences, and sequences encoding epitope tags, export signals, and secretory signals, nuclear localization signals, and plasma membrane localization signals.
  • the recombinant expression vectors comprise a nucleic acid provided herein.
  • the nucleic acid is operatively linked to a promoter.
  • the promoter sequence function to drive expression of the nucleic acids in a mammalian system may be constitutive (driven by any of a variety of promoters including, but not limited to. CMV, SV40, RSV. actin, EF) or inducible (driven by any of a number of inducible promoters including, but not limited to, tetracycline, ecdysone, steroid-responsive).
  • the expression vectors are replicable in the host organisms.
  • the expression vectors comprise one or more selection markers (e.g, an antibiotic resistance gene).
  • prokaryotic cells and eukaryotic cells comprising a recombinant expression vector.
  • the cells are transiently or stably transfected.
  • the cell can be transfected by any technique, including, but not limited to, standard bacterial transformations, calcium phosphate co-precipitation, electroporation, or liposome mediated-delivery, DEAE dextran mediated-delivery, poly cationic mediated- delivery, and viral mediated-transfection.
  • the adenovirus fiber polypeptide provided herein comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to any one of SEQ ID NO: 1 to 66.
  • the adenovirus fiber polypeptide provided herein comprises an amino acid sequence that is at least 65%, at least 70%. at least 75%. at least 80%. at least 85%. at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to any one of SEQ ID NO: 38 to 66.
  • the shaft domains provided herein comprise one or more of: an Ad3 shaft domain, an Ad7 shaft domain, an Adi 1 shaft domain, an Ad 14 shaft domain, an Ad 14a shaft domain, any combinations thereof, and functional equivalents thereof.
  • the adenovirus fiber polypeptides described herein comprise one or more shaft domains.
  • the adenovirus fiber polypeptide comprises at least two shaft domains that are identical.
  • the adenovirus fiber polypeptide comprises at least two shaft domains that are non-identical.
  • the one or more shaft domains comprise 1-22 shaft domains.
  • the adenovirus fiber polypeptides comprise 1-22, 1-21, 1-20, 1- 19, 1-18, 1-17, 1-16, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2,
  • the knob domain provided herein function to induce opening of a tumor tight junction.
  • the knob domain opens a tumor tight junction by interacting with a tumor tight junction protein.
  • the tumor tight junction protein comprises DSG2 protein. The knob domain targets tumors by binding to DSG2, and triggers the transient opening of tumor tight junctions.
  • a functional variant of knob domain is function that enhances the binding affinity of the knob domain to a DSG2, reduces aggregation, increases production, and removes potential conjugatable amino acids.
  • a junction opener protein comprises multimeric knob domains.
  • the junction opener protein comprises a trimeric knob domain.
  • at least two knob domains of the multimeric knob domain are identical.
  • at least two knob domains of the multimeric knob domain are non-identical.
  • the junction opener protein comprises a homo trimeric knob domain.
  • the junction opener protein comprises a heterotrimeric knob domain.
  • the knob domain comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to any one of SEQ ID NOS: 13 to 30
  • the junction opener proteins are in a multimeric form ⁇ e.g., dimer, trimer).
  • a multimer comprises a dimer formed by multimerization through the multimerization domains.
  • a multimer comprises a trimer formed by multimerization through the multimerization domains.
  • the multimer is a heteromultimer. In some embodiments, the multimer is a homomultimer.
  • Non-limiting examples of the numerous multimerization domains known to those of skill in the art and suitable for use in the present invention include, but are not limited to peptide helices containing at least one helix, or a structure formed by a helix, a coil and another helix, etc., coiled coil structures, multimerization domains within, for example, many cell surface signaling receptors, Fc regions or hinge regions of an antibody, leucine zippers, the STAT protein N terminal domain, FK506 binding protein, the LexA protein C-terminal domain, nuclear receptors, the FkpA N-terminal domain, orange carotenoid protein from A. maxima, Ml matrix protein from influenza, neuraminidase from influenza virus, E.
  • the multimerization domain comprises one or more copies of EVSALEK (SEQ ID NO: 36) and/or KVSALKE (SEQ ID NO: 37).
  • the multimerization domain comprises an amino acid sequence that is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%. at least 98%. at least 99% or 100% identical to any one of SEQ ID NOS: 31 to 37.
  • junction opener proteins provided herein comprise one or more conjugatable moieties. In some embodiments, at least two of the one or more conjugatable moieties are identical. In some embodiments, at least two of the one or more conjugatable moieties are non-identical.
  • the conjugatable moiety is linked to or located within a shaft domain. In some embodiments, the conjugatable moiety is linked to or located within a knob domain. In some embodiments, the conjugatable moiety is linked to or located within a multimerization domain. In some embodiments, the conjugatable moiety is linked to or located within one or more of a shaft domain, a knob domain and a multimerization domain.
  • the conjugatable moiety function to conjugate two adenovirus fiber polypeptides to form a multimeric protein. In some embodiments, the conjugatable moiety function to conjugate a shaft domain, a knob domain or a multimerization domain to a lipid carrier. In some embodiments, the conjugatable moiety function to conjugate a shaft domain, a knob domain or a multimerization domain to a bioactive agent.
  • Conjugation can be either covalent or non-covalent.
  • conjugatable moieties include an amino acid with a reactive group (e.g., cysteines), a short run of amino acids (e.g. , poly lysines), biotin and avidin/streptavidin.
  • exemplary non- covalent interaction comprises ionic interaction, hydrogen bond, or combination thereof.
  • junction opener proteins comprise one or more modified amino acid residues.
  • junction opener proteins comprise linker amino acids.
  • the linker amino acids are located between the conjugatable moieties and the junction opener proteins.
  • the linker amino acids can be of any length and sequence.
  • the linker amino acids comprise short sequences of flexible residues (e g, glycine and serine).
  • junction opener proteins comprise a tag.
  • the tag functions for isolation or detection of the junction opener protein.
  • Non-limiting exemplary tags include His. CBP, CYD (covalent yet dissociable NorpD peptide). Strep II. FLAG, GFP, HPC (heavy chain of protein C) peptide tags, GST and MBP affinity tags.
  • junction opener protein is pegylated. In some embodiments, junction opener protein is conjugated to an anionic molecule. In some embodiments, the anionic molecule interacts with cationic lipids of the lipid carriers or nanoparticles. In some embodiments, the anionic molecules are linked to the lipid carriers or the nanoparticles by ionic bond.
  • compositions and methods provided herein comprise lipid carrier complexes or nanoparticle-complexes.
  • compositions and methods provided herein comprise a plurality of lipid carriers or a plurality of nanoparticles interact physically, chemically, and/or covalently with a nucleic acid provided herein and/or other nanoparticles.
  • the specific type of interaction between lipid carriers or between nanoparticles will depend upon the characteristic shapes, sizes, chemical compositions, physical properties, and physiologic properties.
  • nanoparticles include but are not limited to: oil in water emulsions, nanostructured lipid carriers (NLCs), cationic nanoemulsions (CNEs), vesicular phospholipid gels (VPG), polymeric nanoparticles, cationic lipid nanoparticles, PEGylated lipid nanoparticles, liposomes, gold nanoparticles, solid lipid nanoparticles (LNPs or SLNs), mixed phase core NLCs, ionizable lipid carriers, magnetic carriers, polyethylene glycol (PEG)- functionalized carriers, cholesterol-functionalized carriers, polylactic acid (PLA)-functionalized carriers, and polylactic-co-glycolic acid (PLGA)-functionalized lipid carriers.
  • NLCs nanostructured lipid carriers
  • CNEs cationic nanoemulsions
  • VPG vesicular phospholipid gels
  • polymeric nanoparticles cationic lipid nanoparticles
  • the crosslinking agent comprises crosslinking agent, sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane- 1 -carboxylate (Sulfo-SMCC).
  • Sulfo-SMCC sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane- 1 -carboxylate
  • the sulfo-SMCC can be removed from the composition via column purification techniques.
  • FIG. 1A (not to scale)
  • FIG. IB illustrates a nanostructured lipid carrier (NLCs) which can comprise a blend of solid organic lipids (e.g., trimyristin) and liquid oil (e.g., squalene, squalane, dehydroisosqualene, or a combination thereof).
  • NLCs nanostructured lipid carrier
  • the solid lipid is dispersed in the liquid oil.
  • the entire nanodroplet is dispersed in the aqueous (water) phase.
  • the nanoparticle comprises inorganic nanoparticles, as illustrated in FIG. 1C (not to scale), as solid inorganic nanoparticles (e.g., iron oxide nanoparticles) dispersed in liquid oil. The entire nanodroplet is then dispersed as a colloid in the aqueous (water) phase.
  • the nanoparticles provided herein are dispersed in an aqueous solution.
  • aqueous solutions include water (e.g.. sterilized, distilled, deionized, ultra-pure. RNAse-free. etc.), saline solutions (e.g., Kreb’s, Ascaris, Dent’s, Tet’s saline), or 1% (w/v) dimethyl sulfoxide (DMSO) in water.
  • the nanoparticles provided herein comprise a bioactive agent.
  • bioactive agents that are dispersed/dissolved within a liquid core of the nanoparticle, as illustrated in FIG. ID (not to scale).
  • nanoparticles comprising solid inorganic nanoparticles and bioactive agents that are dispersed/dissolved within the liquid oil core, as illustrated in FIG. IE (not to scale).
  • the bioactive agents are within the membrane as illustrated in FIG. IF and FIG. 1H (not to scale), the bioactive agents are bound to the surface as illustrated in FIG. 1G and FIG. II (not to scale), or dispersed/dissolved within the liquid core (FIG. ID).
  • the lipid carrier comprises a membrane and a liquid oil core.
  • the membrane comprises a blend of lipid and surfactant.
  • the lipid comprises pegylated lipid (e.g, DSPE-PEG).
  • the surfactant comprises a blend of sorbitan trioleate and Polysorbate 80.
  • the liquid oil core comprises squalene, squalane, dehydroisosqualene, or a combination thereof. In some embodiments, as illustrated in FIG.
  • the nanoparticle comprises iron oxide nanoparticles dispersed in a liquid oil (e.g, squalene).
  • a liquid oil e.g, squalene
  • the entire nanodroplet can be then dispersed as a colloid in the aqueous (water) phase.
  • the nanoparticles provided herein are dispersed in an aqueous solution.
  • aqueous solutions include water (e.g., sterilized, distilled, deionized, ultra-pure, RNAse-free, etc.), sahne solutions (e.g... Kreb’s, Ascaris. Dent's. Tet’s saline), or 1% (w/v) dimethyl sulfoxide (DMSO) in water.
  • DMSO dimethyl sulfoxide
  • the nanoparticles provided herein comprise a bioactive agent.
  • bioactive agents that are dispersed/dissolved in a liquid oil (e.g, squalene), as illustrated in FIG. IK and 1R (not to scale).
  • nanoparticles comprising solid iron oxide nanoparticles and bioactive agents that are dispersed/dissolved in a liquid oil (e.g., squalene), as illustrated in FIG. IL and IS (not to scale).
  • the bioactive agents are within the membrane as illustrated in FIG. IM, IO, IT, and IV (not to scale), the bioactive agents are bound to the surface as illustrated in FIG.
  • the core comprises a solid lipid.
  • the lipid carriers provided herein comprise a liquid lipid and a solid lipid in the core.
  • nanoparticles provided herein comprise a hydrophilic surface.
  • the hydrophilic surface comprises a cationic lipid.
  • the hydrophilic surface comprises an ionizable lipid.
  • the hydrophilic surface comprises a PEGylated lipid.
  • the nanoparticle comprises a membrane.
  • the membrane comprises a cationic lipid.
  • the membrane comprises an ionizable lipid.
  • the membrane comprises a PEGylated lipid.
  • the membrane comprises a phospholipid (e.g., phosphatidic acid, lysophosphatidic acid, cyclic lysophosphatidic acid, phosphatidylcholine. lysophosphatidylcholine, phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol, lysophosphatidylglycerol, phosphoinositides, lysophosphatidylinositol, phosphatidylserine, lysophosphatidylserine, cardiolipin, sterol- modified phospholipids, and derivatives thereof).
  • a phospholipid e.g., phosphatidic acid, lysophosphatidic acid, cyclic lysophosphatidic acid, phosphatidylcholine. lysophosphatidylcholine, phosphatidylethanolamine, phosphatidylcholine, phosphatidyl
  • the nanoparticles provided herein comprise lipids for inclusion in the hydrophilic surface include, without limitation: l,2-dioleoyloxy-3 (trimethylammonium)propane (DOTAP).
  • DOTAP trimethylammonium
  • DPTAP distearoyltrimethylammonium propane
  • DSTAP distearoyltrimethylammonium propane
  • DOTMA N,N-dioleoyl-N,N- dimethylammonium chloride
  • DOEPC N,N-dioleoyl-sn-glycero-3-ethylphosphocholine
  • DODAP l,2-dioleoyl-3-dimethylammonium-propane
  • DinDMA 1,2- dilinoleyloxy-3- dimethylaminopropane
  • Nl,N3,N5-tris(3-(didodecylamino)propyl)benzene-l,3,5-tricarboxamide (TT3) and any combinations thereof.
  • suitable classes of lipids include, but are not limited to, the phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), phosphatidylglycerol (PGs); and PEGylated lipids including PEGylated version of any of the above lipids (e.g, DSPE-PEGs), and a combination thereof.
  • the PEGylated lid comprises: (R)-2,3-bis(myristoyloxy)propyl-l -(methoxy poly(ethylene glycol)2000) carbamate (also referred to as PEG2000-DMG).
  • the nanoparticle provided herein comprises a cationic lipid.
  • the cationic lipid comprises DOTAP.
  • a junction opener protein provided herein is conjugated to the hydrophilic surface of the nanoparticle. In some embodiments, a junction opener protein provided herein is conjugated to the cationic lipid. In some embodiments, a junction opener protein provided herein is conjugated to the PEGylated lipid. In some embodiments, a junction opener protein provided herein is conjugated to the phospholipid.
  • a nanoparticle provided herein comprises a hydrophobic core.
  • the hydrophobic core comprises an oil.
  • the hydrophobic core comprises a lipid in liquid phase at 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, or 50°C.
  • the nanoparticle provided herein comprises an oil.
  • the oil is in liquid phase.
  • oils that can be used include a-tocopherol, coconut oil, dihydroisosqualene (DHIS), famesene, grapeseed oil, lauroyl polyoxylglyceride, mineral oil, monoacylglycerol, palm kernel oil, olive oil, paraffin oil, peanut oil, propolis, squalene, squalane, squalane, dehydroisosqualene, solanesol, soy lecithin, soybean oil. sunflower oil. a triglyceride, vitamin E, or combinations thereof.
  • the nanoparticle provided herein comprises a triglyceride.
  • Exemplary triglycerides include but are not limited to: capric triglycerides, capry lic triglycerides, a caprylic and capric triglycerides, triglyceride esters, and myristic acid triglycerins.
  • the nanoparticle comprises a triglyceride ester of saturated coconut or palm kernel oil derived caprylic and capric fatty acids and plant derived glycerol, e.g, Miglyol 812 N.
  • the hydrophobic core comprises a lipid in solid phase at 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, or 50°C.
  • the hydrophobic core comprises glyceryl trimyristate-dynasan or a derivative thereof.
  • the nanoparticles provided herein comprise a liquid organic material and a solid inorganic material.
  • the nanoparticle provided herein comprises an inorganic particle.
  • the inorganic particle is a solid inorganic particle.
  • FIG. IE illustrates an embodiment where the solid inorganic particle is within the hydrophobic core.
  • the nanoparticle provided herein comprises the inorganic particle within the hydrophobic core.
  • the nanoparticle provided herein comprises a metal.
  • the nanoparticle provided herein comprises a metal within the hydrophobic core.
  • the metal can be without limitation, a metal salt, a metal oxide, a metal hydroxide, a metal phosphate, or a combination thereof.
  • the nanoparticle provided herein comprises aluminum oxide (AI2O3), aluminum oxyhydroxide, iron oxide (FeaO-i. Fe20s, FeO, or combinations thereof), titanium dioxide, silicon dioxide (SiCh), aluminum hydroxyphosphate (Al(OH) x (PO4) y ), calcium phosphate (Ca3(PO 4 )2), calcium hydroxyapatite (Caio(P0 4 )e(OH)2), iron gluconate, iron sulfate, or a combination thereof.
  • the inorganic particles may be formed from one or more same or different metals (any metals including transition metal).
  • the inorganic particles are formed from iron.
  • the inorganic particles are formed from gadolinium.
  • the inorganic particles e.g. , iron, gadolinium
  • the inorganic particles function as a magnetic resonance imaging contrast agent or computerized tomography contrast agent. Accordingly, in some embodiments, the inorganic particles (e.g.. iron, gadolinium) provide better visualization of nanoparticle biodistribution.
  • the inorganic particle is a transition metal oxide.
  • the transition metal is magnetite (FesOr). maghemite (y-FezCh), wiistite (FeO), hematite (alpha (a)- Fe2O3), or a combination thereof.
  • the metal is aluminum hydroxide or aluminum oxyhydroxide, and a phosphate-terminated lipid or a surfactant, such as oleic acid, oleylamine, SDS, TOPO or DSPA is used to coat the inorganic solid nanoparticle before it is mixed with the liquid oil to form the hydrophobic core.
  • the metal can comprise a paramagnetic, a superparamagnetic, a ferrimagnetic or a ferromagnetic compound. In some embodiments, the metal is a superparamagnetic iron oxide (FeiCh).
  • a junction opener protein provided herein is conjugated to the hydrophobic core. In some embodiments, the junction opener protein is conjugated to the lipid (e.g, cationic lipid, PEGylated lipid, phospholipid). In some embodiments, the junction opener protein is conjugated to the hydrophilic surfactant. In some embodiments, the junction opener protein is conjugated to the hydrophobic surfactant.
  • nanoparticles provided herein comprise a cationic lipid, an oil. and an inorganic particle.
  • the nanoparticle provided herein comprises DOTAP; squalene and/or glyceryl trimyristate-dynasan; and iron oxide.
  • the nanoparticle provided herein further comprises a surfactant.
  • nanoparticles provided herein comprise a cationic lipid, an oil, an inorganic particle, and a surfactant.
  • nanoparticles provided herein comprise a PEGylated lipid, an oil, and an inorganic particle.
  • the nanoparticle provided herein comprises DSPE-PEG; squalene and/or glyceryl trimyristate-dynasan; and iron oxide.
  • the nanoparticle provided herein further comprises a surfactant.
  • nanoparticles provided herein comprise a PEGylated lipid, an oil. an inorganic particle, and a surfactant.
  • Surfactants are compounds that lower the surface tension between two liquids or between a liquid and a solid component of the nanoparticles provided herein.
  • Surfactants can be hydrophobic, hydrophilic, or amphiphilic.
  • the nanoparticle provided herein comprises a hydrophobic surfactant.
  • Exemplar ⁇ ' hydrophobic surfactants that can be employed include but are not limited to: sorbitan monolaurate (SPAN® 20), sorbitan monopalmitate (SPAN® 40), sorbitan monostearate (SPAN® 60), sorbitan tristearate (SPAN® 65), sorbitan monooleate (SPAN® 80), and sorbitan trioleate (SPAN® 85).
  • Suitable hydrophobic surfactants include those having a hydrophilic-lipophilic balance (HLB) value of 10 or less, for instance, 5 or less, from 1 to 5, or from 4 to 5.
  • the hydrophobic surfactant can be a sorbitan ester having an HLB value from 1 to 5, or from 4 to 5.
  • nanoparticles provided herein comprise a ratio of the esters that yields a hydrophilic-lipophilic balance between 8 and 11.
  • HLB is used to categorize surfactants as hydrophilic or lipophilic.
  • the HLB scale provides for the classification of
  • HLB 0: fully lipophilic/hydrophobic carrier
  • HLB between 0 and 6 is an oil soluble carrier
  • HLB between 6 and 9 is a water dispersible carrier
  • HLB between 9 and 20 is a hydrophilic, water soluble carrier
  • HLB 20: fully hydrophilic/lipophobic carrier.
  • a nanoparticle or a lipid carrier provided herein comprises a hydrophilic surfactant, also called an emulsifier.
  • a nanoparticle or a lipid carrier provided herein comprises polysorbate.
  • the hydrophilic surfactant comprises polysorbate.
  • Polysorbates are oily liquids derived from ethoxylated sorbitan (a derivative of sorbitol) esterified with fatty acids.
  • hydrophilic surfactants that can be employed include but are not limited to: polysorbates such as TWEEN®, Kolliphor, Scathes, Alkest, or Canarcel; polyoxyethylene sorbitan ester (polysorbate); polysorbate 80 (polyoxyethylene sorbitan monooleate, or TWEEN® 80); polysorbate 60 (polyoxyethylene sorbitan monostearate, or TWEEN® 60); polysorbate 40 (polyoxyethylene sorbitan monopalmitate, or TWEEN® 40); and polysorbate 20 (polyoxyethylene sorbitan monolaurate, or TWEEN® 20).
  • the hydrophilic surfactant is polysorbate 80.
  • nanoparticles and lipid carriers provided herein comprise a hydrophobic core surrounded by a lipid membrane (e.g., a cationic lipid such as DOTAP, a PEGylated lipid such as DSPE-PEG).
  • a lipid membrane e.g., a cationic lipid such as DOTAP, a PEGylated lipid such as DSPE-PEG.
  • the hydrophobic core comprises: one or more inorganic particles; a phosphate-terminated lipid; and a surfactant.
  • Inorganic solid nanoparticles provided herein can be surface modified before mixing with the liquid oil.
  • the inorganic solid nanoparticle may be coated with hydrophobic molecules (or surfactants) to facilitate the miscibility of the inorganic solid nanoparticle with the liquid oil in the “oil” phase of the nanoemulsion particle.
  • the inorganic particle is coated with a capping ligand, the phosphate-terminated lipid, and/or the surfactant.
  • the hydrophobic core comprises a phosphate-terminated lipid.
  • Exemplary phosphate-terminated lipids that can be employed include but are not limited to: trioctylphosphine oxide (TOPO) or distearyl phosphatidic acid (DSPA).
  • the hydrophobic core comprises a surfactant such as a phosphorous-terminated surfactant, a carboxylate-terminated surfactant, a sulfate-terminated surfactant, or an amine-terminated surfactant.
  • Exemplary carboxylate-terminated surfactants include oleic acid.
  • Typical amine terminated surfactants include oleylamine.
  • the surfactant is distearyl phosphatidic acid (DSPA), oleic acid, oleylamine or sodium dodecyl sulfate (SDS).
  • the inorganic solid nanoparticle is a metal oxide such as an iron oxide, and a surfactant, such as oleic acid, oleylamine, SDS, DSPA, or TOPO, is used to coat the inorganic solid nanoparticle before it is mixed with the liquid oil to form the hydrophobic core.
  • the hydrophobic core comprises: one or more inorganic particles containing at least one metal hydroxide or oxyhydroxide particle optionally coated with a phosphate- terminated lipid, a phosphorous-terminated surfactant, a carboxylate- terminated surfactant, a sulfate-terminated surfactant, or an amine-terminated surfactant; and a liquid oil containing naturally occurring or synthetic squalene; a PEGylated lipid comprising DSPE-PEG; a hydrophobic surfactant comprising a sorbitan ester selected from the group consisting of: sorbitan monostearate, sorbitan monooleate, and sorbitan trioleate; and a hydrophilic surfactant comprising a polysorbate.
  • the hydrophobic core comprises: one or more inorganic nanoparticles containing aluminum hydroxide or aluminum oxyhydroxide nanoparticles optionally coated with TOPO, and a liquid oil containing naturally occurring or synthetic squalene; the PEGylate lipid DSPE-PEG; a hydrophobic surfactant comprising sorbitan monostearate; and a hydrophilic surfactant comprising polysorbate 80.
  • the hydrophobic core comprises one or more inorganic particles containing at least one metal hydroxide or oxyhydroxide particle optionally coated with a phosphate- terminated lipid, a phosphorous-terminated surfactant, a carboxylate- terminated surfactant, a sulfate-terminated surfactant, or an amine-terminated surfactant; and a liquid oil containing naturally occurring or synthetic squalene; a PEGylated lipid comprising DSPE-PEG; a hydrophobic surfactant comprising a sorbitan ester selected from the group consisting of: sorbitan monostearate, sorbitan monooleate, and sorbitan trioleate; and a hydrophilic surfactant comprising a polysorbate.
  • the hydrophobic core comprises one or more inorganic nanoparticles containing aluminum hydroxide or aluminum oxyhydroxide nanoparticles optionally coated with TOPO, and a liquid oil containing naturally occurring or synthetic squalene; the PEGylated lipid DSPE-PEG; a hydrophobic surfactant comprising sorbitan trioleate; and a hydrophilic surfactant comprising polysorbate 80.
  • the nanoparticle provided herein can comprise from about 0.2% to about 40% w/v squalene, from about 0.001% to about 10% w/v iron oxide nanoparticles, from about 0.2% to about 10 % w/v DSPE-PEG, from about 0.25% to about 5% w/v sorbitan trioleate, and from about 0.5% to about 10% w/v polysorbate 80.
  • the nanoparticle provided herein from about 2% to about 6% w/v squalene, from about 0.01% to about 1% w/v iron oxide nanoparticles, from about 0.2% to about 1 % w/v DSPE-PEG, from about 0.25% to about 1% w/v sorbitan trioleate, and from about 0.5%) to about 5% w/v polysorbate 80.
  • the nanoparticle provided herein can comprise from about 0.2% to about 40% w/v squalene, from about 0.001% to about 10% w/v aluminum hydroxide or aluminum oxyhydroxide nanoparticles, from about 0.2% to about 10 % w/v DSPE-PEG, from about 0.25% to about 5% w/v sorbitan trioleate, and from about 0.5% to about 10% w/v polysorbate 80.
  • the nanoparticle provided herein can comprise from about 2% to about 6% w/v squalene, from about 0.01% to about 1% w/v aluminum hydroxide or aluminum oxyhydroxide nanoparticles, from about 0.2% to about 1 % w/v DSPE-PEG, from about 0.25% to about 1% w/v sorbitan trioleate, and from about 0.5%) to about 5% w/v polysorbate 80.
  • the hydrophobic core comprises: one or more inorganic particles containing at least one metal hydroxide or oxyhydroxide particle optionally coated with a phosphate- terminated lipid, a phosphorous-terminated surfactant, a carboxylate- terminated surfactant, a sulfate-terminated surfactant, or an amine-terminated surfactant; and a liquid oil containing naturally occurring or synthetic squalene; a cationic lipid comprising DOTAP; a hydrophobic surfactant comprising a sorbitan ester selected from the group consisting of: sorbitan monostearate, sorbitan monooleate, and sorbitan trioleate; and a hydrophilic surfactant comprising a polysorbate.
  • the hydrophobic core comprises: one or more inorganic nanoparticles containing aluminum hydroxide or aluminum oxyhydroxide nanoparticles optionally coated with TOPO, and a liquid oil containing naturally occurring or synthetic squalene; the cationic lipid DOTAP; a hydrophobic surfactant comprising sorbitan monostearate; and a hydrophilic surfactant comprising polysorbate 80.
  • the hydrophobic core comprises one or more inorganic particles containing at least one metal hydroxide or oxyhydroxide particle optionally coated with a phosphate- terminated lipid, a phosphorous-terminated surfactant, a carboxylate- terminated surfactant, a sulfate-terminated surfactant, or an amine-terminated surfactant; and a liquid oil containing naturally occurring or synthetic squalene; a cationic lipid comprising DOTAP; a hydrophobic surfactant comprising a sorbitan ester selected from the group consisting of: sorbitan monostearate, sorbitan monooleate, and sorbitan trioleate; and a hydrophilic surfactant comprising a polysorbate.
  • the hydrophobic core comprises one or more inorganic nanoparticles containing aluminum hydroxide or aluminum oxyhydroxide nanoparticles optionally coated with TOPO, and a liquid oil containing naturally occurring or synthetic squalene; the cationic lipid DOTAP; a hydrophobic surfactant comprising sorbitan monostearate; and a hydrophilic surfactant comprising polysorbate 80.
  • the nanoparticle provided herein can comprise from about 0.2% to about 40% w/v squalene, from about 0.001% to about 10% w/v iron oxide nanoparticles, from about 0.2% to about 10 % w/v DOTAP, from about 0.25% to about 5% w/v sorbitan monostearate, and from about 0.5% to about 10% w/v polysorbate 80.
  • the nanoparticle provided herein from about 2% to about 6% w/v squalene, from about 0.01% to about 1% w/v iron oxide nanoparticles, from about 0.2% to about I % w/v DOTAP, from about 0.25% to about 1% w/v sorbitan monostearate, and from about 0.5%) to about 5% w/v polysorbate 80.
  • the nanoparticle provided herein can comprise from about 0.2% to about 40% w/v squalene, from about 0.001% to about 10% w/v aluminum hydroxide or aluminum oxyhydroxide nanoparticles, from about 0.2% to about 10 % w/v DOTAP.
  • the nanoparticle provided herein can comprise from about 2% to about 6% w/v squalene, from about 0.01% to about 1% w/v aluminum hydroxide or aluminum oxyhydroxide nanoparticles, from about 0.2% to about 1 % w/v DOTAP, from about 0.25% to about 1% w/v sorbitan monostearate, and from about 0.5%) to about 5% w/v polysorbate 80.
  • a composition provided herein comprises at least one nanoparticle formulation as described in Table 1.
  • a composition provided herein comprises any one of NP-1 to NP-31.
  • a composition provided herein comprises any one of NP-1 to NP-37.
  • nanoparticles provided herein comprise: sorbitan monostearate, polysorbate 80, DOTAP, squalene, and no solid particles. In some embodiments, nanoparticles provided herein comprise: sorbitan monostearate, polysorbate 80, DOTAP, squalene, and iron oxide particles. In some embodiments, nanoparticles provided herein comprise: sorbitan trioleate, polysorbate 80, DSPE-PEG. squalene, and no solid particles. In some embodiments, nanoparticles provided herein comprise: sorbitan trioleate, polysorbate 80, DSPE-PEG, squalene, and iron oxide particles.
  • Nanoparticles provided herein can be of various average diameters in size.
  • nanoparticles provided herein are characterized as having an average diameter (z-average hydrodynamic diameter, measured by dynamic light scattering) ranging from about 20 nanometers (nm) to about 200 nm.
  • the z-average diameter of the nanoparticle ranges from about 20 nm to about 150 nm, from about 20 nm to about 100 nm, from about 20 nm to about 80 nm, from about 20 nm to about 60 nm.
  • the z-average diameter of the nanoparticle ranges from about 40 nm to about 200 nm, from about 40 nm to about 150 nm, from about 40 nm to about 100 nm, from about 40 nm to about 90 nm, from about 40 nm to about 80 nm, or from about 40 nm to about 60 nm.
  • the z- average diameter of the nanoparticle is from about 40 nm to about 150 nm.
  • the z-average diameter of the nanoparticle is from about 40 nm to about 60 nm.
  • the nanoparticle is up to 100 nm in diameter.
  • the nanoparticle is 50 to 70 nm in diameter. In some embodiments, the nanoparticle is 40 to 80 nm in diameter.
  • the inorganic particle (e.g., iron oxide) within the hydrophobic core of the nanoparticle can be an average diameter (number weighted average diameter) ranging from about 3 nm to about 50 nm. For instance, the inorganic particle can have an average diameter of about 5 nm, about 10 nm, about 15 nm, about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 40 nm, about 45 nm, or about 50 nm.
  • the ratio of esters and lipids yield a particle size between 30 nm and 200 nm. In some embodiments, the ratio of esters and lipids yield a particle size between 40 nm and 70 nm.
  • Nanoparticles provided herein may be characterized by the polydispersity index (PDI), which is an indication of their quality with respect to size distribution.
  • PDI polydispersity index
  • average poly dispersity index (PDI) of the nanoparticles provided herein ranges from about 0.1 to about 0.5.
  • the average PDI of the nanoparticles can range from about 0.2 to about 0.5, from about 0.1 to about 0.4, from about 0.2 to about 0.4, from about 0.2 to about 0.3, or from about 0. 1 to about 0.3.
  • compositions comprising: nanoparticles, wherein the nanoparticles comprise: a hydrophobic core comprising lipids in liquid phase at 25 degrees Celsius; and nucleic acids encoding for a protein or an antibody, wherein the nucleic acids are complexed to the nanoparticles to form nucleic acid- nanoparticle complexes, and wherein the nucleic acid- nanoparticle complexes comprise a net positive charge at 37 degrees Celsius.
  • nanoparticles provided herein comprise an oil-to-surfactant molar ratio ranging from about 0.1 : 1 to about 20: 1 , from about 0.5: 1 to about 12: 1 , from about 0.5: 1 to about 9: 1, from about 0.5: 1 to about 5: 1, from about 0.5: 1 to about 3: 1, or from about 0.5: 1 to about 1: 1.
  • nanoparticles provided herein comprise a hydrophilic surfactant-to-lipid ratio ranging from about 0.1: 1 to about 2: 1, from about 0.2: 1 to about 1.5: 1, from about 0.3: 1 to about 1 : 1, from about 0.5: 1 to about 1: 1, or from about 0.6: 1 to about 1 : 1.
  • the nanoparticles provided herein comprise a hydrophobic surfactant- to-lipid ratio ranging from about 0. 1: 1 to about 5: 1, from about 0.2: 1 to about 3: 1, from about 0.3: 1 to about 2: 1, from about 0.5: 1 to about 2: 1, or from about 1 : 1 to about 2: 1.
  • nanoparticles provided herein comprise from about 0.2% to about 40% w/v liquid oil, from about 0.001% to about 10% w/v inorganic solid nanoparticle, from about 0.2% to about 10% w/v lipid, from about 0.25% to about 5% w/v hydrophobic surfactant, and from about 0.5% to about 10% w/v hydrophilic surfactant.
  • the lipid comprises a cationic lipid, and the oil comprises squalene, and/or the hydrophobic surfactant comprises sorbitan ester.
  • the lipid comprises a PEGylated lipid, and the oil comprises squalene, and/or the hydrophobic surfactant comprises sorbitan ester.
  • nanoparticles provided herein are made by homogenization and ultrasonication techniques.
  • a nanoparticle provided herein is admixed with a nucleic acid provided herein to form a nanoparticle-nucleic acid complex.
  • a plurality of nanoparticles provided herein are admixed with a plurality of nucleic acids provided herein to form a plurality of nanoparticle-nucleic acid complexes.
  • compositions comprising nanoparticles (e.g.. NP-30 or NP-1) and a nucleic acid encoding for a protein, an antibody, or a functional variant thereof.
  • nucleic acids provided herein are incorporated, associated with, or complexed a lipid carrier provided herein to form a lipid carrier-nucleic acid complex.
  • the lipid carrier- nucleic acid complex is formed via non-covalent interactions or via reversible covalent interactions.
  • the nucleic acids provided herein are in complex with the surface of the lipid nanoparticle provided herein.
  • the nucleic acid further encodes for an RNA-dependent polymerase.
  • the RNA-dependent polymerase is a viral RNA polymerase.
  • the nucleic acid encoding for the RNA polymerase is on the same nucleic acid strand as the nucleic acid sequence encoding for the protein (e.g. , cis).
  • the nucleic acid encoding for the RNA polymerase is on a different nucleic acid strand as the nucleic acid sequence encoding for the protein (e.g., trans).
  • the nucleic acid encoding for the RNA polymerase is a DNA molecule.
  • nucleic acid sequences encoding for a cancer-associated protein, a tumor antigen, a neoantigen, a cancer therapeutic antibody, or a functional variant thereof are DNA or RNA molecules.
  • cancer-associated proteins and cancer therapeutic antibodies provided herein are encoded by DNA. Nanoparticles for inclusion include, without limitation, any one of NP-1 to NP-31, or any one of NP-1 to NP-37.
  • Nucleic acids for inclusion include, without limitation, comprise a region comprising SEQ ID NO: 176; and/or encodes for an amino acid sequence set forth in any one of SEQ ID NOS: 177-178.
  • the nucleic acids further comprise a region encoding for an RNA polymerase, e.g, a region comprising a sequence of SEQ ID NO: 176.
  • compositions provided herein comprise a plurality of nucleic acids each encoding a protein antigen provided herein. The protein antigen encoded by each nucleic acid can be the same or different.
  • a molar ratio of the lipid carrier to the nucleic acid can be chosen to increase the delivery efficiency of the nucleic acid, increase the ability' of the nucleic acid-carrying nanoemulsion composition to elicit an immune response to the antigen, increase the ability' of the nucleic acid-cartying nanoemulsion composition to elicit the production of antibody titers to the antigen in a subject.
  • compositions provided herein have a molar ratio of the lipid carrier to the nucleic acid can be characterized by the nitrogen-to-phosphate molar ratio, which can range from about 0.01 : 1 to about 1000:1, for instance, from about 0.2: 1 to about 500: 1, from about 0.5: 1 to about 150: 1, from about 1: 1 to about 150: 1, from about 1: 1 to about 125:1, from about 1: 1 to about 100: 1, from about 1 : 1 to about 50: 1, from about 1: 1 to about 50: 1, from about 5: 1 to about 50: 1. from about 5: 1 to about 25: 1, or from about 10: 1 to about 20: 1.
  • the molar ratio of the lipid carrier to the nucleic acid characterized by the nitrogen-to- phosphate (N:P) molar ratio, ranges from about 1: 1 to about 150: 1, from about 5: 1 to about 25: 1, or from about 10: 1 to about 20: 1. In some embodiments, the N:P molar ratio of the nanoemulsion composition is about 15: 1. In some embodiments, the nanoparticle comprises a nucleic acid provided herein covalently attached to the membrane.
  • compositions provided herein can be characterized by an oil-to-surfactant molar ratio.
  • the oil-to-surfactant ratio is the molar ratio of squalene: DOTAP, hydrophobic surfactant, and hydrophilic surfactant.
  • the oil-to-surfactant ratio is the molar ratio of squalene: DOTAP, sorbitan monostearate, and polysorbate 80.
  • the oil-to-surfactant ratio is the molar ratio of squalene: DSPE-PEG, hydrophobic surfactant, and hydrophilic surfactant.
  • the oil-to-surfactant ratio is the molar ratio of squalene: DSPE-PEG. sorbitan trioleate, and polysorbate 80.
  • the oil-to surfactant molar ratio ranges from about 0.1: 1 to about 20:1. from about 0.5: 1 to about 12: 1, from about 0.5: 1 to about 9: 1, from about 0.5: 1 to about 5: 1, from about 0.5: 1 to about 3:1, or from about 0.5:1 to about 1:1.
  • the oil-to- surfactant molar ratio is at least about 0.1: 1, at least about 0.2:1, at least about 0.3: 1, at least about 0.4: 1, at least about 0.5: 1, at least about 0.6: 1, at least about 0.7: 1. In some embodiments, the oil-to surfactant molar ratio is at least about 0.4: 1 up to 1 : 1.
  • Compositions provided herein can be characterized by hydrophilic surfactant-to- lipid (e.g, cationic lipid. PEGylated lipid) ratio.
  • the hydrophilic surfactant-to-lipid ratio ranges from about 0.1: 1 to about 2: 1, from about 0.2: 1 to about 1.5: 1, from about 0.3: 1 to about 1 : 1, from about 0.5: 1 to about 1: 1, or from about 0.6: 1 to about 1 : 1.
  • Compositions provided herein can be characterized by hydrophobic surfactant-to-lipid (e.g, cationic lipid, PEGylated lipid) ratio ranging.
  • the hydrophobic surfactant-to-lipid ratio ranges from about 0.1: 1 to about 5: 1, from about 0.2: 1 to about 3: 1. from about 0.3: 1 to about 2: 1, from about 0.5: 1 to about 2: 1, or from about 1 : 1 to about 2: 1.
  • a dried composition comprising a sorbitan fatty acid ester, an ethoxylated sorbitan ester, a cationic lipid or a PEGylated lipid, an immune stimulant, and, optionally, an RNA.
  • dried compositions wherein the dried composition comprises sorbitan monostearate, polysorbate 80, DOTAP, and, optionally, an RNA.
  • dried compositions wherein the dried composition comprises sorbitan trioleate, polysorbate 80, DSPE-PEG, and, optionally, an RNA
  • compositions and methods provided herein comprise one or more bioactive agents. In some embodiments, the compositions and methods comprise at least two similar bioactive agents. In some embodiments, the compositions and methods comprise at least two non-identical bioactive agents. In some embodiments, the one or more bioactive agents are associated with, conjugated to, or complexed with ajunction opener protein provided herein. In some embodiments, the one or more bioactive agents are associated/conjugated/complexed with the adenovirus fiber polypeptide. In some embodiments, the one or more bioactive agents are associated/conjugated/complexed with the knob domain. In some embodiments, the one or more bioactive agents are associated/conjugated/complexed with the multimerization domain.
  • one or more bioactive agents are not associated/ conjugated/ complexed with the nanoparticles or the junction opener protein. Accordingly, in some embodiments, the one or more bioactive agents are administered at a different time relative to the administration of the junction opener protein. In some embodiments, the one or more bioactive agents administered on a different day relative to the administration of the junction opener protein. In some embodiments, the one or more bioactive agents are administered at a different time and/or day relative to the administration of the junction opener protein. In some embodiments, the one or more bioactive agents are coadministered with the junction opener protein.
  • the one or more bioactive agents provided herein are associated with, conjugated to, or complexed with the nanoparticles provided herein.
  • the one or more bioactive agents are associated/conjugated/complexed with cationic lipids of the nanoparticles.
  • the one or more bioactive agents are associated/conjugated/complexed with hydrophilic surfactants of the nanoparticles.
  • the one or more bioactive agents are associated/conjugated/complexed with hydrophobic surfactants of the nanoparticles.
  • the one or more bioactive agents are present in hydrophobic core of the nanoparticles.
  • the one or more bioactive agents are associated/conjugated/complexed with liquid oil of the hydrophobic core.
  • the liquid oil comprises tumor suppressant or tumor killing property.
  • the liquid oil comprises squalene, squalane, dehydroisosqualene, or a combination thereof.
  • the one or more bioactive agents comprise nucleic acids.
  • the nucleic acids comprise DNA, RNA, or combinations thereof.
  • the nucleic acid may be linear or include a secondary- structure (e.g., a hair pin).
  • the nucleic acid is a polynucleotide comprising modified nucleotides or bases, and/or their analogs.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs.
  • modification to the nucleotide structure is imparted before or after assembly of compositions provided herein.
  • the nucleic acid comprises one or more of phosphorami dite. phosphorothioate and methylphosphonate linkages.
  • the bioactive agent is an RNA.
  • the RNA comprises a nucleotide sequence encoding the junction opener proteins provided herein.
  • the RNA comprises a replicon RNA, an RNA that modulates innate immune responses, an RNA that encodes a protein or an antigen, non-coding RNA. a silencing RNA, a microRNA, a tRNAs, a self-replicating RNA, or a combination thereof.
  • the RNA is modified to increase translation efficacy.
  • the RNA is codon optimized increasing translation efficacy.
  • the RNA is modified to increase half-life of the RNA.
  • the RNA is modified by attaching a poly A tail (e.g., of about 30 adenosine residues or more) to the 3' end of the RNA increasing its half-life.
  • a poly A tail e.g., of about 30 adenosine residues or more
  • the 5 ' end of the RNA is capped with a modified ribonucleotide with the structure m7G (5') ppp (5') N (cap 0 structure) or a derivative thereof, which is incorporated during RNA synthesis or is enzy matically engineered after RNA transcription (e.g., by using Vaccinia Virus Capping Enzyme (VCE) consisting of mRNA triphosphatase, guanylyl-transferase and guanine-7-methyltransferase, which catalyzes the construction of N7-monomethylated cap 0 structures).
  • VCE Vaccinia Virus Capping Enzyme
  • the Cap structure provides stability and translational efficacy to the RNA molecule.
  • the 5' cap of the RNA molecule is further modified by a 2'-O-Methyltransferase resulting in the generation of a cap 1 structure (m7Gppp [m2'-O] N).
  • a cap 1 structure may increase translation efficacy and/or in vivo potency.
  • the bioactive agent is a self-replicating nucleic acid.
  • the self-replicating nucleic acid encodes an RNA polymerase.
  • the RNA polymerase comprises a viral RNA polymerase.
  • the self-replicating RNA e.g, a replicon RNA (repRNA)
  • repRNA replicon RNA
  • the self-replicating RNA comprises any genetic element, for example, a plasmid, cosmid, bacmid, phage or virus that is capable of replication largely under its own control.
  • the self-replicating RNA is single stranded.
  • the self-repli eating RNA is double stranded.
  • RNA polymerase examples include: an alphavirus RNA polymerase, an Eastern equine encephalitis virus (EEEV) RNA polymerase, a Western equine encephalitis virus (WEEV), Venezuelan equine encephalitis virus (VEEV), Chikungunya virus (CHIKV), Semliki Forest virus (SFV). or Sindbis virus (SINV).
  • EEEV Eastern equine encephalitis virus
  • WEEV Western equine encephalitis virus
  • VEEV Venezuelan equine encephalitis virus
  • CHKV Chikungunya virus
  • SFV Semliki Forest virus
  • Sindbis virus Sindbis virus
  • a self-replicating nucleic acid provided herein comprises repRNA, RAR or a combination thereof.
  • the repRNA encodes for one or more structural proteins from a non-enveloped virus.
  • the repRNA encodes a protease.
  • the repRNA encodes a 3CD protease.
  • the structural protein and the protease are co-expressed.
  • the repRNA comprises one or more open reading frames.
  • the open reading frames are separated by an internal ribosomal entry site (IRES).
  • the open reading frames are separated by a ribosomal skipping peptide sequence.
  • the ribosomal skipping peptide sequence is from Thosea asigna virus (T2A).
  • the RNA comprises a nucleotide sequence encoding the junction opener protein provided herein and the self-replicating nucleic acid provided herein.
  • the nucleotide sequence encoding the junction opener protein is located 5’ to the self-replicating nucleic acid sequence.
  • the nucleotide sequence encoding the junction opener protein is located 3’ to the self-replicating nucleic acid sequence.
  • the nucleotide sequence encoding the junction opener protein is located 5’ to the self-replicating nucleic acid sequence encoding the VEEV RNA polymerase.
  • the nucleotide sequence encoding the junction opener protein is located 3’ to the self-replicating nucleic acid sequence encoding the VEEV RNA polymerase.
  • compositions comprising: a lipid carrier provided herein; and one or more nucleic acids, wherein the one or more nucleic acids comprises a sequence encoding for an antigen.
  • the antigen is a cancer-associated protein (also referred to as a tumor protein antigen or tumor antigen).
  • nucleic acids provided herein encode for a cancer-associated protein.
  • the cancer-associated protein is a surface protein, a cytosolic protein, or a transmembrane protein.
  • the cancer-associated protein is a protein that is expressed by a cancer cell.
  • the cancer-associated protein is a protein that is expressed by a microbial organism that causes a cancer (e.g. viral proteins).
  • nucleic acids provided herein encode for a protein expressed by a solid cancer cell or a blood cancer cell.
  • the solid cancer cell is a melanoma cell.
  • the protein expressed by the melanoma cell is not expressed by a non-cancer cell.
  • the protein expressed by a melanoma cell comprises a mutation in the amino acid sequence relative to a comparable amino acid sequence in anon-cancer cell.
  • nucleic acids provided herein encode for MAGE-A1 (SEQ ID NO: 70, SEQ ID NO: 73) or a functional fragment thereof.
  • nucleic acids provided herein encode for MAGE- A3 (SEQ ID NO: 71, SEQ ID NO: 74) or a functional fragment thereof. In some embodiments, nucleic acids provided herein encode for TRP-1 (SEQ ID NO: 72, SEQ ID NO: 75) or a functional fragment thereof. In some embodiments, nucleic acids provided herein encode for TRP-1 and MAGE-A1. In some embodiments, nucleic acids provided herein encode for TRP-1 and MAGE- A3. In some embodiments, nucleic acids provided herein encode for a tyrosinase. In some embodiments, nucleic acids provided herein comprise a sequence that is at least 85% identical to SEQ ID NOS: 70-72.
  • nucleic acids provided herein encode for an amino acid sequence listed in Table 2. In some embodiments, nucleic acids provided herein encode for an amino acid sequence that is at least 85% identical to any one of SEQ ID NOS: 73-124. In some embodiments, compositions provided herein comprise two or more, three or more, four or more, five or more, six or more, or up to seven or more nucleic acids coding different sequences listed in Table 2. In some embodiments, nucleic acids provided herein encoding for a protein sequence listed in Table 2 is used as part of a treatment or prevention of melanoma. In some embodiments, a nucleic acid provided herein encodes for a cancer-associated protein listed in Table 2.
  • compositions provided herein comprise two or more nucleic acids encoding for different sequences listed in Table 2.
  • nucleic acids provided herein encode for a cancer-associated protein sequence comprising at least 80%, 85%, 90%. 95%. 96%. 97%. 98%. or 99% sequence identity to a sequence listed in Table 2.
  • compositions provided herein comprise two or more nucleic acids encoding different sequences listed in Table 2.
  • the nucleic acid provided herein encodes for a cancer-associated protein or a functional fragment thereof comprising at least 80%. 85%. 90%. 95%. 96%, 97%, 98%, or 99% sequence similarity to a sequence listed Table 2.
  • Percent (%) sequence identity for a given sequence relative to a reference sequence is defined as the percentage of identical residues identified after aligning the two sequences and introducing gaps if necessary, to achieve the maximum percent sequence identity. Percent identity can be calculated using alignment methods known in the art, for instance alignment of the sequences can be conducted using publicly available software such as BLAST. Align. ClustalW2. Those skilled in the art can determine the appropriate parameters for alignment, but the default parameters for BLAST are specifically contemplated.
  • AAGIGILTV (SEQ ID NO: 91) is also recognized by HLA B45-1- restricted cytotoxic T lymphocyte.
  • ⁇ Phenylalanine (F) at position 9 is the result of mutation.
  • the wild-type sequence is SYLDSGIHS (SEQ ID NO: 122) ⁇ Glutamine (Q) at position 6 is the result of somatic mutation.
  • the wild-type sequence is ETVSEESNV (SEQ ID NO: 123).
  • 'Isolcucinc (I) at position 5 is the result of mutation.
  • the wild-type sequence is EEKLSVVLF (SEQ ID NO: 124).
  • a cancer-associated protein encoded by a nucleic acid provided herein comprises a cell membrane-contacting domain or functional fragment thereof.
  • the cell membrane-contacting domain comprises a transmembranebinding domain, an outer cell membrane-contacting domain, or an inner cell membranecontacting domain.
  • the cell membrane-contacting domain comprises a transmembrane-binding domain, an outer cell membrane-contacting domain, and an inner cell membrane-contacting domain.
  • the cancer-associated protein is a protein expressed by a melanoma cancer cell, a prostate cancer cell, a colon cancer cell, an ovarian cancer cell, a breast cancer cell, a pancreatic cancer cell, or a blood cell.
  • nucleic acid provided herein comprises a sequence encoding a dimer, trimer, or multimer of a cancer-associated protein provided herein. In some embodiments, nucleic acid provided herein comprises a sequence encoding an amino acid sequence that is at least about 500 amino acids in length or more. In some embodiments, nucleic acid provided herein comprises a sequence encoding an amino acid sequence that is at least about 200, 300, 400, 500, 750, 1000 or more amino acids in length or more.
  • nucleic acid provided herein comprises a sequence encoding one or more cancer- associated protein, wherein the one or more cancer-associated protein comprises a molecular weight of at least about 50 kilodaltons (kDa) or more, at least about 100 kilodaltons (kDa) or more, at least about 150 kilodaltons (kDa) or more, at least about 200 kilodaltons (kDa) or more, at least about 250 kilodaltons (kDa) or more, at least about 300 kilodaltons (kDa) or more, at least about 350 kilodaltons (kDa) or more, at least about 400 kilodaltons (kDa) or more, at least about 450 kilodaltons (kDa) or more, at least about 500 kilodaltons (kDa) or more, up to 1000 kDa or more.
  • nucleic acids at least about 50 kil
  • nucleic acids provided herein comprise a sequence encoding a cancer-associated protein associated with prostate cancer.
  • the cancer-associated protein with prostate cancer comprises prostem.
  • the cancer-associated protein is prostein.
  • the cancer-associated protein is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of full length prostein.
  • the prostein is human prostein.
  • the cancer-associated protein comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to any one of SEQ ID NOS:
  • the one or more bioactive agents comprise anticancer agents.
  • an anticancer agent described herein comprise: (a) an anti cancer antibody, a function variant thereof, or a nucleic acid encoding the anticancer antibody, (b) an immunomodulator, a functional variant thereof, or a nucleic acid encoding the immunomodulator, (c) a chemotherapeutic agent, or (d) a combination thereof.
  • An Anticancer Antibody a function variant thereof or a Nucleic Acid encoding the Anticancer Antibody
  • the bioactive agents provided herein comprise an anticancer antibody or a nucleic acid encoding the anticancer antibody.
  • the anticancer antibody comprises trastuzumab, cetuximab, pertuzumab. apomab, conatumumab, lexatumumab, bevacizumab, bevacizumab.
  • denosumab zanolimumab, lintuzumab, edrecolomab, rituximab, ticilimumab, tositumomab, alemtuzumab, epratuzumab, mitumomab, gemtuzumab ozogamicin, oregovomab, pemtumomab daclizumab, panitumumab, catumaxomab, ofatumumab and ibritumomab+3F8, Abagovomab, Abituzumab, Adecatumumab, Altumomab pentetate, Amatuximab, Anatumomab mafenatox.
  • Necitumumab Nesvacumab. Nimotuzumab. Nofetumomab merpentan, Obinutuzumab, Ocaratuzumab, Olaratumab, Onartuzumab, Ontuxizumab, Oportuzumab monatox, Otlertuzumab, Parsatuzumab, Patritumab, Pinatuzumab vedotin, Pintumomab, Polatuzumab vedotin, Pritumumab, Racotumomab, Radretumab, Ramucirumab, Rilotumumab, Robatumumab, Satumomab pendetide.
  • Seribantumab Sibrotuzumab, Siltuximab, Simtuzumab, Sofituzumab vedotin, Solitomab, Tacatuzumab tetraxetan, Tamtuvetmab, Taplitumomab paptox, Tarextumab, Tenatumomab, Teprotumumab, Tigatuzumab, Tovetumab, Tucotuzumab celmoleukin, Ublituximab, Vandortuzumab vedotin, Vantictumab, Vanucizumab, Veltuzumab, Vesencumab, Vorsetuzumab mafodotin, Votumumab.
  • the anticancer antibody comprises an amino acid sequence that targets any one of the tumor antigen described in Table 3.
  • the anticancer antibody comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to any one of the heavy chain amino acid sequences listed in Table 3.
  • the anticancer antibody comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% similar to any one of the heavy chain amino acid sequences listed in Table 3.
  • the bioactive agents provided herein comprise an immunomodulator, a functional variant thereof, or a nucleic acid encoding the immunomodulator.
  • An immune system modulator is an agent, cytokine, or protein that changes the level of an immune cell (e.g, B-cell, T-cell, antigen presenting cell, activated B-cell, activated T-cell, activated macrophage), changes the level of immunomodulatory molecules (e.g, inflammatory cytokines, chemokines), or a combination thereof.
  • Modulation of immune response by such an immune system modulator can be a suppression of the immune response (immunosuppression or anti-inflammatory ) in a subject or an increase the immune response (immunostimulatory or pro-inflammatory) in a subject relative to a subject that has not been administered a composition provided herein.
  • an immunomodulator comprises a pattern recognition receptor (PRR) agonist.
  • the PRR agonist comprises a nucleic acid.
  • the nucleic acid is a single-stranded nucleic acid or a double-stranded nucleic acid.
  • the nucleic acid is a RNA, a DNA or a combination thereof.
  • the nucleic acid is a linear nucleic acid.
  • the nucleic acid comprises a hairpin nucleic acid.
  • the PRR is an endosomal nucleic acid sensor.
  • the endosomal nucleic acid sensor is toll-like receptor (TLR).
  • Exemplary TLR PRRs include TLR3, TLR7, TLR8, and TLR9.
  • the TLR PRR is TLR3.
  • the TLR3 agonist is RIBOXXOL, poly(I:C), or Hiltonol®.
  • the PRR is a DNA sensor.
  • Exemplary' DNA sensor PRRs include cyclic GMP-AMP synthase (cGAS).
  • the PRR is a retinoic acid-inducible gene I (RIG-I)-like receptor (RLR).
  • the RLR is RIG-I, melanoma differentiation-associated protein 5 (MDA5), or laboratory' of genetics phy siology 2 (LGP2).
  • the PRR agonist is a viral RNA sequence. In some embodiments, the PRR agonist comprises a triphosphate (PPP) group at the 5’ end. In some embodiments, the PRR agonist comprising a triphosphate (PPP) group at the 5’ end is an RNA molecule. In some embodiments, the PRR agonist comprises an uncapped diphosphate (PP) group at the 5’ end. In some embodiments, the PRR agonist comprises an uncapped diphosphate (PP) group at the 5’ end is an RNA molecule. In some embodiments, the PRR agonist comprises a 5 ’-terminal nucleotide having an unmethylated 2’-0 position.
  • the PRR agonist binds to a carboxy-terminal domain (CTD) of an RLR.
  • CTD carboxy-terminal domain
  • the PRR agonist comprises nucleic acid base pairs which contact the helicase domain of an RLR.
  • the PRR agonist is an RLR agonist.
  • the RLR agonist is a RIG-I agonist.
  • the RIG-I agonist comprises a uridine rich stretch.
  • the RIG-I agonist comprises an RNA sequence derived from hepatitis C virus (HVC).
  • the RIG-I agonist comprises an RNA sequence derived from Sendai virus RNA.
  • the PRR agonist is a non-coding RNA, a TLR agonist, a RIG-I agonist, a saponin, a peptide, a protein, a carbohydrate, a carbohydrate polymer, a conjugated carbohydrate, a whole viral particle, a virus-like particle, viral fragments, cellular fragments, and combinations thereof.
  • the nucleic acid is a TLR agonist or a RIG-I agonist.
  • Exemplary TLR agonists include a TLR2, TLR3. TLR4, TLR7, TLR8, or TLR9 agonist.
  • TLR agonist for inclusion in a composition provided herein is, without limitation, is a TLR3 agonist, such as RIBOXXOL, poly(I:C) (Polyinosinic:polycytidylic acid, sodium salt, ((CioHioN4Na07P)x «(C9HnN3Na07P)x)), or Hiltonol®.
  • TLR3 agonist such as RIBOXXOL, poly(I:C) (Polyinosinic:polycytidylic acid, sodium salt, ((CioHioN4Na07P)x «(C9HnN3Na07P)x)), or Hiltonol®.
  • a PRR agonist comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to any one of the amino acid sequences listed in Table 4.
  • an immunomodulator comprises a TLR agonist (e.g., imidazoquinoline), a STING agonist, an inflammasome agonist, a RIG-I agonist or a combination thereof.
  • the immunomodulator comprises imiquimod, resiquimod, RAR, or a combination thereof.
  • immunomodulators target cytokine receptors to modulate immune responses, cell growth, and other cellular functions.
  • Immune cells secrete cytokines and interferons to signal to other immune cells, e.g., to promote phagocytosis of a microorganism or infected cells, or induce inflammation at the site of an injury.
  • the cytokine comprises a pro-inflammatory cytokine.
  • pro-inflammatory cytokines include: interleukin- 12 (IL-12), IL-18, IL-17.
  • IL-1J3 TNF-alpha (TNF-a) TNF-alpha
  • the cytokine comprises an anti-inflammatory cytokine.
  • anti-inflammatory cytokines include: IL-4, IL-10, IL-11, IL-13, and IL-35.
  • a nucleic acid provided herein encodes for an IL-12 family cytokine.
  • IL-12 is generally secreted from B-cells and macrophages.
  • IL-12 can induce proliferation of natural killer (NK) cells, increase interferon (IFN) production, and promote cell-mediated immune functions.
  • IL- 12 can induce naive CD4+ T cells to differentiate into Thl cells.
  • the interleukin 12 (IL-12) family is comprised of 4 members, IL-12, IL-23, IL-27, and IL-35.
  • a nucleic acid provided herein encodes for an alpha and/or a beta chain of an IL- 12 family cytokine.
  • nucleic acids provided herein encode for an IL-12p40.
  • nucleic acids provided herein encode for a Ebi3.
  • nucleic acids provided herein encode for an IL-12p35.
  • nucleic acids provided herein encode for an IL-23pl9.
  • nucleic acids provided herein encode for an IL-27p28.
  • anucleic acid provided herein encodes for (1) IL-12p35, IL-23pl9, or IL-27p28; and (2) IL-12p40 or Ebi3.
  • a nucleic acid provided herein encodes for IL-12, or a functional variant thereof.
  • a nucleic acid provided herein encodes a human IL-12A subunit, or a functional variant thereof.
  • a nucleic acid provided herein encodes a human IL-12B subunit, or a functional variant thereof.
  • a nucleic acid provided herein encodes a mouse IL- 12a subunit, or a functional variant thereof.
  • anucleic acid provided herein encodes a mouse IL-12b subunit or a functional variant thereof.
  • a nucleic acid provided herein further comprises a sequence encoding a linker.
  • the linker comprises an amino acid sequence comprising VPGVGVPGVG (SEQ ID NO: 67), a fragment, or a variant thereof.
  • a nucleic acid provided herein encodes a human IL-12B, a linker, and a human IL12A subunit. In some embodiments, a nucleic acid provided herein encodes a mouse IL- 12b, a linker, and a mouse IL 12a subunit. In some embodiments, a nucleic acid provided herein encodes for a mouse IL-12 comprising an amino acid sequence that is at least 85% identical to any one of SEQ ID NOS: 159-164, and any combination thereof.
  • the nucleic acid encodes for a fusion protein comprising a mouse IL- 12b (p40) and a mouse IL-12a (p35) subunit, wherein the fusion protein further comprises an elastin linker between subunits.
  • a nucleic acid provided herein encodes for a mouse IL-12 fusion protein, wherein the nucleic acid comprises a sequence that is at least 85% identical to one of SEQ ID NOS: 165-166.
  • a nucleic acid provided herein encodes for interferon gamma (IFNy), or a functional variant thereof.
  • IFNy interferon gamma
  • a nucleic acid provided herein encodes for a human IFNy comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 167. In some embodiments, a nucleic acid provided herein encodes for a mouse IFNy comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 168.
  • Interferon is generally secreted from activated Thl T cells and natural killer (NK) cells.
  • IFNy can induce expression of class I MHC molecule on the surface of somatic cells, induce class II MHC expression on antigen presenting cells (APCs) and somatic cells. IFNy can induce activation of macrophages, neutrophils, and NK cells. In addition, IFNy promotes cell-mediated immunity and antiviral responses.
  • a nucleic acid provided herein encodes for IL-2, or a functional variant thereof. In some embodiments, a nucleic acid provided herein encodes for a human IL-2 comprising an amino acid sequence that is at least 85% identical to SEQ ID NO:
  • IL-2 is generally secreted or expressed by activated Thl cells and NK cells. IL-2 can induce and enhance proliferation of B cells and activated T cells. IL-2 can also modulate NK cellular functions.
  • a nucleic acid provided herein encodes for IL-15, or a functional variant thereof.
  • a nucleic acid provided herein encodes for a human IL-15 comprising an amino acid sequence that is at least 85% identical to SEQ ID NO:
  • IL-15 is generally expressed or secreted by mononuclear phagocytes (e.g., macrophages, monocytes, Kupffer cells, histiocytes, microglia, osteoclasts, dust cells, Langerhans cells, Hofbauer cells, intraglomerular mesangial cells sinusoidal lining cells, etc.). IL-15 induces proliferation of NK cells among other functions.
  • mononuclear phagocytes e.g., macrophages, monocytes, Kupffer cells, histiocytes, microglia, osteoclasts, dust cells, Langerhans cells, Hofbauer cells, intraglomerular mesangial cells sinusoidal lining cells, etc.
  • a nucleic acid provided herein encodes for IL-18, or a functional variant thereof.
  • a nucleic acid provided herein encodes for a human IL-18 comprising an amino acid sequence that is at least 85% identical to SEQ ID NO:
  • IL-18 is generally expressed or secreted by macrophages and can indue interferon production and expression by T cells and NK cells.
  • a nucleic acid provided herein encodes for IL-21, or a functional variant thereof. In some embodiments, a nucleic acid provided herein encodes for a human IL-21 comprising an amino acid sequence that is at least 85% identical to SEQ ID NO:
  • IL-21 is generally secreted by T cells such as Th2 cells, T follicular cells, and NK T cells. IL-12 induces cell proliferation and activates CD8+ T cell effector activity.
  • a nucleic acid provided herein encodes for IL-23, or a functional variant thereof.
  • a nucleic acid provided herein encodes for a human IL-23 comprising an amino acid sequence that is at least 85% identical to SEQ ID NO:
  • IL-23 is generally secreted from or expressed by activated dendritic cells, macrophages, monocytes; innate lymphoid cells, y5 T cells; and B cells. IL-23 induces the development and differentiation of effector Thl7 cells, and stimulates IL- 17 production and expression, among other functions.
  • a nucleic acid provided herein encodes for IL-27, or a functional variant thereof. In some embodiments, a nucleic acid provided herein encodes for a human IL-27 comprising an amino acid sequence that is at least 85% identical to SEQ ID NO:
  • IL-27 is composed of an a chain p28 and [3 chain Epstein-Ban induce gene-3 (EBI3).
  • the p28 subunit is also called IL-30.
  • IL-27 is generally expressed or secreted by antigen presenting cells. IL-27 can induce differentiation of T cells and upregulate IL- 10 secretion.
  • a nucleic acid provided herein encodes for IL-35, or a functional variant thereof.
  • IL-35 is a dimeric protein composed of IL-12a and IL-27J3 chains, which are encoded by two separate genes - IL12A and EBI3 (Epstein-Barr virus-induced gene 3), respectively.
  • a nucleic acid provided herein encodes for a human IL- 27 comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 159, SEQ ID NOS: 174, or a combination thereof.
  • IL-35 is an immunosuppressive cytokine that blocks the development of Thl and Thl 7 cells by limiting early T cell proliferation in a subject.
  • a nucleic acid provided herein encodes for IL-39, or a functional variant thereof.
  • IL-39 is a heterodimer of IL-23pl9 and Epstein-Barr induced gene- 3 (EBB).
  • EBB Epstein-Barr induced gene- 3
  • IL-39 is a cytokine secreted by stimulated and activated B cells.
  • IL-39 induces and/or expands neutrophils and can increase the secretion of B cell activation factor (BAFF), stimulating inflammation in a subject.
  • BAFF B cell activation factor
  • nucleic acids provided herein encode for a cytokine listed in Table 5.
  • compositions provided herein comprise two or more nucleic acids encoding for different sequences listed in Table 5.
  • nucleic acids provided herein encode for a cancer-associated protein sequence comprising at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a sequence listed in Table 5.
  • compositions provided herein comprise two or more nucleic acids encoding different sequences listed in Table 5.
  • the nucleic acid provided herein encodes for a cancer-associated protein or a functional fragment thereof comprising at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence similarity to a sequence listed Table 5.
  • Percent (%) sequence identity for a given sequence relative to a reference sequence is defined as the percentage of identical residues identified after aligning the two sequences and introducing gaps if necessary, to achieve the maximum percent sequence identity. Percent identity can be calculated using alignment methods known in the art, for instance alignment of the sequences can be conducted using publicly available software such as BLAST, Align, ClustalW2. Those skilled in the art can determine the appropriate parameters for alignment, but the default parameters for BLAST are specifically contemplated.
  • Table 5 lists cytokines and sequences that can be encoded by the nucleic acids provided herein.
  • a nucleic acid provided herein comprises a nucleic acid encoding for an amino acid sequence that is least 85% identical to any one of SEQ ID NOS: 159-164, 167-175. In some embodiments, a nucleic acid provided herein comprises a nucleic acid encoding for an amino acid sequence that is least 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOS: 159-164, 167-175. In some embodiments, a nucleic acid provided herein comprises a nucleic acid encoding for any one of SEQ ID NOS: 159-164, 167-175
  • a nucleic acid provided herein encodes for interferon alpha (IFN-a) and/or interferon beta (IFN-
  • IFN-a and IFN-P are generally secreted or expressed by macrophages, neutrophils, and somatic cells. Interferons can induce antiviral effects, induce expression of class I MHC molecules on the surface of somatic cells, activate NK cells and macrophages.
  • a nucleic acid provided herein encodes for granulocytemacrophage colony-stimulating factor (GM-CSF) or a functional variant thereof.
  • GM-CSF is generally secreted or expressed by Th cells.
  • GM-CSF induces the growth and differentiation of monocytes and dendritic cells.
  • a nucleic acid provided herein encodes for IL-1 a or a functional variant thereof.
  • IL- la is generally secrete or expressed by macrophages and other APCs.
  • IL-1 a co-stimulates APCs and T cells and induces inflammation, fever acute phase response, and hematopoiesis, among other functions.
  • a nucleic acid provided herein encodes for IL-3 and/or IL- 4, or a functional variant thereof.
  • IL-3 and IL-4 are secreted by activated T cells.
  • IL-3 induces the growth of hematopoietic progenitor cells.
  • IL-4 can induce B-cell proliferation, eosinophil and mast cell growth, induces eosinophil and mast cell function, induces IgE and class II MHC molecule expression on B cells, and can inhibit cytokine production by monocytes and macrophages.
  • a nucleic acid provided herein encodes for IL-5 or a functional variant thereof.
  • IL-5 is generally secreted or expressed by Th2 cells and mast cells to induce eosinophil growth and function.
  • a nucleic acid provided herein encodes for IL-6 or a functional variant thereof.
  • IL-6 is generally secreted or expressed by activated Th2 cells, APCs, and somatic cells. IL-6 induces acute phase responses, B-cell proliferation, thrombopoiesis. IL- 6 works synergistically with IL-1 and TNF on T cell activation.
  • a nucleic acid provided herein encodes for IL-7 or a functional variant thereof. IL-7 is generally secreted or expressed by thymic stromal cells and marrow stromal cells to induce T cell and B cell lymphopoiesis.
  • a nucleic acid provided herein encodes for IL-8 or a functional variant thereof.
  • IL-8 is generally secreted or expressed by macrophages and somatic cells. IL-8 to function as a chemoattractant for neutrophils and T cells.
  • a nucleic acid provided herein encodes for IL-9 or a functional variant thereof.
  • IL-9 is generally secreted or expressed by T cells to induce hematopoiesis and can also have thymopoeitic effects.
  • a nucleic acid provided herein encodes for IL- 10 or a functional variant thereof.
  • IL- 10 is generally secreted or expressed by activated Th2 cells, CD8+ T cells, B cells, and macrophages.
  • IL-10 inhibits cytokine production, promotes B cell proliferation and antibody production.
  • IL- 10 also suppresses cellular immunity and mast cell growth.
  • a nucleic acid provided herein encodes for IL- 13 or a functional variant thereof.
  • IL- 13 is generally secreted or expressed by Th2 cells and can act synergistically with IL-4 to induce B-cell proliferation.
  • a nucleic acid provided herein encodes for IL- 17 (also called IL-17a) or a functional variant thereof.
  • IL-17 is generally secreted or expressed by T- helper 17 (Thl7) cells, a subset of CD4+ T-cell that secrete IL-17.
  • Thl7 cells T- helper 17 (Thl7) cells, a subset of CD4+ T-cell that secrete IL-17.
  • IL-17 acts as a chemotaxis signal for monocytes and neutrophils to a site of inflammation.
  • IL- 17 mediates effects on stromal cells, resulting in production of inflammatory cytokines and recruitment of leukocytes (e.g, neutrophils), creating a link between innate and adaptive immunity.
  • a nucleic acid provided herein encodes for IL-22 or a functional variant thereof.
  • IL-22 is generally secreted by Thl. Th22, Thl7. and y5 T cells; NK T cells; innate lymphoid cells (ILC3), neutrophils; and macrophages.
  • IL-22 can improve cell survival and proliferation.
  • IL-22 can also promote the synthesis of anti-microbial peptides such as S100, regenerating islet-derived protein 3-beta (Reg3
  • a nucleic acid provided herein encodes for IL-25 (also called IL-17e) or a functional variant thereof.
  • IL-25 is generally secreted by T cells, dendritic cells, macrophages, mast cells, basophils, eosinophils, epithelial cells and Paneth cells.
  • IL-25 can induce NF -KB activation, the production of IL-8, and a neutrophil chemotaxis.
  • IL-25 also activates eosinophil expansion.
  • a nucleic acid provided herein encodes for macrophage inflammatory protein (MlP)-la and/or MIP-ip. or a functional variant thereof.
  • MIP-la also called chemokine (C-C motif) ligand 3 (CCL3)
  • MIP-ip also called chemokine (C-C motif) ligands 4 (CCL4) is secreted by lymphocytes and macrophages.
  • MIP-la and MIP-ip can activate granulocytes (e.g.. neutrophils, eosinophils and basophils) to induce acute inflammation.
  • a nucleic acid provided herein encodes for transforming growth factor beta (TGF-P) or a functional variant thereof.
  • TGF-P is a multifunctional cytokine belonging to the transforming growth factor superfamily that includes three different mammalian isoforms (TGF-P 1 to 3, HGNC symbols TGFB1, TGFB2, TGFB3) and many other signaling proteins.
  • TGF-P proteins are generally produced and secreted by leukocytes, including T cells and monocytes. TGF-P can induce chemotaxis, IL-1 synthesis, IgA synthesis, and inhibit cell proliferation.
  • a nucleic acid provided herein encodes for a tumor necrosis factor family protein.
  • a nucleic acid provided herein encodes for tumor necrosis factor-alpha (TNF-a) or a functional variant thereof.
  • TNF-a is generally secreted by macrophages, mast cells, NK cells, and sensory neurons. TNF-a can induce cell death, induce inflammation, and activate pain signaling.
  • a nucleic acid provided herein encodes for tumor necrosis factor-beta (TNF-P) or a functional variant thereof.
  • TNF-p also called lymphotoxin-alpha (LT-a) is produced and secreted by lymphocytes.
  • TNF-P has a number of different functions depending on the form that is secreted or expressed by a cell (e.g., a soluble homotrimer or as a cell surface protein heterotrimer- LTP). TNF-P can induce cell death and induce inflammation.
  • a nucleic acid provided herein encodes for a linker.
  • the linker is between an alpha and a beta chain of a cytokine.
  • the linker is betw een an alpha and a beta chain of an IL- 12 family cytokine.
  • the linker is about 14 to 18 amino acids long.
  • the bioactive agents provided herein comprise chemotherapeutic agents.
  • the chemotherapeutic agents comprise tumor suppressant or tumor killing properties.
  • chemotherapeutic agents comprise one or more of doxorubicin and cyclophosphamide.
  • the one or more bioactive agents comprise antiinflammatory agents.
  • the anti-inflammatory agent comprises a corticosteroid.
  • the corticosteroid is dexamethasone.
  • the corticosteroid is prednisone.
  • compositions comprising a dried or lyophilized composition provided herein that is reconstituted in a suitable diluent and a pharmaceutically acceptable carrier.
  • the diluent is aqueous.
  • the diluent is water.
  • dried compositions and lyophilized compositions provided herein comprise (a) a lipid carrier, wherein the lipid carrier is a nanoemulsion comprising: (i) a hydrophobic core; (li) optionally, one or more inorganic nanoparticles; (iii) and one or more lipids; (b) one or more nucleic acids; and (c) at least one cryoprotectant.
  • the cry oprotectant is selected from the group consisting of: sucrose, maltose, trehalose, mannitol, glucose, and any combinations thereof.
  • cry oprotectants include but are not limited to: dimethyl sulfoxide (DMSO), glycerol, propylene gly col, ethylene glycol, 3-O-methyl-D-glucopyranose (3-OMG), olyethylene glycol (PEG), 1,2-propanediol, acetamide, trehalose, formamide, sugars, proteins, and carbohydrates.
  • DMSO dimethyl sulfoxide
  • glycerol propylene gly col
  • ethylene glycol 3-O-methyl-D-glucopyranose (3-OMG)
  • PEG olyethylene glycol
  • 1,2-propanediol 1,2-propanediol
  • acetamide 1,2-propanediol
  • trehalose 1,2-propanediol
  • formamide 1,2-propanediol
  • sugars 1,2-propanediol
  • proteins 1,2-propanediol
  • compositions and methods provided herein comprise at least one cryoprotectant.
  • cryoprotectants for inclusion are, but not limited to, sucrose, maltose, trehalose, mannitol, or glucose, and any combinations thereof.
  • additional or alternative cryoprotectant for inclusion is sorbitol, ribitol, erythritol, threitol, ethylene glycol, or fructose.
  • cryoprotectant for inclusion is dimethyl sulfoxide (DMSO), glycerol, propylene glycol, ethylene glycol, 3-O-methyl-D-glucopyranose (3-OMG), polyethylene glycol (PEG), 1,2- propanediol, acetamide, trehalose, formamide, sugars, proteins, and carbohydrates.
  • DMSO dimethyl sulfoxide
  • PEG polyethylene glycol
  • 1,2- propanediol 1,2- propanediol
  • acetamide trehalose
  • formamide sugars, proteins, and carbohydrates.
  • the cryoprotectant is present at about 1% w/v to at about 20% w/v. preferably about 10% w/v to at about 20% w/v, and more preferably at about 10% w/v.
  • the cryoprotectant is sucrose.
  • the cryoprotectant is maltose.
  • the cryoprotectant is trehalose. In some aspects of the disclosure, the cryoprotectant is mannitol. In some aspects of the disclosure, the cryoprotectant is glucose. In some embodiments, the cryoprotectant is present in an amount of about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 325, 350, 375, 400, 450, 500 mg or more. In some embodiments, the cryoprotectant is present in an amount of about 50 to about 500 mg.
  • the cryoprotectant is present in an amount of about 200 to about 300 mg. In some embodiments, the cryoprotectant is present in an amount of about 250 mg. In some embodiments, the cry oprotectant is present in amount of a lyophilized composition by weight of at least about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or more percent. In some embodiments, the cryoprotectant is present in amount of a lyophilized composition by weight of about 95%. In some embodiments, the cryoprotectant is present in amount of a lyophilized composition by weight of 80 to 98%, 85 to 98%, 90 to 98%, or 94 to 96%. In some embodiments, the cryoprotectant is a sugar.
  • the sugar is sucrose, maltose, trehalose, mannitol, or glucose. In some embodiments, the sugar is sucrose. In some embodiments, the sucrose is present in an amount of about 10. 20. 30. 40. 50. 60. 70. 80. 90. 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 325, 350, 375, 400, 450, 500 or more mg. In some embodiments, the sucrose is present in an amount of about 50 to about 500 mg. In some embodiments, the sucrose is present in an amount of about 200 to about 300 mg.
  • the sucrose is present in an amount of about 250 mg. In some embodiments, the sucrose is present in amount of a lyophilized composition by weight of at least about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or more percent. In some embodiments, the sucrose is present in amount of a lyophilized composition by weight of about 95%. In some embodiments, the sucrose is present in amount of a lyophilized composition by weight of 80 to 98%, 85 to 98%, 90 to 98%, or 94 to 96%.
  • the cryoprotectant is sucrose. In some embodiments, the cryoprotectant is at a concentration of at least about 0.1% w/v. In some embodiments, the cryoprotectant is at a concentration of about 1% w/v to at about 20% w/v. In some embodiments, the cryoprotectant is at a concentration of about 10% w/v to at about 20% w/v. In some embodiments, the cryoprotectant is at a concentration of about 10% w/v.
  • compositions provided herein are thermally stable.
  • a composition is considered thermally stable when the composition resists the action of heat or cold and maintains its properties, such as the ability to protect a nucleic acid molecule from degradation at given temperature.
  • compositions provided herein are thermally stable at about 25 degrees Celsius (°C) or standard room temperature.
  • compositions provided herein are thermally stable at about 45 °C.
  • compositions provided herein are thermally stable at about - 20 °C.
  • compositions provided herein are thermally stable at about 2 °C to about 8 °C.
  • compositions provided herein are thermally stable at a temperature of at least about -80 °C, at least about- 20 °C, at least about 0 °C, at least about 2 °C, at least about 4 °C. at least about 6 °C. at least about 8 °C, at least about 10 °C. at least about 20 °C. at least about 25 °C, at least about 30 °C, at least about 37 °C, up to 45 °C.
  • compositions provided herein are thermally stable for at least about 5 day, at least about 1 week, at least about 2 weeks, at least about 1 month, up to 3 months.
  • compositions provided herein are stored at a temperature of at least about 4° C up to 37 °C for at least about 5 day, at least about 1 week, at least about 2 weeks, at least about 1 month, up to 3 months. In some embodiments, compositions provided herein are stored at a temperature of at least about 20 °C up to 25 °C for at least about 5 day, at least about 1 week, at least about 2 weeks, at least about 1 month, up to 3 months.
  • Also provided herein are methods for preparing a lyophilized composition comprising obtaining a lipid carrier, wherein the lipid carrier is a nanoemulsion comprising a hydrophobic core, one or more inorganic nanoparticles and one or more lipids; incorporating one or more nucleic acid into the lipid carrier to form a lipid carrier- nucleic acid complex; adding at least one cryoprotectant to the lipid carrier-nucleic acid complex to form a formulation; and lyophilizing the formulation to form a lyophilized composition.
  • lipid carrier is a nanoemulsion comprising a hydrophobic core, one or more inorganic nanoparticles and one or more lipids; incorporating one or more nucleic acid into the lipid carrier to form a lipid carrier- nucleic acid complex; adding at least one cry oprotectant to the lipid carrier-nucleic acid complex to form a formulation; and spray drying the formulation to form a spray-dried composition.
  • lipid carrier is a nanoemulsion comprising a hydrophobic core, one or more inorganic nanoparticles, and one or more lipids; incorporating one or more nucleic acid into the said lipid carrier to form a lipid carrier-nucleic acid complex; adding at least one cryoprotectant to the lipid carrier-nucleic acid complex to form a formulation; lyophilizing the formulation to form a lyophilized composition; and reconstituting the lyophilized composition in a suitable diluent.
  • lipid carrier is a nanoemulsion comprising a hydrophobic core, one or more inorganic nanoparticles, and one or more lipids, incorporating one or more nucleic acid into the said lipid carrier to form a lipid carrier-nucleic acid complex; adding at least one cryoprotectant to the lipid carrier-nucleic acid complex to form a formulation; spray drying the formulation to form a spray-dried composition; and reconstituting the spray -dried composition in a suitable diluent.
  • compositions provided herein e.g, NP-1 or NP-30 complexed with nucleic acids.
  • a suspension comprising a composition provided herein.
  • suspensions provided herein comprise a plurality of nanoparticles or compositions provided herein.
  • compositions provided herein are in a suspension, optionally a homogeneous suspension.
  • compositions provided herein are in an emulsion form.
  • compositions provided herein are combined with pharmaceutically acceptable salts, excipients, and/or carriers to form a pharmaceutical composition.
  • Pharmaceutical salts, excipients, and carriers may be chosen based on the route of administration, the location of the target issue, and the time course of delivery of the drug.
  • a pharmaceutically acceptable carrier or excipient may include solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., compatible with pharmaceutical administration.
  • the pharmaceutical composition is in the form of a solid, semi-solid, liquid or gas (aerosol).
  • injectable preparations for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution, suspension, or emulsion in a nontoxic parenterally acceptable diluent or solvent.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P., and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.
  • the encapsulated or unencapsulated conjugate is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or (a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, (c) humectants such as glycerol, (d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, (e) solution retarding agents such as paraffin, (f) absorption accelerators such as quaternary ammonium compounds, (g) wetting agents such
  • compositions provided herein may be formulated in dosage unit form for ease of administration and uniformity of dosage.
  • a dosage unit form is a physically discrete unit of a composition provided herein appropriate for a subject to be treated. It will be understood, however, that the total usage of compositions provided herein w ill be decided by the attending physician within the scope of sound medical judgment.
  • the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, such as mice, rabbits, dogs, pigs, or non-human primates. Dosing may be for veterinary or human therapeutic uses. The animal model is also used to achieve a desirable concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • compositions provided herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose is therapeutically effective in 50% of the population) and LD50 (the dose is lethal to 50% of the population).
  • the dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50.
  • Pharmaceutical compositions which exhibit large therapeutic indices may be useful in some embodiments.
  • the data obtained from cell culture assays and animal studies may be used in formulating a range of dosage for human and non-human animal use.
  • compositions and pharmaceutical compositions for administering to a subject in need thereof are provided herein.
  • the subject is a human.
  • the subject is a non-human animal.
  • the animal is a domesticated animal or livestock.
  • compositions and pharmaceutical compositions for veterinary and therapeutic use in non- human animals are provided herein.
  • Subjects include, without limitation, domesticated animals, farmed animals and insects (including without limitation pigs, cows, horses, donkeys, mules, buffalo, bison, goats, sheep, pigs, ducks, geese, chicken, turkey, fish, camels, alpacas, llamas, rabbits, zebu, deer, guinea pigs, yaks, ferrets, birds, hedgehogs, rodents, turtles, amphibians, bees), wild animals, as well as humans.
  • pharmaceutical compositions provided here are in a form which allows for compositions provided herein to be administered to a subject.
  • the administering is local administration or systemic administration.
  • a composition provided herein is formulated for administration/for use in administration via an intratumoral, subcutaneous, intradermal, intramuscular, inhalation, intravenous, intraperitoneal, intracranial, intranasal, oral, intrathoracic, or intrathecal route.
  • the administering is every 1, 2, 4, 6, 8, 12, 24, 36, or 48 hours.
  • the administering is daily, weekly, or monthly.
  • the administering is repeated at least about every 28 days or at least about every 56 days.
  • a composition or pharmaceutical composition provided herein is administered to the subject by two doses.
  • a second dose of a composition or pharmaceutical composition provided herein is administered about 28 days after the first dose or about 56 after the first dose.
  • a third dose of a composition or pharmaceutical composition provided herein is administered to a subject. In some embodiments, the third dose of a composition or pharmaceutical composition provided herein is administered about 56 days after the second dose.
  • a method of treating cancer in a subject comprising administering to the subject a composition provided herein (e.g., NP-1 or NP-30 complexed with nucleic acids), wherein the composition comprises one or more markers of a favorable safety profile.
  • the compositions provided herein are processed to remove endotoxin.
  • a favorable safety profile is a set of parameters that reduce the incidence of adverse events in a population of subjects that are administered the composition relative to a comparable composition that has at least one difference in the composition.
  • Adverse events are any untoward medical occurrence caused by a medical treatment, which injures a patient.
  • adverse events are severe adverse events that result in a hospital stay longer than 24 hours, a life-threatening medical event, or death.
  • Nonlimiting examples of adverse events include myocarditis, pericarditis, convulsions, blood dyscrasias, or allergic bronchospasm.
  • the safety profile of a composition provided herein e.g. , NP-1 or NP-30 complexed with nucleic acids
  • a favorable safety profile can include one or more parameters including but not limited to: a low or no reported toxicity 7 ; high tolerability 7 at nucleic acid doses above 10 pg; a low or no reported incidence of adverse events in the subject following administration of the composition; a low severity of any reported adverse events in the subject following administration of the composition: a low or no reported incidence of pain at the injection site, redness, and swelling; a low or no reported incidence of fever, fatigue, headache, chills, vomiting, diarrhea, new or worsened muscle pain, and new or worsened joint pain; a low or no reported incidence of abnormal hematology; a low or no reported incidence of cardiovascular events; a low or no reported incidence of chest pain; low or no reported incidence of myocarditis; low or no reported incidence of cardiac inflammation; low or no reported incidence of myocardial injury; a low or no reported incidence of seizure; a low or no reported incidence of liver injury; or a low or no reported incidence of kidney failure, as reported by the subject, a
  • a favorable safety profile of a composition provided herein can include alow expression of reactogenic biomarkers relative to comparable compositions or a reference level.
  • a reference level is the level of the reactogenic biomarker in a subject with systemic inflammation.
  • Reactogenicity is the physical manifestation of the systemic inflammatory response induced by a pharmaceutical composition. Reactogenicity results in both local and systemic inflammation. The systemic inflammation is the cause of adverse events associated with the composition, including anaphylactic reactions, and autoimmune events.
  • a composition provided herein when administered to a subject reduces the level or activity' of at least one reactogenic biomarker relative to the level or activity of the reactogenic biomarker in a subject with systemic inflammation.
  • reactogenic biomarkers include: type-I interferons, interferon-gamma (IFN-y), interferon- a2. interleukin-6 (IL-6), interleukin- 1(3 (IL- 1 P), IL-27p28. MyD88, toll-like receptor 7 (TLR7).
  • TLR8 caspase- 1, NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3), IL-18, IL-33, Monocyte chemoattractant protein-1 (MCP1) or CCL2, CXCL10, C-reactive protein (CRP), sera soluble ST2, creatine kinase-MB (CK-MB), troponin I (cTNI), and troponin T (cTNT).
  • Methods of detecting and quantifying the level of a reactogenic biomarker include, e.g, immunoassays, PCR, absorbance assays, sequencing, digital ELISA (single molecular array, Simoa), or mass spectrometry.
  • cardiac troponins are detected in the serum of a subject by the use of monoclonal antibodies to epitopes of cTnl and cTnT.
  • Cardiac troponin is a serological marker of cardiac damage.
  • a composition provided herein e.g, NP-1 or NP-30 complexed with nucleic acids
  • a composition provided herein generates a local immune response in a subject and maintains cardiac troponin I levels below a reference level for at least about 24 hours following administration.
  • the reference level for cTNI is 0.04 ng/mL.
  • a composition provided herein maintains cardiac troponin I levels below the reference level for at least about 48 hours following administration or more.
  • interferon alpha2 can be measured in a blood sample or a cerebral spinal fluid sample from a subject.
  • a composition provided herein (e.g., NP-1 or NP-30 complexed with nucleic acids) generates a local immune response in a subject and maintains IFN-a2 levels in the serum below 10,000 femtograms/mL (fg/mL) for a period of at least about 24 hours following administration.
  • a composition provided herein maintains interferon alpha2 (IFN-a2) levels below the reference level for at least about 24 hours following administration or more, at least about 48 hours following administration or more, 72 hours following administration or more, 96 hours following administration or more, 120 hours following administration or more.
  • IFN-a2 interferon alpha2
  • compositions e.g., NP-1 or NP-30 complexed with nucleic acids on the surface of the nanoparticles
  • Reactogenic nanoparticles are any nanoparticle composition that comprises one or more of the following characteristics or functional properties: (1) a neutral net charge (e g., 0 net charge) at 37 degrees Celsius; (2) comprise nucleic acids encapsulated within the core of the reactogenic nanoparticle; (3) comprise an average Z- diameter greater than 60 nm; (4) when administered to a subject, the reactogenic nanoparticle compositions has a higher level of a nucleic acid biodistribution ratio relative a composition provided herein; (5) when administered to a subject, the reactogenic nanoparticle increases the level of cardiac troponin in the subject relative to a composition provided herein or relative to a control subject that has not been administered the reactogenic nanoparticle; (6) when administered to a subject, the reactogenic nanoparticle increases the
  • compositions that have a reduced reactogenicity relative to reactogenic nanoparticles (e.g., nanoparticles that comprise nucleic acids within the core).
  • the reduced reactogenicity of a composition provided herein is characterized by a lower level or activity of systemic interferons (e.g. , in the serum of a subject) relative to the level of systemic interferons in a subject that has been administered a reactogenic nanoparticle composition.
  • the reduced reactogenicity comprises a lower level or activity of cardiac troponin in the subject relative to a level or activity of cardiac troponin in a subject that has been administered a reactogenic nanoparticle provided herein.
  • the cardiac troponin level is determined by obtaining a blood sample from a subject and performing an immunoassay.
  • the method comprises administering to a tissue in a subject a composition provided herein (e.g., NP-1 or NP-30 complexed with nucleic acids).
  • a composition provided herein (e.g., NP-1 or NP-30 complexed with nucleic acids).
  • the composition comprises: nanoparticles, wherein the nanoparticles comprise: a hydrophobic core comprising lipids in liquid phase at 25 degrees Celsius; and nucleic acids, wherein the nucleic acids are complexed to the nanoparticles to form nucleic acid-nanoparticle complexes.
  • the administering of the composition generates a local immune response within a tissue.
  • the tissue is at the site of administration (e.g., an intramuscular injection site or skeletal muscle tissue).
  • the tissue is not within or not adjacent to the tissue administered the composition (e.g.. a liver tissue, a spleen tissue, an ovarian tissue, atestis tissue, a lung tissue, a brain tissue, or a heart tissue).
  • compositions provided herein can be administered to any appropriate tissue in the subject.
  • the tissue is a muscle tissue, an epithelial tissue, an endothelial tissue, a mucosal tissue, a thecal tissue, or a tumor.
  • the administering is intramuscular administration, intradermal administration, transdermal administration, sublingual administration, buccal administration, intranasal administration, inhalation administration, intrathecal administration, or intratumoral administration.
  • the local immune response can be characterized by various parameters in the tissue of administration provided herein, including, but not limited to interferon levels, immunostimulatory biomarkers, cytokines, and cells.
  • the local immune response generated by a composition provided herein is characterized by an increase in the level of an immunostimulatory marker in a tissue relative to a reference level. In some embodiments, the local immune response is characterized by an increase in the level or activity of interferon in the tissue relative to the level or activity of interferon in a spleen or a liver of the subject. In some embodiments, the tissue is not within or adjacent to the tissue administered the composition (e.g., a liver tissue, a spleen tissue, an ovarian tissue, a testis tissue, a lung tissue, a brain tissue, or a heart tissue). In some embodiments, the local immune response generated by a composition provided herein is characterized by immune cell recruitment to the tissue.
  • the local immune response is characterized by localization of the nucleic acids to a tissue that is within or adjacent to a site of administration. In some embodiments, the local immune response is characterized by the nucleic acid biodistribution ratio.
  • the level of nucleic acids is determined by polymerase chain reaction (PCR), real time PCR (RT-PCR), spectrophotometry (e.g., NanoDrop®, ThermoFisher), microarray (e.g, Counter® Analysis System, NanoString), or an in vivo imaging assay.
  • PCR polymerase chain reaction
  • RT-PCR real time PCR
  • spectrophotometry e.g., NanoDrop®, ThermoFisher
  • microarray e.g, Counter® Analysis System, NanoString
  • an in vivo imaging assay e.g., a fluorescent dye can be conjugated to a composition provided herein, followed by administration of the composition to a subject (e.g., a test animal).
  • An in vivo imaging system can be used to determine the biodistribution of the composition by tracking the fluorescent dye in various organs in the subject.
  • a composition provided herein comprises a nucleic acid biodistribution ratio between 0 and 0.01 (e.g., 0 ⁇ nucleic acid biodistribution ratio ⁇ 0.01) when the composition is administered to a tissue in a subject.
  • a composition provided herein comprises a nucleic acid biodistribution ratio between 0 and 0.1 (e.g., 0 ⁇ nucleic acid biodistribution ratio ⁇ 0.1) when the composition is administered to a tissue in a subject.
  • a composition provided herein comprises a nucleic acid biodistribution ratio between 0 and 1 (e.g., 0 ⁇ nucleic acid biodistribution ratio ⁇ 1) when the composition is administered to a tissue in a subject. Therefore, a biodistribution ratio between 0 and 1 indicates that a composition provided herein has a favorable safety profile, reduces reactogenicity, and produces a local immune response in a subject relative to a comparable nucleic acid composition (e.g., a reactogenic nanoparticle composition, lipid nanoparticles (LNPs)).
  • a comparable nucleic acid composition e.g., a reactogenic nanoparticle composition, lipid nanoparticles (LNPs)
  • a composition provided herein comprises a nucleic acid biodistribution ratio that the lower than a reactogenic nanoparticle composition.
  • a composition provided herein comprises a nucleic acid biodistribution ratio of 0.01 or more. 0.1 or more, 0.2 or more, 0.3 or more, 0.4 or more, 0.5 or more, 0.6 or more, 0.7 or more, 0.8 or more. 0.9 or more, up to 1.0.
  • compositions provided herein can increase the level or activity of an immunostimulatory biomarker in the tissue of a subject relative to the level or activity of the immunostimulatory biomarker in the tissue prior to administration or the level or activity of the immunostimulatory biomarker in a healthy subject.
  • the immunostimulatory biomarker has an increased expression in the muscle tissue when the composition provided herein is administered intramuscularly.
  • immunostimulatory biomarkers include: CD86, H2-abl, H2-ebl, ITGAL/Cdl la, ITGAM/Cdl lb, Fey receptor, CD28, and Class I MHC-mediated antigen processing and presentation pathway proteins.
  • the local immune response can also be determined by the immune cell composition within the tissue.
  • pDCs monocyte dendritic cells, conventional dendritic cells, neutrophils, macrophages, and NK cells can be recruited to the tissue following administration of a composition provided herein.
  • Methods of detecting or quantifying an immune response can be used to determine the safety and efficacy of a composition administered to a subject provided herein.
  • Immunoassays e.g., ELISA, ELISPOT assays, Western blots, immunohistology', flow cytometry', or microscopy can be used to detect and/or quantify immune cells, antibodies, cytokines, and any other factor associated with an immune response.
  • flow cytometry can be used to detect and/or quantify CD4 + T cells and CD8 + T cells in a sample or specific subsets of immune cells provided herein.
  • Flow cytometry can also be used to determine the cellular composition in a tissue from a subject provided herein.
  • the cellular composition can be used to determine whether a composition generates a local or a systemic immune response in a subject in a particular tissue (e.g.. muscle, liver, or spleen).
  • a composition provided herein increases CD8 + T cell proliferation in a subject in response to an antigen provided herein.
  • Methods of detecting and quantifying gene expression can also be used to determine the efficacy and safety of a compositions provided herein.
  • Methods for assessing the presence of antibody neutralization of a microbial antigen or a tumor can be accomplished, e.g., by cellular impedance, live cell imaging assays, or immunoassays.
  • Cellular impedance assays include wells or plates with gold impedance biosensor arrays that measure the flow of electric current within a well that has been seeded with cells. Impedance is measured before, during, and after infection (e.g., a viral infection).
  • Antibody-mediated suppression of the CPE is readily detected as changes in both the kinetics and magnitude of the impedance signal. Plotting the value of the impedance signal at various time points as a function of antibody concentration can produce a dose response curve to yield IC50 measurements and determine the percentage of neutralization relative to control readings.
  • the methods comprise: administering to a subject having the cancer a composition provided herein.
  • the methods comprise: administering to a subject a first composition comprising a junction opener protein described herein, and administering to the subject a second composition comprising nanoparticles, wherein the nanoparticles comprise squalene, squalane, dehydroisosqualene, or a combination thereof.
  • the first composition and the second composition are administered together.
  • the first composition and the second composition are administered at a different time and/or day.
  • the methods comprise administering to a subject a composition, a suspension, or a pharmaceutical composition provided herein.
  • the compositions further comprise an inorganic particle.
  • the inorganic particle comprises an iodine-based contrast agent.
  • the inorganic particle comprises a metal.
  • the metal comprises a metal salt, a metal oxide, a metal hydroxide, or a metal phosphate.
  • the metal oxide comprises aluminum oxide, aluminum oxyhydroxide, iron oxide, titanium dioxide, or silicon dioxide.
  • the metal comprises iron or gadolinium-based metal salt, metal oxide, metal hydroxide or metal phosphate.
  • the subject has an overexpression of DSG2 protein on a surface of a cancer cell.
  • the cancer is a blood cancer or a solid tumor.
  • the blood cancer is lymphoma or leukemia.
  • the solid tumor is a carcinoma, a melanoma, or a sarcoma.
  • the subject has, is at risk for developing, or is suspected of having a skin cancer.
  • the skin cancer is a basal cell cancer, a melanoma, a Merkel cell cancer, a squamous cell carcinoma, a cutaneous lymphoma, a Kaposi sarcoma, or a skin adnexal cancer.
  • the subject has, is at risk for developing, or is suspected of having a pancreatic cancer.
  • the pancreatic cancer is a pancreatic adenocarcinoma, a pancreatic exocrine cancer, a pancreatic neuroendocrine cancer, an islet cell cancer, or a pancreatic endocrine cancer.
  • the subject has, is at risk for developing, or is suspected of having a colon cancer, a prostate cancer, an ovarian cancer, or a breast cancer.
  • kits comprising a composition provided herein.
  • a formulation or a composition provided herein is prepared in a single container for administration.
  • a formulation of a composition provided herein is prepared in two containers for administration, separating the nucleic acid, the protein, and/or the bioactive agent provided herein from the nanoparticle carrier.
  • container includes vessel, vial, ampule, tube, cup, box, bottle, flask, jar. dish, well of a single-well or multi- well apparatus, reservoir, tank, or the like, or other device in which the herein disclosed compositions may be placed, stored and/or transported, and accessed to remove the contents.
  • containers include glass and/or plastic sealed or re-sealable tubes and ampules, including those having a rubber septum or other sealing means that is compatible with withdrawal of the contents using a needle and syringe.
  • the containers are RNase free.
  • kit comprising: a first container comprising: a composition comprising nanoparticles, wherein the nanoparticles comprise a hydrophobic core, wherein the hydrophobic core comprises an oil in liquid phase at 25 degrees Celsius; and a second container comprising: a nucleic acid encoding for a protein or a functional variant thereof, a junction opener protein provided herein, or any bioactive agent provided herein.
  • the nanoparticles comprise a hydrophobic core comprising lipids in liquid phase at 25 degrees Celsius.
  • the nanoparticles comprise a cationic lipid, an oil, surfactants, and optionally, an inorganic particle.
  • the nucleic acid further codes for a RNA polymerase.
  • the RNA polymerase is a Venezuelan equine encephalitis virus (VEEV) RNA polymerase.
  • the nucleic acid sequence encoding for the RNA polymerase comprises the sequence of SEQ ID NO: 176.
  • the first container is lyophilized.
  • the kit further comprises an adjuvant provided herein.
  • compositions wherein the junction opener protein is conjugated to an anionic molecule, wherein the anionic molecule interacts with the lipid.
  • the junction opener protein comprises an amino acid sequence that is at least 80% identical to any one of SEQ ID NOS: 1 to 66
  • the junction opener protein comprises an amino acid sequence that binds to a tumor tight junction protein.
  • the tumor tight junction protein comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 68.
  • compositions, wherein the composition comprises tumor suppressant or tumor killing property 7 .
  • the bioactive agent comprises an anticancer agent.
  • compositions wherein the immunomodulator comprises a TLR agonist, a STING agonist, an inflammasome agonist, a RIG-I agonist, or a combination thereof.
  • TLR agonist is an imidazoquinoline.
  • compositions wherein the lipid comprises L2-dioleoyloxy-3 (trimethylammonium)propane (DOTAP), 30- [N — (N',N'-dimethylaminoethane) carbamoyl] cholesterol (DC Cholesterol), dimethyldioctadecylammonium (DDA); 1,2-dimyristoyl 3- trimethylammoniumpropane(DMTAP),dipalmitoyl(C 16: 0)trimethyl ammonium propane (DPTAP), distearoyltrimethylammonium propane (DSTAP), N-[l-(2,3- dioleyloxy)propyl]N,N,Ntrimethylammonium, chloride (DOTMA), N,N-dioleoyl-N,N- dimethylammonium chloride (DODAC), l,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), l,
  • DOTAP
  • compositions wherein the inorganic particle comprises a metal.
  • the metal comprises a metal salt, a metal oxide, a metal hydroxide, or a metal phosphate.
  • the metal oxide comprises aluminum oxide, aluminum oxyhydroxide, iron oxide, titanium dioxide, or silicon dioxide.
  • the metal comprises iron or gadolinium-based metal salt, metal oxide, metal hydroxide or metal phosphate.
  • the hydrophobic surfactant is sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, or sorbitan trioleate.
  • compositions wherein the nanoparticle is characterized as having a z-average diameter particle size measurement of about 20 nm to about 60 nm when measured using dynamic light scattering.
  • compositions wherein the hydrophilic surfactant is a polysorbate.
  • compositions for imaging or tracking nanoparticle conjugated to junction opener proteins wherein the compositions comprise any one of the compositions provided herein.
  • compositions wherein the multimerization domain comprises an amino acid sequence that is at least 80% identical to any one of SEQ ID NOS: 31 to 37.
  • compositions wherein the conjugatable moiety is linked to the shaft domain.
  • compositions, wherein the conjugatable moiety is linked to the multimerization domain.
  • compositions, wherein the junction opener protein is conjugated to the lipid.
  • compositions, wherein the junction opener protein is conjugated to the hydrophilic surfactant.
  • compositions wherein the immunomodulator comprises a TLR agonist, a STING agonist, an inflammasome agonist, a RIG-I agonist, or a combination thereof.
  • the TLR agonist is an imidazoquinoline.
  • compositions, wherein the immunomodulator comprises an imiquimod, a resiquimod, a RAR, or a combination thereof.
  • the chemotherapeutic agent comprises doxorubicin.
  • compositions wherein the composition further comprises a nucleic acid.
  • nucleic acid comprises repRNA, RAR or a combination thereof.
  • compositions, wherein hpids present in the hydrophobic core are in liquid phase at 25 degrees Celsius.
  • compositions, wherein the hydrophobic core comprises a liquid oil.
  • compositions wherein the liquid oil comprises a-tocopherol, coconut oil, grapeseed oil, lauroyl polyoxylglyceride, mineral oil, monoacylglycerol, palm kernel oil, olive oil, paraffin oil, peanut oil, propolis, squalene, squalane, dehydroisosqualene, soy lecithin, soybean oil, sunflower oil, a triglyceride, or vitamin E.
  • the triglyceride is capric triglyceride, caprylic triglyceride, a caprylic and capric triglyceride, a triglyceride ester, or myristic acid triglycerin.
  • compositions wherein the liquid oil comprises tumor suppressant or tumor killing property. Further provided herein are compositions, wherein the liquid oil comprises squalene, squalane, dehydroisosqualene, or a combination thereof. Further provided herein are compositions, wherein the lipid comprises l,2-dioleoyloxy-3 (trimethylammonium)propane (DOTAP), 3p-[N — (N',N'- dimethylaminoethane) carbamoyl] cholesterol (DC Cholesterol), dimethyldioctadecylammonium (DDA); 1,2-dimyristoyl 3- trimethylammoniumpropane(DMTAP),dipalmitoyl(C16:0)trimethyl ammonium propane (DPTAP), distearoyltrimethylammonium propane (DSTAP), N-[l-(2,3- dioleyloxy)propyl]N,N,Ntrimethylammonium, chloride (DOTAP), l,
  • compositions wherein the nanoparticle comprises an inorganic particle. Further provided herein are compositions, wherein the inorganic particle is within the hydrophobic core. Further provided herein are compositions, wherein the inorganic particle comprises an iodine-based contrast agent. Further provided herein are compositions, wherein the inorganic particle comprises a metal. Further provided herein are compositions, wherein the metal comprises a metal salt, a metal oxide, a metal hydroxide, or a metal phosphate. Further provided herein are compositions, wherein the metal oxide comprises aluminum oxide, aluminum oxyhydroxide, iron oxide, titanium dioxide, or silicon dioxide.
  • compositions wherein the metal comprises iron or gadolinium-based metal salt, metal oxide, metal hydroxide or metal phosphate.
  • the hydrophobic surfactant is sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, or sorbitan trioleate.
  • the nanoparticle is characterized as having a z-average diameter particle size measurement of about 20 nm to about 60 nm when measured using dynamic light scattering.
  • the hydrophilic surfactant is a polysorbate.
  • compositions for imaging or tracking nanoparticle conjugated to junction opener proteins wherein the compositions comprise any one of the compositions provided herein.
  • compositions wherein the pharmaceutical compositions comprise any one of the compositions provided herein; and a pharmaceutically acceptable excipient.
  • kits for treating a cancer in a subject comprise: administering to a subject any one of the compositions provided herein, any one of the suspensions provided herein, or any one of the pharmaceutical compositions provided herein.
  • the composition, the suspension or the pharmaceutical composition is administered via intramuscular injection, intranasal administration, oral administration, subcutaneous administration, intratumoral administration, intrathecal administration, or intravenous injection.
  • the subject is a mammal.
  • methods, wherein the subject is a human.
  • methods wherein the subject has a cancer, and wherein the subject has an overexpression of DSG2 protein on a surface of a cancer cell. Further provided herein are methods, wherein the subject has a solid tumor, wherein the subject has an overexpression of DSG2 protein on a surface of a cancer cell. Further provided herein are methods, wherein the solid tumor is a carcinoma, a melanoma, or a sarcoma. Further provided herein are methods, wherein the subject has a skin cancer, a pancreatic cancer, a colon cancer, a prostate cancer, an ovarian cancer, or a breast cancer.
  • the skin cancer is a basal cell cancer, a melanoma, a Merkel cell cancer, a squamous cell carcinoma, a cutaneous lymphoma, a Kaposi sarcoma, or a skin adnexal cancer.
  • the pancreatic cancer is a pancreatic adenocarcinoma, a pancreatic exocrine cancer, a pancreatic neuroendocrine cancer, an islet cell cancer, or a pancreatic endocrine cancer.
  • kits for imaging/tracking nanoparticles conjugated to junction opener proteins comprise: administering to a subject any one of the compositions provided herein, any one of the suspensions provided herein, or any one of the pharmaceutical compositions provided herein.
  • the composition, the suspension or the pharmaceutical composition is administered via intramuscular injection, intranasal administration, oral administration, subcutaneous administration, intratumoral administration, intrathecal administration, or intravenous injection.
  • the subject is a mammal.
  • methods, wherein the subject is a human.
  • the methods comprise: a) administering to a subject a first composition comprising a junction opener protein, wherein the junction opener protein comprises: (i) a shaft domain; (ii) a knob domain; and (iii) a multimerization domain; and b) administering to the subject a second composition comprising nanoparticles, wherein the nanoparticles comprise squalene, squalane, dehydroisosqualene, or a combination thereof. Further provided herein are methods, wherein the methods comprise administering the first composition and the second composition together.
  • the methods comprise administering the first composition and the second composition at a different time and/or day.
  • the shaft domain is selected from the group consisting of an Ad3 fiber polypeptide shaft domain motif, an Ad7 fiber polypeptide shaft domain motif, an Adi l fiber polypeptide shaft domain motif, an Ad 14 fiber polypeptide shaft domain motif, an Adl4a fiber polypeptide shaft domain motif, and combinations thereof.
  • the shaft domain comprises an amino acid sequence that is at least 80% identical to any one of SEQ ID NOS: 1 to 12.
  • the knob domain comprises an amino acid sequence that is at least 80% identical to any one of SEQ ID NOS: 13 to 30.
  • the multimerization domain comprises an amino acid sequence that is at least 80% identical to any one of SEQ ID NOS: 31 to 37.
  • the junction opener protein comprises an amino acid sequence that is at least 80% identical to any one of SEQ ID NOS: 1 to 66.
  • the junction opener protein comprises an amino acid sequence that binds to a tumor tight junction protein.
  • the tumor tight junction protein comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 68.
  • the methods further comprise an anticancer agent.
  • the anticancer agent comprises an anticancer antibody, an immunomodulator, a chemotherapeutic agent, or a combination thereof.
  • the anticancer antibody comprises one or more of atezolizumab, avelumab, bevacizumab, cemiplimab, cetuximab, daratumumab, dinutuximab, durvalumab. elotuzumab, ipilimumab, isatuximab, mogamulizumab.
  • the immunomodulator comprises a TLR agonist, a STING agonist, an inflammasome agonist, a RIG-I agonist, or a combination thereof.
  • the TLR agonist is an imidazoquinoline.
  • the immunomodulator comprises an imiquimod, a resiquimod, a RAR, or a combination thereof.
  • the chemotherapeutic agent comprises doxorubicin.
  • the composition further comprises a nucleic acid.
  • the nucleic acid comprises repRNA, RAR or a combination thereof.
  • the nanoparticle comprises a lipid carrier.
  • the lipid carrier comprises surfactants forming a hydrophobic core
  • the hydrophobic core comprises: a) a lipid for inclusion in a hydrophilic surface of the nanoparticle, wherein the lipid comprises a cationic lipid, a PEGylated lipid, a phospholipid or a combination thereof; b) a hydrophilic surfactant; and c) a hydrophobic surfactant.
  • lipids present in hydrophobic core are in liquid phase at 25 degrees Celsius.
  • the hydrophobic core comprises a liquid oil.
  • the liquid oil comprises a-tocopherol, coconut oil, grapeseed oil, lauroyl poly oxy Iglyceride, mineral oil, monoacylglycerol, palm kernel oil, olive oil, paraffin oil, peanut oil, propolis, squalene, squalane, dehydroisosqualene, soy lecithin, soybean oil, sunflower oil, a triglyceride, or vitamin E.
  • the triglyceride is capric triglyceride, caprylic triglyceride, a caprylic and capric triglyceride, a triglyceride ester, or myristic acid triglycerin.
  • the liquid oil comprises tumor suppressant or tumor killing property'. Further provided herein are methods, wherein the liquid oil comprises squalene, squalane, dehydroisosqualene, or a combination thereof. Further provided herein are methods, wherein the lipid comprises l,2-dioleoyloxy-3 (trimethylammonium)propane (DOTAP), 3P-[N — (N'.N'-dimethylaminoethane) carbamoyl] cholesterol (DC Cholesterol), dimethyldioctadecylammonium (DDA): 1,2-dimyristoyl 3- trimethylammoniumpropane(DMTAP),dipalmitoyl(C 16:0)trimethyl ammonium propane (DPTAP), distearoyltrimethylammonium propane (DSTAP), N-[l-(2,3- dioleyloxy)propyl]N,N,Ntrimethylammonium, chloride (DOTAP), 3P-[N
  • the hydrophobic core comprises an inorganic particle. Further provided herein are methods, wherein the inorganic particle is within the hydrophobic core. Further provided herein are methods, wherein the inorganic particle comprises a metal. Further provided herein are methods, wherein the metal comprises a metal salt, a metal oxide, a metal hydroxide, or a metal phosphate. Further provided herein are methods, wherein the metal oxide comprises aluminum oxide, aluminum oxyhydroxide, iron oxide, titanium dioxide, or silicon dioxide.
  • the hydrophobic surfactant is sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, or sorbitan trioleate.
  • the nanoparticle is characterized as having a z-average diameter particle size measurement of about 20 nm to about 60 nm when measured using dynamic light scattering.
  • the hydrophilic surfactant is a polysorbate.
  • the composition, the suspension or the pharmaceutical composition is administered via intramuscular injection, intranasal administration, oral administration, subcutaneous administration, intratumoral administration. intrathecal administration, or intravenous injection.
  • methods wherein the subject is a mammal. Further provided herein are methods, wherein the subject is a human. Further provided herein are methods, wherein the subject has a cancer, and wherein the subject has an overexpression of DSG2 protein on a surface of a cancer cell. Further provided herein are methods, wherein the subject has a solid tumor, wherein the subject has an overexpression of DSG2 protein on a surface of a cancer cell. Further provided herein are methods, wherein the solid tumor is a carcinoma, a melanoma, or a sarcoma.
  • the subject has a skin cancer, a pancreatic cancer, a colon cancer, a prostate cancer, an ovarian cancer, or a breast cancer.
  • the skin cancer is a basal cell cancer, a melanoma, a Merkel cell cancer, a squamous cell carcinoma, a cutaneous lymphoma, a Kaposi sarcoma, or a skin adnexal cancer.
  • the pancreatic cancer is a pancreatic adenocarcinoma, a pancreatic exocrine cancer, a pancreatic neuroendocrine cancer, an islet cell cancer, or a pancreatic endocrine cancer.
  • nucleic acids wherein the shaft domain comprises an amino acid sequence that is at least 80% identical to any one of SEQ ID NOS: 1 to 12
  • the knob domain comprises an amino acid sequence that is at least 80% identical to any one of SEQ ID NOS: 13 to 30.
  • the nucleic acids, wherein the multimerization domain comprises an amino acid sequence that is at least 80% identical to any one of SEQ ID NOS: 31 to 37
  • EXAMPLE 1 Manufacturing Junction Opener Proteins Conjugated to a Nanoparticle.
  • Sulfo-SMCC Maleimide portion of the Sulfo-SMCC was then used to react with single cysteine residue of Junction Opener Protein (SEQ ID NO: 61), between 1-10 molar excess, resulting in covalent linkage between the Junction Opener Protein and the nanoparticles.
  • the conjugated formulations, NP-043-JOC, NP-044-JOC, NP-045-JOC, NP-046-JOC, NP-047-JOC, NP-048- JOC and NP-141-JOC. were purified using size exclusion chromatography. JOC concentration was verified by a DSG-2 binding ELISA assay.
  • EXAMPLE 3 In Vitro Assay of Nanoparticle- Junction Opener Protein Compositions in Human DSG2 + BT-474 Tumor Spheroids.
  • JOC Junction Opener Protein Conjugated to Nanoparticle
  • human DSG2 + BT-474 tumor spheroids were treated with eleven different formulations, each contained squalene-based nanoparticles with or without Junction Opener Proteins.
  • the eleven formulations included NP-043-JOC, NP-044-JOC, NP-046-JOC, NP-047- JOC, NP-048-JOC, NP-043, NP-044, NP-045, NP-046, NP-047 and NP-048.
  • Two sets of data were generated using 0.25 pg/mL and 0.0625 pg/rnL dose of squalene, respectively.
  • FIGS. 4A-4E The results of the assay are shown in FIGS. 4A-4E.
  • An analysis of FIGS. 4A- 4E indicates that the primary functional component for cytotoxic effect in tumors is the combination of squalene and Junction Opener Protein, wherein Junction Opener Protein reduces tumor volume via tumor disruption and squalene having tumor killing property.
  • spheroid disruption was due to the NP formulation alone especially NP-047 (squalene emulsion with DOPC) and NP-044 (squalene emulsion control).
  • NP-JOC formulations showed significant increase in disruption of tumor spheroids, especially for NP- 044 and NP-047 at a lower squalene concentration.
  • NP-046 and NP-048, which both contain PEG show ed relatively low disruption levels indicating PEG may reduce tumor cell killing.
  • EXAMPLE 4 In Vitro Assay of Nanoparticle- Junction Opener Protein Compositions in Human DSG2 + BT-474 Tumor Spheroids Transduced with NucLight red [mKate2].
  • NP-JOC squalene formulations exhibited a significant increase in cell death of tumor spheroids, especially for NP-047-JOC and NP-048- JOC.
  • NP-141 which contained miglyol in place of squalene, showed no tumor cell killing. These results indicated that the tumor cell death was squalene mediated cell death.
  • Relative levels of JOC per well varied between formulations NP-044-JOC, NP-047-JOC, NP-048-JOC, and NP-141-JOC have 1.38, 1.34, 3.5. 1.70 ng/pL JOC, respectively.
  • SEQ ID NO: 70 MAGE-A1 RNA-encoding sequence augccgcuggagcagcgaucccaacauugcaaaccagaggaaggccucgaggcaagaggugaggccuggggcuc gucggagcucaggcaccggcaacggaggagcaagaagcggccucuagcagcuccacacucgucgaagugacacuc ggcgagguccccgcugcggaaucucccgauccuccgcaaagcccucaaggggcuuccucccuccccacuacuaug aacuauccguuguggagccaaagcuaugaggauuccucaaaccaagaggaggaaggccccuccacauucccggac cucgaaucugaguuccaggcagcgcucagcaggaaaguggcggacuuguaaaaaguggcggacuuguaaaaaguggcgg
  • SEQ ID NO: 72 TRP-1 RNA-encoding Sequence augaaaucuuacaacguccucccccuagccuauaucucccuuuuccugaugcuguuuuuaucagguuugggcucag uuuccacgagagugugccaauauugaggcucugagacgugggguguguugcccagaccugcucccuuccucugga ccggggacugacccuuguggcucaucaucaggaagaggcagguguguggcugugauugcagacucccgaccccac
  • SEQ ID NOS: 77-124 See Table 2. Cancer-Associated Proteins.
  • SEQ ID NOS: 125-147 See Table 3. Cancer Therapeutic Antibodies.
  • SEQ ID NO: 160 Human IL-12B (p40 subunit) Amino Acid Sequence- NP 002178.2 interleukin- 12 subunit beta precursor [Homo sapiens ⁇ SEQ ID NO: 178 VEEV RNA polymerase Amino Acid Sequence (NCBI Accession:

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Abstract

Les divulgations concernent des compositions comprenant des nanoparticules et une protéine d'ouverture de jonction; et des nanoparticules comprenant un ou plusieurs agents bioactifs et une protéine d'ouverture de jonction qui est conjuguée à la surface des nanoparticules. Divers agents bioactifs sont décrits, tels que le squalène, le squalane et le déhydroisosqualène, entre autres. L'invention concerne différentes compositions de nanoparticules, comprenant des supports lipidiques comprenant un noyau hydrophobe. L'invention concerne en outre des méthodes de traitement d'un sujet atteint d'un cancer par l'administration des compositions décrites dans la description.
PCT/US2024/051011 2023-10-12 2024-10-11 Compositions de protéine d'ouverture de jonction et de nanoparticules et leurs utilisations Pending WO2025081013A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030022257A1 (en) * 2000-07-21 2003-01-30 Macina Roberto A. Compositions and methods relating to lung specific genes
US20110305634A1 (en) * 2010-06-10 2011-12-15 University Of Washington Through Its Center For Commercialization Methods and systems for adenovirus interaction with desmoglein 2 (DSG2)
WO2018213631A1 (fr) * 2017-05-18 2018-11-22 The Regents Of The University Of California Immunothérapie anticancéreuse nano-activée
US20200347101A1 (en) * 2018-01-25 2020-11-05 Pai Life Sciences, Inc. Conjugatable desmoglein 2 (dsg2) binding proteins and uses therefor
US20210338789A1 (en) * 2015-09-23 2021-11-04 Massachusetts Institute Of Technology Compositions and methods for modified dendrimer nanoparticle delivery
WO2023049777A2 (fr) * 2021-09-22 2023-03-30 Hdt Bio Corp. Compositions agonistes de capteur d'acide nucléique et leurs utilisations

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030022257A1 (en) * 2000-07-21 2003-01-30 Macina Roberto A. Compositions and methods relating to lung specific genes
US20110305634A1 (en) * 2010-06-10 2011-12-15 University Of Washington Through Its Center For Commercialization Methods and systems for adenovirus interaction with desmoglein 2 (DSG2)
US20210338789A1 (en) * 2015-09-23 2021-11-04 Massachusetts Institute Of Technology Compositions and methods for modified dendrimer nanoparticle delivery
WO2018213631A1 (fr) * 2017-05-18 2018-11-22 The Regents Of The University Of California Immunothérapie anticancéreuse nano-activée
US20200347101A1 (en) * 2018-01-25 2020-11-05 Pai Life Sciences, Inc. Conjugatable desmoglein 2 (dsg2) binding proteins and uses therefor
WO2023049777A2 (fr) * 2021-09-22 2023-03-30 Hdt Bio Corp. Compositions agonistes de capteur d'acide nucléique et leurs utilisations

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