Nucleic acid-based cancer vaccines and methods thereof

Abstract

Compositions and methods of use thereof are described herein. The compositions described herein may comprise a single-stranded trimeric nucleic acid encoding a first T cell epitope, a beta2-microglobulin, and an MHC class I heavy chain sequence. The methods described herein can be used to activate and / or expand antigen presenting cells. The methods described herein may also be used to treat or prevent viral infections, bacterial infections, parasitic infections, and / or cancer in a subject.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to and the benefit of U.S. Provisional Application 63 / 431,571, filed December 9, 2022, the contents of which are hereby incorporated by reference in their entirety.

[0003] References to sequence listings

[0004] The Sequence Listing in the text file named “11538-006WO1_ST26.xml” created on December 8, 2023 and having a file size of 679,893 bytes was submitted on December 11, 2023, is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5). BACKGROUND

[0005] Tumor-specific antigens (TSAs) or tumor-associated antigens (TAAs) are the key targets of cancer vaccines that activate host anti-tumor CD8 T cells to specifically attack and destroy cancer cells that overexpress the same antigens. However, cancer vaccines have not achieved the expected clinical effects, and the main reasons for their ineffectiveness are at least the following:

[0006] 1) These tumor antigen-derived peptides have low MHC binding affinity and / or abundance, and they must compete with a large number of endogenous self-antigen-derived peptides or other pathogen-derived peptides to bind to MHC I molecules. Since MHC I molecules cannot distinguish between self- and non-self-derived peptides, the processing and MHC I presentation efficiency of these tumor peptides are usually low, which leads to ineffective anti-tumor CD8 T cell responses in the clinic.

[0007] 2) Most cancer vaccines aim to activate antigen-specific CD8 T cell responses, but increasing evidence supports that CD4 T cells not only play a key role in cellular immunity, but also provide essential help for tumor-specific cytotoxic T lymphocytes (CTLs), including activating antigen-presenting cells, promoting CD8 T cell homing to tumor tissues, and generating effective memory CD8 T cells.

[0008] 3) The vaccine delivery platform is insufficient to express tumor antigens, or cannot deliver antigens to antigen-presenting cells (APCs), which are specialized cells that effectively activate T cells.

[0009] Therefore, there remains a need for compositions and methods for treating and preventing bacterial infections, viral infections, parasitic infections, and / or cancer.

[0010] The compositions and methods disclosed herein address these and other needs. SUMMARY

[0011] Provided herein are single-stranded nucleic acid trimers, compositions comprising the single-stranded trimer nucleic acids described herein, and methods of using the same.

[0012] Described herein are single-chain trimer nucleic acids encoding a first T cell epitope, β2-microglobulin, and an MHC class I heavy chain sequence. In some embodiments, the compositions described herein comprise single-chain trimer nucleic acids encoding a first T cell epitope, β2-microglobulin, and an MHC class I heavy chain sequence.

[0013] In some embodiments, the single-stranded trimer nucleic acid may further comprise a second T cell epitope. In some embodiments, the second T cell epitope may be an MHC class II restricted epitope. In some embodiments, the second T cell epitope may include SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, or an epitope listed in Table 5.

[0014] In some embodiments, the single-chain trimer nucleic acid may comprise nucleic acids encoding a T cell epitope, β2 microglobulin, and an MHC heavy chain sequence in order from amino to carboxyl terminus.

[0015] In some embodiments, the single-chain trimer nucleic acid may further comprise a flexible first linker between the first T cell epitope and the β2 microglobulin and a second linker between the β2 microglobulin and the MHC class I heavy chain sequence.

[0016] In some embodiments, the composition may further comprise a pharmaceutically acceptable carrier. In some aspects, disclosed herein is a pharmaceutical composition that may be a single-stranded trimer nucleic acid (e.g., mRNA) encoding a first T cell epitope, β2-microglobulin, and an MHC heavy chain sequence; and a pharmaceutically acceptable carrier. In some embodiments, the composition may be a nanoparticle, a lipid nanoparticle dispersion, a liposomal formulation, a lipid emulsion, a vaccine, a carrier, or any combination thereof.

[0017] In some embodiments, a pharmaceutical composition may comprise a lipid nanoparticle comprising a single-stranded trimeric nucleic acid (eg, mRNA) encoding a first T cell epitope, β2-microglobulin, and an MHC class I heavy chain sequence.

[0018] Also described herein are cells comprising the compositions described herein. In some embodiments, the cells can include dendritic cells, B cells, macrophages, or other cells derived from a subject that can be used for immunotherapy.

[0019] Also described herein are methods for activating and / or expanding antigen-presenting cells, comprising co-culturing antigen-presenting cells and cells comprising single-chain trimer nucleic acids, compositions, lipid nanoparticles, or pharmaceutical compositions as described herein. In some embodiments, the antigen-presenting cells may be dendritic cells, B cells, macrophages, or other cells derived from a subject that can be used for immunotherapy.

[0020] Also described herein are methods of treating a subject having a viral infection, a bacterial infection, a parasitic infection, and / or cancer, comprising administering to the subject a therapeutically effective amount of a single-stranded trimeric nucleic acid described herein, a composition described herein, a lipid nanoparticle described herein, a cell described herein, or a pharmaceutical composition described herein.

[0021] Also described herein are methods for treating subjects with viral infections, bacterial infections, parasitic infections, and / or cancers, comprising obtaining antigen-presenting cells from a subject; contacting the antigen-presenting cells from the subject with a single-chain trimer nucleic acid, a composition as described herein, a lipid nanoparticle as described herein, or a pharmaceutical composition as described herein, thereby activating the antigen-presenting cells; and administering the activated antigen-presenting cells to the subject. In some embodiments, the antigen-presenting cells may include dendritic cells, B cells, macrophages, or other cells derived from the subject that can be used for immunotherapy.

[0022] The details of one or more embodiments of the present disclosure are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the present disclosure are apparent from the description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figures 1A to 1F Demonstrating the design and in vitro efficacy of Kb-E6-SCT mRNA. Figure 1A ) shows the design of SCT-E6 mRNA. ( Figure 1B ) shows a linearized plasmid template; and ( Figure 1C ) shows in vitro transcription of SCT-E6 mRNA. ( Figures 1D to 1E ) shows the physicochemical properties of lipid nanoparticle (LNP) formulations and the encapsulation efficacy of L2M6 (L2M6 represents LNP formulation of Kb-E6-SCT mRNA using the ionizable lipid ARV-T1). Figure 1D Displays particle size and polydispersity index. Figure 1E Shows zeta potential and encapsulation efficiency. ( Figure 1F ) shows the expression of SCT-E6 in 293T cells after transfection.

[0024] Figures 2A to 2CIn vivo validation was shown. C57BL / 6 mice were immunized twice intramuscularly with 1 μg of the indicated LNP-formulated vaccine mRNA at two-week intervals. Seven days after the last immunization, the E6-CD8 T cell peptide was used for ELISPOT ( Figures 2A to 2B )(2A) images and (2B) graphs; and intracellular IFN-γ staining ( Figures 2C to 2D ) to determine the E6-specific T cell population.

[0025] Figures 3A to 3C Demonstrating in vivo antitumor efficacy of mRNA-L2M6 prophylactic vaccination. Figure 3A Figure 5: Five C57BL / c mice were injected intramuscularly with 1 μg of mRNA / SCT-E6 on days 1 and 14, and subcutaneously with 1×10^5 TC1 tumor cells on day 21. Tumor growth was monitored ( Figure 3B ) and survival ( Figure 3C ).

[0026] Figures 4A to 4C Demonstrated in vivo efficacy of therapeutically administered mRNA-L2M6 vaccine. Figure 4A The experimental protocol was for 10 female C57BL / 6 mice. 2×10^5 TC-1 tumor cells were injected subcutaneously in the flank on days 1, 8, and 15 after tumor challenge. The mice received three doses of L2M6 or PBS immunization. Figure 4B It is a tumor progression chart. Figure 4C is the long-term survival graph of each experimental group.

[0027] Figure 5 In vitro synthesis of HPV mRNA vaccines is shown. HPV mRNAs 1-3 were synthesized by in vitro transcription using T7 RNA polymerase and run on a 0.8% MOPS agarose gel.

[0028] Figures 6A to 6C In vitro validation of the HPV mRNA vaccine is shown. HPV mRNAs 1-4 were transfected into 293T cells, and expression was measured 48 hours later. (6A to 6B) Flow cytometry was used to detect the expression levels of HPV mRNA 1 (6A) and mRNAs 2-4 (6B) using anti-Sars-S2 antibodies (6A) and anti-HLA-A2 antibodies (6B), respectively. (6C) Western blotting was used to detect the expression of HPV mRNA-1 using the E7 antibody.

[0029] Figures 7A to 7IHPV mRNA vaccines were shown to elicit robust T cell and antibody responses in hHLA-A2 transgenic mice. (7C to 7D) Splenocytes were isolated from mouse spleens and stimulated with a pool of E6 mixed peptides, a pool of E7 mixed peptides, and a pool of E6 single peptides for ELISPOT assays. (7C) is a representative image of an ELISpot plate readout, and IFNγ-producing T lymphocytes were quantified, as shown in 7D. (7E to 7I) Splenocytes were isolated from mouse spleens and stimulated with a pool of E6+E7 mixed peptides (7F to 7G) or a pool of E6 single peptides (7H to 7I) for flow cytometry intracellular staining.

[0030] Figures 8A to 8C HPV mRNA was shown to induce a strong cell-mediated immune response. hHLA-A2 transgenic mice were immunized intramuscularly with 5ug LNP-formulated mRNA vaccine (HPV mRNA1+3) on days 0 and 14. The spleens of the mice were collected on day 28. Splenocytes were isolated from the spleens of the mice. (8A to 8B) Mouse CD8 T cells were isolated using a kit and detected by flow cytometry using anti-mouse CD8 T cell antibodies. (8C) T cell-mediated immune responses were evaluated by cytotoxic T lymphocyte assay (CTL assay). Tumor target cells (T) and TA2-Luc stable cells (TC-1 cell line overexpressing hHLA-A2 and luciferase) were seeded in 96-well plates on day 0, with a cell suspension of 2×10 4 cells / well. Assume that cancer cells double in number overnight. On day 1, CD8 T cells isolated from A were used as effector cells (E) at a concentration of 1×10 5 100 μg / well (E:T=2.5:1) were added to the tumor target cells. On day 3, the cells were evaluated for cytotoxicity based on flow cytometry using Annexin V and Helix NP.

[0031] Like reference symbols in the various drawings indicate like elements. DETAILED DESCRIPTION

[0032] A number of embodiments of the present disclosure have been described. However, it will be appreciated that various modifications may be made without departing from the spirit and scope of the present invention. Accordingly, other embodiments are within the scope of the following claims.

[0033] definition

[0034] For the purposes of the present disclosure, certain terms are defined below. As used in the present disclosure, and except as otherwise expressly provided herein, all technical and scientific terms have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Publications cited herein and the material for which they are cited are specifically incorporated by reference.

[0035] General Definitions

[0036] As used in the specification and the appended claims, the terms "comprise" (and any form of comprise, including "comprising" and "comprises") and "comprise" (and any form of comprise, including "comprising" and "comprises") are inclusive (i.e., open-ended) and do not exclude additional, unrecited elements or steps. For example, as used in the specification and appended claims, the terms "comprise" and / or "comprising," when used in the specification and appended claims, specify the presence of stated features, integers, steps, operations, elements, and / or components but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. Except as otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, physical properties of materials and / or sizes of the structural components shown herein are to be understood as modified in all instances by the term "about," to the extent that such term has been used herein. Nothing in the specification should be construed as indicating any element, feature or step is essential.

[0037] Accordingly, such terms are intended to cover not only the recited elements or steps, but also other elements or steps not expressly recited. Furthermore, as used herein, the use of the terms "a," "an" and "the" to describe elements or items of the disclosure are not intended to be construed as excluding particular embodiments having more than one of such element or item, unless otherwise indicated. Thus, for example, a formulation having "a" or "an" active agent can include more than one active agent, and a formulation having "the" active agent can include more than one active agent, unless otherwise indicated.

[0038] In this article, ranges can be expressed as from "about" a specific value and / or to "about" another specific value. "About" means within 5% of the value, for example, within 4%, 3%, 2% or 1% of the value. When expressing such a range, another aspect includes from the specific value and / or to the specific value. Similarly, when a value is expressed as an approximation by using the antecedent "about", it should be understood that the specific value forms another aspect. It should be further understood that the endpoints of each range are meaningful both relative to the other endpoint and independently of the other endpoint. It should also be understood that many values ​​are disclosed herein, and in addition to the value itself, each value is also disclosed herein as "about" the specific value. For example, if the value "10" is disclosed, then "about 10" is also disclosed. A range can be interpreted as including the beginning and end of the range. For example, a range of 10% to 20% (i.e., a range of 10% to 20%) can include 10% and also include 20%, and include the percentages between 10% and 20%, unless otherwise expressly stated herein.

[0039] As used herein, the terms "may," "optionally," and "may optionally" are used interchangeably and are meant to include instances where a condition occurs as well as instances where the condition does not occur. Thus, for example, the statement that a formulation "may include an excipient" is meant to include instances where the formulation includes the excipient as well as instances where the formulation does not include the excipient.

[0040] It should be understood that when combinations, subsets, groups, etc. of elements are disclosed (e.g., combinations of components in a composition or combinations of steps in a method), each element is specifically contemplated and described herein, even though specific reference to each of the various individual and collective combinations and arrangements of such elements may not be explicitly disclosed.

[0041] " using " includes introducing or delivering any approach of medicament to the subject.Using can be carried out by any suitable approach, including oral, local, percutaneous, through corium, intra-articular, intra-arteriole, intra-dermal, intra-ventricular, intra-lesion, intranasal, rectal, vaginal, by suction, by implanted depot, parenteral (such as subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intraperitoneal, intra-hepatic, intra-lesion and intracranial injection or infusion technology) etc. As used herein, " using simultaneously (concurrent administration) ", " using in combination ", " using simultaneously (simultaneous administration) " or " using simultaneously " means compound uses at the same time point or basically one immediately after one uses.In the latter case, the time of administration of two kinds of compounds is close enough, so that the result obtained when the observed result and the compound use at the same time point is difficult to distinguish. "Systemic administration" refers to the introduction or delivery of a pharmaceutical agent to a subject by a route that introduces or delivers the agent to a wide area of ​​the subject's body (e.g., greater than 50% of the body), such as by entering the circulatory or lymphatic systems. In contrast, "local administration" refers to the introduction or delivery of a pharmaceutical agent to a subject by a route that introduces or delivers the agent to the area or area immediately adjacent to the point of administration and does not introduce the agent systemically in therapeutically significant amounts. For example, a locally administered pharmaceutical agent may be readily detected in the local vicinity of the point of administration, but may not be detected or may be detected in negligible amounts in distal portions of the subject's body. Administration includes self-administration and administration by another person.

[0042] As used herein, the terms "beneficial agent" and "active agent" are used interchangeably herein to refer to a chemical compound or composition that has a beneficial biological effect. Beneficial biological effects include therapeutic effects, i.e., treating a disease or other undesirable physiological condition, and prophylactic effects, i.e., preventing a disease or other undesirable physiological condition. The terms also encompass pharmaceutically acceptable pharmacologically active derivatives of the beneficial agents specifically mentioned herein, including but not limited to salts, esters, amides, prodrugs, active metabolites, isomers, fragments, analogs, and the like. When the terms "beneficial agent" or "active agent" are used, or when a particular agent is specifically identified, it should be understood that the terms include the agent itself as well as pharmaceutically acceptable pharmacologically active salts, esters, amides, prodrugs, conjugates, active metabolites, isomers, fragments, analogs, and the like.

[0043] "Reduce" can refer to any change that causes a smaller amount of symptoms, diseases, compositions, symptoms or activity. When the genetic output of the gene product utilizing a substance is lower than the output of the gene product not utilizing the substance, the substance should also be understood to reduce the genetic output of the gene. For example, reducing can be a change in the symptoms of a disease so that the symptoms are less than previously observed. Reducing can be any individual, median or average reduction of a symptom, activity, or composition with statistically significant amounts. Therefore, reducing can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% reduction, as long as reducing is statistically significant.

[0044] "Inhibit," "inhibiting," and "inhibition" mean a reduction in an activity, response, condition, disease, or other biological parameter. This can include, but is not limited to, complete ablation of an activity, response, condition, or disease. This can also include, for example, a 10% reduction in an activity, response, condition, or disease compared to a natural or control level. Thus, a reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount therebetween, compared to a natural or control level.

[0045] "Inactivate," "inactivating," and "inactivation" refer to the reduction or elimination of an activity, response, condition, disease, or other biological parameter due to chemistry (covalent bond formation) between a ligand and its biological target.

[0046] "Reduce" or other forms of the word, such as "reducing" and "reduction", refers to a decrease in an event or characteristic (e.g., tumor growth). It will be understood that this is usually relative to a standard or expected value, in other words, it is relative, but it does not always require reference to a standard or relative value. For example, "reduce tumor growth" means reducing the rate of tumor growth relative to a standard or control.

[0047] As used herein, the terms "treating" or "treatment" of a subject include administering a drug to a subject for the purpose of preventing, curing, rehabilitating, alleviating, mitigating, altering, remedying, improving, ameliorating, stabilizing, or affecting a disease or condition, or a symptom of a disease or condition. The terms "treating" and "treatment" may also refer to reducing the severity and / or frequency of symptoms, eliminating symptoms and / or underlying causes, preventing the occurrence of symptoms and / or their underlying causes, and ameliorating or remedying damage.

[0048] "Prevent" or other forms of the word such as "preventing" or "prevention" means to prevent a particular event or characteristic, stabilize or delay the development or progression of a particular event or characteristic, or minimize the chance of a particular event or characteristic occurring. Prevention does not need to be compared to a control because it is generally more absolute than, for example, reduction. As used herein, something can be reduced but not prevented, but something that is reduced can also be prevented. Similarly, something can be prevented but not reduced, but something that is prevented can also be reduced. It should be understood that unless otherwise expressly stated, where reduction or prevention is used, the use of other words is also expressly disclosed. For example, the term "prevent" or "inhibit" can refer to a treatment that prevents or slows the onset of a disease or condition in advance or reduces the severity of a disease or condition. Therefore, if a treatment can treat a disease in a subject with symptoms of the disease, it can also prevent or inhibit the disease in a subject who has not yet suffered some or all of the symptoms. As used herein, the term "preventing" a condition or adverse physiological event in a subject specifically refers to preventing the occurrence of symptoms and / or their underlying causes, wherein the subject may or may not show a high sensitivity to the condition or event.

[0049] The term "effective amount" of a therapeutic agent refers to an amount of a beneficial agent that is non-toxic but sufficient to provide the desired effect. The amount of an "effective" beneficial agent will vary from subject to subject, depending on the subject's age and general condition, the specific beneficial agent or agents, etc. Therefore, it is not always possible to specify an exact "effective amount." However, one of ordinary skill in the art can determine an appropriate "effective amount" for any subject using routine experimentation. Moreover, as used herein and unless expressly stated otherwise, an "effective amount" of a beneficial agent may also refer to an amount that covers both a therapeutically effective amount and a prophylactically effective amount.

[0050] The "effective amount" of a drug necessary to achieve a therapeutic effect can vary according to factors such as the age, sex, and weight of the subject. The dosage regimen can be adjusted to provide the optimal therapeutic response. For example, several divided doses can be administered daily, or the dose can be proportionally reduced as indicated by the exigencies of the therapeutic situation.

[0051] As used herein, a "therapeutically effective amount" of a therapeutic agent refers to an amount effective to achieve a desired therapeutic result and a "prophylactically effective amount" of a therapeutic agent refers to an amount effective to prevent an adverse physiological condition. The therapeutically effective amount and prophylactically effective amount of a given therapeutic agent will generally vary according to factors such as the type and severity of the condition or disease being treated and the age, gender, and weight of the subject. The term "therapeutically effective amount" can also refer to an amount of a therapeutic agent or a rate of delivery of a therapeutic agent (e.g., amount as a function of time) that is effective to promote a desired therapeutic effect. The precise desired therapeutic effect will vary according to the condition being treated, the subject's tolerance, the drug and / or drug formulation being administered (e.g., potency of the therapeutic agent (drug), concentration of the drug in the formulation, etc.), and various other factors as understood by one of ordinary skill in the art.

[0052] As used herein, the term "pharmaceutically acceptable" component can refer to a component that is not biologically or otherwise undesirable, i.e., the component can be incorporated into a pharmaceutical formulation of the present application and administered to a subject as described herein without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the formulation in which it is contained. The term "pharmaceutically acceptable" when referring to an excipient generally means that the component has been tested and meets the required standards for toxicology and manufacturing testing, or the component is included in the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration.

[0053] A "pharmaceutically acceptable carrier" (sometimes referred to as a "carrier") means a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe, non-toxic, and neither biologically nor otherwise undesirable, and includes a carrier or vehicle that is acceptable for veterinary and / or human pharmaceutical or therapeutic use. The term "carrier" or "pharmaceutically acceptable carrier" can include, but is not limited to, phosphate buffered saline solution, water, emulsions (such as oil / water or water / oil emulsions), and / or various types of wetting agents. The term "carrier" as used herein encompasses, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material known in the art to be useful in pharmaceutical formulations and as described further herein.

[0054] As used herein, "pharmaceutically acceptable salts" are derivatives of the disclosed compounds wherein the parent compound is modified by making inorganic and organic, non-toxic, acid or base addition salts thereof. The salts of the compounds of the present application can be synthesized from the parent compound that contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting free acid forms of these compounds with a stoichiometric amount of the appropriate base (such as Na, Ca, Mg, or K hydroxide, carbonate, bicarbonate, or the like) or by reacting free base forms of these compounds with an equivalent amount of the appropriate acid. Such reactions typically are carried out in water or in an organic solvent, or in a mixture of both. Generally, non-aqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are typical, where feasible. Salts of the compounds of the present application further include solvates of compounds and compound salts.

[0055] Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali metal or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts include the conventional non-toxic salts of the parent compound as well as quaternary ammonium salts. For example, conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, sulfonic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, HOOC-(CH2)n-COOH where n is 0-4, and the like, or using different acids that result in the same counterion. Lists of additional suitable salts can be found, e.g., in Remington's Pharmaceutical Sciences, 17th Ed., Mack Publishing Company, Easton, Pa., p. 1418 (1985).

[0056] Further, as used herein, the term "pharmacologically active" (or simply "active") as in "pharmacologically active" derivative or analog can refer to a derivative or analog (e.g., salt, ester, amide, conjugate, metabolite, isomer, fragment, etc.) that has the same type of pharmacological activity as the parent compound and roughly equal in degree.

[0057] A "control" is an alternative subject or sample used in an experiment for comparison purposes. A control can be "positive" or "negative."

[0058] As used herein, “subject” means an individual. Thus, a “subject” can include domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cows, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mice, rabbits, rats, guinea pigs, etc.), and birds. A “subject” can also include a mammal, such as a primate or a human. Thus, a subject can be a human or a veterinary patient. The term “patient” refers to a subject under treatment by a clinician, e.g., a physician. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the subject is a veterinary patient. Administration of a therapeutic agent can be at a dosage and for a duration effective to treat the subject. In some embodiments, the subject is a human.

[0059] Genetic definitions

[0060] The term “nucleic acid” as used herein refers to a polymer composed of nucleotides, e.g., deoxyribonucleotides or ribonucleotides.

[0061] The terms “ribonucleic acid” and “RNA” as used herein refer to a polymer composed of ribonucleotides.

[0062] The terms “deoxyribonucleic acid” and “DNA” as used herein refer to a polymer composed of deoxyribonucleotides.

[0063] The term “oligonucleotide” denotes a single- or double-stranded nucleotide polymer of about 2 to up to about 100 nucleotides in length. Suitable oligonucleotides can be prepared by the phosphoramidite method described by Beaucage and Carruthers, Tetrahedron Lett., 22: 1859-1862 (1981) or by the trister method according to Matteucci, et al., J. Am. Chem. Soc, 103: 3185 (1981), both of which are incorporated herein by reference, or by using other chemical methods such as those described in VLSIPS TM When an oligonucleotide is referred to as being “double-stranded,” one skilled in the art will appreciate that a pair of oligonucleotides exists in the usual array of hydrogen bonds associated with, for example, DNA. In addition to the 100% complementary form of double-stranded oligonucleotides, the term “double-stranded” as used herein also means to refer to those forms that include such structural features as bulges and loops, which are more fully described in such biochemistry texts as Stryer, Biochemistry, Third Edition, (1988), which is incorporated herein by reference for all purposes.

[0064] The term "polynucleotide" refers to a single-stranded or double-stranded polymer composed of nucleotide monomers. The polynucleotide sequence can be modified by using, for example, base modification or end-capping, for example, to enhance efficacy and / or reduce immunoreactivity. In other embodiments, an unmodified polynucleotide sequence is used. For example, the polynucleotide can be an RNA sequence or a DNA sequence. In some embodiments, the mRNA can include optimized codons. Through codon optimization, the formation of secondary structures can be reduced and translation efficiency can be improved. In certain embodiments, codon optimization includes GC enrichment of the coding region. In certain embodiments, codon optimization includes codon quality enrichment of the coding region. In some aspects, the mRNA can include one or more regions or portions that act or play the role of the untranslated region (UTR) of the gene. The UTR is transcribed but not translated. In the mRNA, the 5'UTR starts at the transcription start site and extends to the start codon, but does not include the start codon. The 3'UTR starts immediately after the stop codon and continues until the transcription termination signal. The use of human UTR can promote the expression of the polypeptide in the cell. In some embodiments, the polynucleotide includes at least one chemically modified nucleotide. In some embodiments, at least one chemically modified nucleotide comprises a chemically modified nucleobase, a chemically modified ribose, a chemically modified phosphodiester bond, or a combination thereof. In some embodiments, the polynucleotide sequence used comprises modified nucleosides, such as 5-methylcytosine or pseudouridine.

[0065] As used herein, "modified" refers to the state or structure of the present invention molecule changes. Molecule can be modified in a variety of ways, including chemical modification, structural modification and functional modification. In one embodiment, the polynucleotide of the present invention is "chemically modified" by introducing non-natural nucleosides and / or nucleotides, for example, when it relates to natural ribonucleotides A, U, G and C. The modification of the nucleosides used in the present invention and / or nucleotides can be naturally occurring (i.e., including nucleotides and / or nucleosides other than natural ribonucleotides A, U, G and C) or may be artificial. Although non-canonical nucleotides (such as cap structures) are different from the chemical structures of A, G, C and U ribonucleotides, they are not considered to be "modified". As used herein, "structure" modification is a modification in which two or more connected nucleosides are inserted, deleted, replicated, inverted or randomized in a polynucleotide without significant chemical modification of the nucleotide itself. Because chemical bonds will inevitably be destroyed and reformed to realize structural modification, structural modification has chemical properties, and is therefore chemically modified. However, structural modification will result in different nucleotide sequences. When a polynucleotide of the present invention is chemically and / or structurally modified, the polynucleotide may be referred to as a "modified nucleotide."

[0066] In some embodiments, the nucleic acids disclosed herein may include at least one chemically modified nucleotide. In some embodiments, the at least one chemically modified nucleotide includes a chemically modified nucleobase, a chemically modified ribose, a chemically modified phosphodiester bond, or a combination thereof.

[0067] In one embodiment, the at least one chemically modified nucleotide is a chemically modified nucleobase.

[0068] In one embodiment, the chemically modified nucleobase is selected from 5-formylcytidine (5fC), 5-methylcytidine (5meC), 5-methoxycytidine (5moC), 5-hydroxycytidine (5hoC), 5-hydroxymethylcytidine (5hmC), 5-formyluridine (5fU), 5-methyluridine (5-meU), 5-methoxyuridine (5moU), 5-carboxymethyl ester uridine (5camU), pseudouridine (Ψ), N 1 -methyl pseudouridine (me 1 Ψ), N 6 -methyladenosine (me 6 A) or guanosine ( th G).

[0069] In some embodiments, the chemically modified nucleobase is 5-methoxyuridine (5moU). In some embodiments, the chemically modified nucleobase is pseudouridine (Ψ). In some embodiments, the chemically modified nucleobase is N 1 -methyl pseudouridine (me 1 Ψ).

[0070] The structures of these modified nucleobases are shown below:

[0071]

[0072] In one embodiment, the at least one chemically modified nucleotide is a chemically modified ribose sugar.

[0073] In one embodiment, the chemically modified ribose is selected from 2'-O-methyl (2'-O-Me), 2'-fluoro (2'-F), 2'-deoxy-2'-fluoro-β-D-arabino-nucleic acid (2'F-ANA), 4'-S, 4'-SFANA, 2'-azido, UNA, 2'-O-methoxy-ethyl (2'-O-ME), 2'-O-allyl, 2'-O-ethylamine, 2'-O-cyanoethyl, locked nucleic acid (LAN), methylene-cLAN, N-MeO-amino BNA or N-MeO-aminooxy BNA. In one embodiment, the chemically modified ribose is 2'-O-methyl (2'-O-Me). In one embodiment, the chemically modified ribose is 2'-fluoro (2'-F).

[0074] The structures of these modified riboses are shown below:

[0075]

[0076] In one embodiment, the at least one chemically modified nucleotide is a chemically modified phosphodiester linkage.

[0077] In one embodiment, the chemically modified phosphodiester linkage is selected from phosphorothioate (PS), boranophosphate, phosphorodithioate (PS2), 3',5'-amide, N3'-phosphoramide (NP), phosphodiester (PO), or 2',5'-phosphodiester (2',5'-PO). In one embodiment, the chemically modified phosphodiester linkage is phosphorothioate.

[0078] The structures of these modified phosphodiester linkages are shown below:

[0079]

[0080] In some embodiments, the mRNA can include a heterologous 5' untranslated region (5' UTR). In some embodiments, the mRNA can include a heterologous 3' untranslated region (3' UTR).

[0081] The term "polypeptide" refers to a compound composed of a single chain of D- or L-amino acids, or a mixture of D- and L-amino acids, joined by peptide bonds. Polypeptides are composed of about twenty standard naturally occurring amino acids, but can also use natural amino acids that are not members of the twenty standard amino acid members, and synthetic amino acids. The twenty standard amino acids include alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (lie, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V). The term "polypeptide sequence" or "amino acid sequence" is a polypeptide molecule represented in alphabetical order.

[0082] Conservative substitutions of amino acids in proteins and polypeptides are known in the art. For example, it is known to those skilled in the art that the substitution of one amino acid residue by another biologically and / or chemically similar amino acid residue is a conservative substitution. For example, a conservative substitution can be the substitution of one hydrophobic residue by another hydrophobic residue, or one polar residue by another polar residue. Substitutions include combinations such as, for example, Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr. Such conservatively substituted variants of each explicitly disclosed sequence are encompassed by the polypeptides provided herein.

[0083] Substantial changes in protein function or immunological identity are achieved by selecting less conservative substitutions, i.e., residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of ​​the substitution, e.g., as a sheet or helical conformation, (b) the charge or hydrophobicity of the target site molecule, or (c) the bulk of the side chain. In general, substitutions expected to produce the greatest changes in protein properties will be those in which (a) a hydrophilic residue (e.g., seryl or threonyl) replaces (or is replaced by) a hydrophobic residue (e.g., leucyl, isoleucyl, phenylalanyl, valyl, or alanyl); (b) a cysteine ​​or proline replaces (or is replaced by) any other residue; (c) a residue with a positively charged side chain (e.g., lysyl, arginyl, or histidyl) replaces (or is replaced by) a negatively charged residue (e.g., glutamyl or aspartyl); or (d) a residue with a bulky side chain (e.g., phenylalanine) replaces (or is replaced by) a residue without a side chain (e.g., glycine), in which case (e) by increasing the number of sites of sulfation and / or glycosylation.

[0084] "Variant" refers to a molecule with a substantially similar structure. Therefore, in one embodiment, a variant refers to a protein whose amino acid sequence is similar to a reference amino acid sequence, but does not have 100% identity with the corresponding reference sequence. The variant protein has a changed sequence, wherein one or more of the amino acids in the reference sequence are deleted or substituted, or one or more amino acids are inserted into the reference amino acid sequence. Due to these changes, the variant protein has an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90% or 95% identical to the reference sequence. For example, a variant sequence with at least 95% identity has no more than 5 changes (i.e., any combination of deletions, insertions or substitutions) per 100 amino acids of the reference sequence.

[0085] The term "complementary" refers to the topological compatibility or matching of the interaction surfaces of the probe molecule and its target. Thus, a target and its probe can be described as complementary, and further the contact surface properties complement each other.

[0086] The term "hybridization" refers to the process of forming a non-covalent, sequence-specific interaction between two or more complementary strands of nucleic acids to form a single hybridization complex, which in the case of two strands is referred to as a duplex.

[0087] In the context of two or more nucleic acid or polypeptide sequences, the terms "identical" or percent "identity," mean that two or more sequences or subsequences that are the same with respect to the identification of amino acid residues or nucleotides, when compared and aligned for maximum correspondence, as measured using the BLAST or BLAST 2.0 sequence comparison algorithms, with the default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site, etc.). Such sequences are then referred to as "substantially identical." This definition also refers to the complement of a test sequence. The definition also includes sequences that have deletions and / or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, the identity exists over a region that is at least about 10 amino acids or 20 nucleotides in length, or more preferably over a region that is 10 to 50 amino acids or 20 to 50 nucleotides in length. As used herein, percent (%) amino acid sequence identity is defined as the percentage of amino acids in a candidate sequence that are identical with the amino acids in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art will appreciate that optimal alignment of sequences will take into account the identity of residues and the conservation of amino acid families. Any alignment program that considers these parameters will be suitable.

[0088] For sequence comparison, typically one sequence acts as the reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the program parameters.

[0089] An example of an algorithm suitable for determining sequence identity and sequence similarity percentages is the BLAST and BLAST 2.0 algorithms described in, respectively, Altschul et al. (1977) Nuc.Acids Res.25:3389-3402, and Altschul et al. (1990) J.Mol.Biol.215:403-410. Software for performing BLAST analysis can be publicly available from the National Center for Biotechnology Information. The algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short characters of length W in the query sequence that match or meet a positive threshold score T when aligned with a character of the same length in the database sequence. T is referred to as a neighborhood word score threshold (Altschul et al. (1990) J.Mol.Biol.215:403-410). These initial neighborhood word hits serve as seeds for initiating searches to find longer HSPs containing the initial neighborhood word hits. The word hits are extended in both directions along each sequence until the cumulative alignment score can be increased. For nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatched residues; always <0) are used. For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. The word hit extension in each direction will stop when the following occurs: the cumulative alignment score drops by the number X from its maximum achieved value; the cumulative score becomes zero or lower due to the accumulation of one or more negative scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses a word length (W) of 11, an expectation (E) or 10, M=5, N=-4, and a comparison of both chains as default values. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, an expectation (E) of 10, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915), an alignment (B) of 50, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0090] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of a test nucleic acid to a reference nucleic acid is less than about 0.2, more preferably less than about 0.01.

[0091] The term "nucleobase" refers to the portion of a nucleotide that carries out Watson / Crick base pairing. The most common natural nucleobases, adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T), carry out hydrogen bonding, binding one nucleic acid strand to another in a sequence-specific manner.

[0092] Reference will now be made in detail to specific aspects of the disclosed materials, compounds, compositions, articles, and methods, examples of which are illustrated in the accompanying Examples and Figures.

[0093] single-stranded trimer nucleic acid

[0094] Described herein are single-chain trimer nucleic acid sequences encoding a first T cell epitope, β2-microglobulin, and an MHC class I heavy chain sequence.

[0095] In some embodiments, the single-stranded trimer nucleic acid may comprise a nucleic acid sequence selected from any one of the following: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 498, or SEQ ID NO: 500; and a nucleic acid sequence having the same sequence as SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 498, or SEQ ID NO: 500. A nucleic acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8% or 99.9%) identical to the nucleic acid sequence of NO:500.

[0096] In some embodiments, the single-stranded trimer nucleic acid can encode an amino acid selected from any one of the following: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 497, or SEQ ID NO: 499; and a nucleic acid having a sequence similar to that of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 497, or SEQ ID NO: 499. An amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8% or 99.9%) identical to the amino acid sequence of NO:499.

[0097] In some embodiments, the first T cell epitope can include an epitope listed in Tables 1 to 3 or a homolog having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8% or 99.9%) identity with an epitope of Table 2.

[0098] In some embodiments, the first T cell epitope can include an epitope listed in Table 2 or a homolog having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8% or 99.9%) identity to an epitope of Table 2.

[0099] In some embodiments, the single-chain trimer nucleic acid may comprise nucleic acids encoding a T cell epitope, β2 microglobulin, and an MHC heavy chain sequence in order from amino to carboxyl terminus.

[0100] DNA and protein sequences of exemplary cancer vaccine constructs are listed in Table 1. Table 1 also includes SEQ ID NO: 21 and SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26 universal CD4 T cell epitopes.

[0101] SEQ ID NO: 21 and SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 or the epitopes listed in Table 5 may be universal CD4 T cell epitopes capable of activating pre-existing memory CD4 T cells, which can effectively promote the anti-tumor activity of CD8 T cells.

[0102] Table 1. DNA and protein sequences in the constructs

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118] Table 2: List of T cell epitopes from human mutant genes.

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126] Table 3: List of T cell epitopes from viral genes.

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135] MHC HLA-A1:01, HLA-A2:01, HLA-A2:03, HLA-A2:06, HLA-A2:07, HLA-A3:01, HLA-A9, HLA-A1:01:01 、HLA-A23:01、HLA-A24:02、HLA-A25:01、HLA-A26:01、HLA-A29:02、HLA-A30:01、HLA-A30:02、HLA-A31:01-HLA-HLA A32:02、HLA-A33:03、HLA-A34:01、HLA-A34:02、HLA-A68:01、HLA-A68:02、HLA-A74:01、HLA-B5、HLA-B7:02、HLA -B8:01、HLA-B-12、HLA-B13:01、HLA-B14:02、HLA-B15:01、HLA-B15:02、HLA-B15:03、HLA-B15:18、HLA-B18:01、HLA LA-B35:01、HLA-B38:02、HLA-B40:01、HLA-B40:02、HLA-B42:01、HLA-B44:02、HLA-B44:03、HLA-B45:01、HLA-B4 6:01、HLA-B49:01、HLA-B51:01、HLA-B52:01、HLA-B53:01、HLA-B54:01、HLA-B55:02、HLA-B57:01、HLA-B58:01、HLA LA-B67:01、HLA-C1:02、HLA-C2:02、HLA-C3:02、HLA-C3:03、HLA-C3:04、HLA-C4:01、HLA-C5:01、HLA-C6:02、HLA-C3:02 -C7:01, HLA-C7:02, HLA-C8:01, HLA-C8:02, HLA-C12:03, HLA-C14:02, HLA-C16:01, HLA-C17:01 and HLA-C18:01.

[0136] There are 4 different types of HLA types.

[0137]

[0138]

[0139]

[0140] In some embodiments, the single-stranded trimeric nucleic acid can further comprise a second T cell epitope. In some embodiments, the second T cell epitope can be an MHC class II restricted epitope. In some embodiments, the second T cell epitope comprises SEQ ID NO: 21 and SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, or an epitope listed in Table 5 or a homolog having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity to a sequence selected from SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 and an epitope listed in Table 5.

[0141] Table 5 is a list of the most important MHC class II restricted epitopes (universal CD4 T cell epitope sequences).

[0142]

[0143]

[0144]

[0145]

[0146]

[0147] In some embodiments, the single-stranded trimeric nucleic acid can further comprise a flexible first linker between the first T cell epitope and the beta 2 microglobulin and a second linker between the beta 2 microglobulin and the MHC class I heavy chain sequence.

[0148] In some embodiments, the nucleic acid can comprise a plasmid DNA, a minicircle DNA, a microRNA, an mRNA, a self-amplifying RNA, a circular RNA, a DNA primed self- amplifying RNA, or a viral vector. In some embodiments, the T cell epitope can comprise a viral, bacterial, parasitic, or cancer T cell epitope. In some embodiments, the T cell epitope can comprise a T cell epitope of a tumor specific antigen (TSA), a tumor associated antigen (TAA), or a viral derived cancer antigen.

[0149] Compositions

[0150] Described herein are compositions comprising a single-stranded trimeric nucleic acid encoding a first T cell epitope, a beta 2-microglobulin, and a MHC class I heavy chain sequence.

[0151] In some embodiments, the single-stranded trimer nucleic acid may comprise a nucleic acid sequence selected from any one of the following: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 498, or SEQ ID NO: 500; and a nucleic acid sequence having the same sequence as SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 498, or SEQ ID NO: 500. A nucleic acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8% or 99.9%) identical to the nucleic acid sequence of NO:500.

[0152] In some embodiments, the single-stranded trimer nucleic acid can encode an amino acid selected from any one of the following: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 497, or SEQ ID NO: 499; and a nucleic acid having a sequence similar to that of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 497, or SEQ ID NO: 499. An amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8% or 99.9%) identical to the amino acid sequence of NO:499.

[0153] In some embodiments, the first T cell epitope can include an epitope listed in Tables 1 to 3 or a homolog having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8% or 99.9%) identity to an epitope in Tables 1 to 3.

[0154] In some embodiments, the first T cell epitope can include an epitope listed in Table 2 or a homolog having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8% or 99.9%) identity to an epitope of Table 2.

[0155] In some embodiments, the single-chain trimer nucleic acid may comprise nucleic acids encoding a T cell epitope, β2 microglobulin, and an MHC heavy chain sequence in order from amino to carboxyl terminus.

[0156] In some embodiments, the single-stranded trimer nucleic acid may further comprise a second T cell epitope. In some embodiments, the second T cell epitope may be an MHC class II restricted epitope. In some embodiments, the second T cell epitope includes SEQ ID NO: 21 and SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 or an epitope listed in Table 5 or having a sequence selected from SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8% or 99.9%) homologues and the epitopes listed in Table 5.

[0157] In some embodiments, the single-chain trimer nucleic acid may further comprise a flexible first linker between the first T cell epitope and the β2 microglobulin and a second linker between the β2 microglobulin and the MHC class I heavy chain sequence.

[0158] About MHC The results showed that HLA-A1:01, HLA-A2:01, HLA-A2:03, HLA- A2:06、ALL-A2:07、ALL-A3:01、ALL-A9、ALL-A10、ALL-A1 1:01、HLA-A23:01、HLA-A24:02、HLA-A25:01、HLA-A26:01、HLA-A29:02、HLA-A30:01、HLA-A30:02、H301-A LA-A32:02、HLA-A33:03、HLA-A34:01、HLA-A34:02、HLA-A68:01、HLA-A68:02、HLA-A74:01、HLA-B5、H2A-B7 LA-B8:01、HLA-B-12、HLA-B13:01、HLA-B14:02、HLA-B15:01、HLA-B15:02、HLA-B15:03、HLA-B15:18:0HLA-12 、HLA-B35:01、HLA-B38:02、HLA-B40:01、HLA-B40:02、HLA-B42:01、HLA-B44:02、HLA-B44:03、HLA-B.45:01 46:01、HLA-B49:01、HLA-B51:01、HLA-B52:01、HLA-B53:01、HLA-B54:01、HLA-B55:02、HLA-B57:01、5:HLA-8 HLA-B67:01、HLA-C1:02、HLA-C2:02、HLA-C3:02、HLA-C3:03、HLA-C3:04、HLA-C4:01、HLA-C5:01、HLA-C6:02 -C7:01、HLA-C7:02、HLA-C8:01、HLA-C8:02、HLA-C12:03、HLA-C14:02、HLA-C17:01

[0159] MHC II antibodies also express HLA-DP, HLA-DQ, and HLA-DR.

[0160] In some embodiments, the composition can further comprise a pharmaceutically acceptable carrier. In some aspects, disclosed herein is a pharmaceutical composition, which can comprise a single chain trimeric nucleic acid (e.g., mRNA) encoding a first T cell epitope, a beta 2-microglobulin, and an MHC heavy chain sequence; and a pharmaceutically acceptable carrier. In some embodiments, the composition can be a nanoparticle, a lipid nanoparticle dispersion, a liposomal formulation, a lipid emulsion, a vaccine, a vector, or any combination thereof.

[0161] In some embodiments, the pharmaceutical composition can comprise a lipid nanoparticle comprising a single chain trimeric nucleic acid (e.g., mRNA) encoding a first T cell epitope, a beta 2-microglobulin, and an MHC class I heavy chain sequence.

[0162] Also described herein are cells comprising the compositions described herein. In some embodiments, the cells can include dendritic cells, B cells, macrophages, or other cells derived from a subject that can be used for immunotherapy. In some embodiments, the T cell epitope can include a viral, bacterial, parasitic, or cancer T cell epitope. In some embodiments, the T cell epitope can include a T cell epitope of a tumor specific antigen (TSA), a tumor associated antigen (TAA), or a viral derived cancer antigen.

[0163] Delivery methods

[0164] Suitable nucleic acid delivery vehicles are well known in the art and can include, but are not limited to, lipid-based delivery vehicles (e.g., liposomal formulations, lipoplexes, or lipid nanoparticles (LNP)), viral-based delivery vehicles, or physical methods such as injection, microinjection, electroporation, ultrasound, gene gun, hydrodynamic application, or any combination thereof.

[0165] Nanoparticles

[0166] Described herein are nanoparticles comprising a single chain trimeric nucleic acid (e.g., mRNA) encoding a first T cell epitope, a beta 2-microglobulin, and an MHC class I heavy chain sequence.

[0167] In some embodiments, the nanoparticle can be a lipid nanoparticle. In some embodiments, the nanoparticle can be a lipid-poly cation complex, referred to as a cationic lipid nanoparticle. As non-limiting examples, the poly cation can include a cationic peptide or polypeptide such as, but not limited to, polylysine, polyornithine, and / or polyarginine. In some embodiments, the lipid nanoparticle can comprise a non-cationic lipid such as, but not limited to, cholesterol or dioleoyl phosphatidyl ethanolamine (DOPE).

[0168] In some embodiments, described herein are lipid nanoparticles comprising a single chain trimeric nucleic acid (e.g., mRNA) encoding a first T cell epitope, a beta 2-microglobulin, and a MHC class I heavy chain sequence.

[0169] The lipid nanoparticle formulation can be influenced by, but not limited to, the selection of cationic lipid component, the degree of saturation of the cationic lipid, the nature of the PEGylation, the ratio of all components, and biophysical parameters such as size. In one example Semple et al. (Nature Biotech. 2010 28: 172-176), the lipid nanoparticle can further comprise 57% cationic lipid, 7% dipalmitoyl phosphatidyl choline, 34% cholesterol, and 1.5% PEG-c-DMA. As another example, varying the composition of the cationic lipid can more efficiently deliver siRNA to various antigen presenting cells (Basha et al. Mol Ther. 2011 19:2186-2200).

[0170] In some embodiments, the lipid nanoparticle formulation can comprise 35% to 45% cationic lipid, 40% to 50% cationic lipid, 50% to 60% cationic lipid, and / or 55% to 65% cationic lipid. In some embodiments, the ratio of lipid to nucleic acid (e.g., mRNA) in the lipid nanoparticle can be 5: 1 to 20: 1, 10: 1 to 25: 1, 15: 1 to 30: 1, and / or at least 30: 1.

[0171] The lipid nanoparticle formulation typically comprises lipids, in particular ionizable cationic lipids, and further comprises neutral lipids, sterols, and molecules capable of reducing particle aggregation, such as PEG or PEG-modified lipids.

[0172] In some embodiments, the lipid nanoparticle formulation comprises at least one ionizable lipid, a cationic lipid, or any combination thereof; a neutral lipid selected from DSPC, DPPC, POPC, DOPE, and SM; (iii) a sterol, such as cholesterol; and (iv) a PEG-lipid, such as PEG-DMG or PEG-cDMA, in a molar ratio of 20%-60% cationic lipid: 5%-25% neutral lipid: 25%-55% sterol; 0.5%-15% PEG-lipid.

[0173] In some embodiments, the lipid nanoparticle formulation comprises 25-75% by mole (e.g., 35-65%, 45-65%, 60%, 57.5%, 50%, or 40% by mole) of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319).

[0174] In some embodiments, the lipid nanoparticle formulation comprises, on a molar basis, 25-75% cationic lipid, 0.5-15% neutral lipid, 5-50% sterol, and 0.5-20% PEG or PEG-modified lipid.

[0175] In some embodiments, the lipid nanoparticle formulation comprises, on a molar basis, 35-65% cationic lipid, 3-12% neutral lipid, 15-45% sterol, and 0.5-10% PEG or PEG-modified lipid.

[0176] In some embodiments, the lipid nanoparticle formulation comprises, on a molar basis, 45-65% cationic lipid, 5-10% neutral lipid, 25-40% sterol, and 0.5-10% PEG or PEG-modified lipid.

[0177] In some embodiments, the lipid nanoparticle formulation comprises, on a molar basis, 60% cationic lipid, 7.5% neutral lipid, 31% sterol, and 1.5% PEG or PEG-modified lipid.

[0178] In some embodiments, the lipid nanoparticle formulation comprises, on a molar basis, 50% cationic lipid, 10% neutral lipid, 38.5% sterol, and 1.5% PEG or PEG-modified lipid.

[0179] In some embodiments, the lipid nanoparticle formulation comprises, on a molar basis, 50% cationic lipid, 10% neutral lipid, 35% sterol, 4.5% or 5% PEG or PEG-modified lipid, and 0.5% targeting lipid.

[0180] In some embodiments, the lipid nanoparticle formulation comprises, on a molar basis, 40% cationic lipid, 15% neutral lipid, 40% sterol, and 5% PEG or PEG-modified lipid.

[0181] In some embodiments, the lipid nanoparticle formulation comprises, on a molar basis, 57.2% cationic lipid, 7.1% neutral lipid, 34.3% sterol, and 1.4% PEG or PEG-modified lipid.

[0182] In some embodiments, the lipid nanoparticle formulation comprises 57.5% by mole of a cationic lipid selected from PEG lipids that are PEG-cDMA (PEG-cDMA is further discussed in Reyes et al. J. Controlled Release, 107, 276-287 (2005), the contents of which are incorporated herein by reference in their entirety), 7.5% by mole of a neutral lipid, 31.5% by mole of a sterol, and 3.5% by mole of a PEG or PEG-modified lipid.

[0183] In some embodiments, the lipid nanoparticle formulation comprises a lipid mixture having a molar ratio of 20%-70% cationic lipid: 5%-45% neutral lipid: 20%-55% cholesterol: 0.5%-15% PEG-modified lipid. In some embodiments, the lipid nanoparticle formulation comprises a lipid mixture having a molar ratio of 20%-60% cationic lipid: 5%-25% neutral lipid: 25%-55% cholesterol: 0.5%-15% PEG-modified lipid.

[0184] In some embodiments, the lipid molar ratio is 50 / 10 / 38.5 / 1.5 (mol% cationic lipid / neutral lipid, e.g., DSPC / Chol / PEG-modified lipid, e.g., PEG-DMG, PEG-DSG, or PEG-DPG), 57.2 / 7.1 / 34.3 / 1.4 (mol% cationic lipid / neutral lipid, e.g., DPPC / Chol / PEG-modified lipid, e.g., PEG-cDMA), 40 / 15 / 40 / 5 (mol% cationic lipid / neutral lipid, e.g., DSPC / Chol / PEG-modified lipid, e.g., PEG-DMG), 50 / 10 / 35 / 4.5 / 0.5 (mol% cationic lipid / neutral lipid, e.g., DSPC / Chol / PEG-modified lipid, e.g., , e.g., PEG-DSG), 50 / 10 / 35 / 5 (cationic lipid / neutral lipid, e.g., DSPC / Chol / PEG-modified lipid, e.g., PEG-DMG), 40 / 10 / 40 / 10 (mol% cationic lipid / neutral lipid, e.g., DSPC / Chol / PEG-modified lipid, e.g., PEG-DMG or PEG-cDMA), 35 / 15 / 40 / 10 (mol% cationic lipid / neutral lipid, e.g., DSPC / Chol / PEG-modified lipid, e.g., PEG-DMG or PEG-cDMA), or 52 / 13 / 30 / 5 (mol% cationic lipid / neutral lipid, e.g., DSPC / Chol / PEG-modified lipid, e.g., PEG-DMG or PEG-cDMA).

[0185] For example, non-limiting examples of lipid nanoparticle compositions and methods for their preparation are described in Semple et al. (2010) Nat. Biotechnol. 28: 172-176; Jayarama et al. (2012), Angew. Chem. Int. Ed., 51: 8529-8533; and Maier et al. (2013) Molecular Therapy 21, 1570-1578 (the contents of each of which are incorporated herein by reference in their entirety).

[0186] In certain embodiments, the lipid nanoparticle formulation can include cationic lipids, PEG lipids and structural lipids, and optionally include non-cationic lipids. As a non-limiting example, the lipid nanoparticle can include 40%-60% cationic lipids, 5%-15% non-cationic lipids, 1%-2% PEG lipids and 30%-50% structural lipids. As another non-limiting example, the lipid nanoparticle can include 50% cationic lipids, 10% non-cationic lipids, 1.5% PEG lipids and 38.5% structural lipids. As another non-limiting example, the lipid nanoparticle can include 55% cationic lipids, 10% non-cationic lipids, 2.5% PEG lipids and 32.5% structural lipids.

[0187] In some embodiments, the lipid nanoparticle formulations described herein can be 4-component lipid nanoparticles. The lipid nanoparticles can include cationic lipids, non-cationic lipids, PEG lipids, and structural lipids. As a non-limiting example, the lipid nanoparticles can include 40%-60% cationic lipids, 5%-15% non-cationic lipids, 1%-2% PEG lipids, and 30%-50% structural lipids. As another non-limiting example, the lipid nanoparticles can include 50% cationic lipids, 10% non-cationic lipids, 1.5% PEG lipids, and 38.5% structural lipids. As another non-limiting example, the lipid nanoparticles can include 55% cationic lipids, 10% non-cationic lipids, 2.5% PEG lipids, and 32.5% structural lipids.

[0188] In some embodiments, the lipid nanoparticle formulations described herein may comprise cationic lipids, non-cationic lipids, PEG lipids, and structural lipids. As a non-limiting example, the lipid nanoparticles comprise 50% cationic lipid DLin-KC2-DMA, 10% non-cationic lipid DSPC, 1.5% PEG lipid PEG-DOMG, and 38.5% structural lipid cholesterol. As a non-limiting example, the lipid nanoparticles comprise 50% cationic lipid DLin-MC3-DMA, 10% non-cationic lipid DSPC, 1.5% PEG lipid PEG-DOMG, and 38.5% structural lipid cholesterol. As a non-limiting example, the lipid nanoparticles comprise 50% cationic lipid DLin-MC3-DMA, 10% non-cationic lipid DSPC, 1.5% PEG lipid PEG-DMG, and 38.5% structural lipid cholesterol. As another non-limiting example, the lipid nanoparticles comprise 55% cationic lipid L319, 10% non-cationic lipid DSPC, 2.5% PEG lipid PEG-DMG, and 32.5% structural lipid cholesterol.

[0189] In some embodiments, the nanoparticles may be such as those described in U.S. Patent No. 10,933,127, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the nanoparticles may be such as those described in U.S. Patent No. 10,933,127, for example, nanoparticles comprising a compound according to Formula (I), (Ia), (II), (IIa), (IIb), (IIc), (IId), or (IIe) as described in columns 101 to 187.

[0190] In some embodiments, nanoparticles (e.g., lipid nanoparticles) have an average diameter of 10-500 nm, 20-400 nm, 30-300 nm, 40-200 nm. In some embodiments, nanoparticles (e.g., lipid nanoparticles) have an average diameter of 50-150 nm, 50-200 nm, 80-100 nm, or 80-200 nm.

[0191] The lipid nanoparticles (LNPs) described herein can be manufactured in a sterile environment.

[0192] In some embodiments, the LNP formulations can be formulated as nanoparticles, such as nucleic acid-lipid particles. As non-limiting examples, the lipid particles can include one or more active or therapeutic agents; one or more cationic lipids, accounting for 50 mol% to 85 mol% of the total lipids present in the particle; one or more non-cationic lipids, accounting for 13 mol% to 49.5 mol% of the total lipids present in the particle; and one or more conjugated lipids that inhibit particle aggregation, accounting for 0.5 mol% to 2 mol% of the total lipids present in the particle.

[0193] The nanoparticle formulation may include a phosphate conjugate. The phosphate conjugate may increase in vivo circulation time and / or increase targeted delivery of the nanoparticle. As a non-limiting example, the phosphate conjugate may include a compound of any formula described in International Application No. WO2013033438, the contents of which are incorporated herein by reference in their entirety.

[0194] The nanoparticle formulation may comprise a polymer conjugate. The polymer conjugate may be a water-soluble conjugate. The polymer conjugate may have a structure as described in U.S. Patent Application No. 20130059360, the contents of which are incorporated herein by reference in their entirety. In some embodiments, polymer conjugates with polynucleotides of the present disclosure may be prepared using the methods and / or segmented polymer reagents described in U.S. Patent Application No. 20130072709, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the polymer conjugate may have a side group comprising a ring portion, such as, but not limited to, the polymer conjugate described in U.S. Patent Application No. US20130196948, the contents of which are incorporated herein by reference in their entirety.

[0195] Nanoparticle formulations may include conjugates to enhance delivery of the disclosed nanoparticles in a subject.

[0196] In some embodiments, the composition comprises a polynucleotide of the present disclosure and a conjugate that may have a degradable bond. Non-limiting examples of conjugates include aromatic moieties comprising ionizable hydrogen atoms, spacer moieties, and water-soluble polymers. As non-limiting examples, pharmaceutical compositions comprising conjugates having degradable bonds and methods for delivering such pharmaceutical compositions are described in U.S. Patent Publication No. US20130184443, the contents of which are incorporated herein by reference in their entirety.

[0197] In some embodiments, the lipid nanoparticles may comprise 20% to 80% ionizable lipids, cationic lipids, or any combination thereof; greater than 0% to 5% polyethylene glycol-lipids; greater than 0% to 20% helper lipids; 20% to 80% sterols; and a single-chain trimeric nucleic acid encoding a first T cell epitope, β2-microglobulin, and an MHC class I heavy chain sequence encapsulated in the lipid nanoparticles.

[0198] In some embodiments, the lipid nanoparticles may comprise 20% to 80% ionizable lipids, cationic lipids, or any combination thereof; greater than 0% to 5% polyethylene glycol-lipids; greater than 0% to 20% helper lipids; 20% to 80% sterols; and a single-stranded trimeric nucleic acid (e.g., mRNA) encoding a first T cell epitope, β2-microglobulin, and an MHC class I heavy chain sequence encapsulated in the lipid nanoparticles.

[0199] In one embodiment, the nanoparticles may comprise 40% to 60% ionizable lipids, cationic lipids, or any combination thereof; 1% to 2% polyethylene glycol-lipids; 8% to 12% helper lipids; 35% to 40% sterols; and a single-stranded trimeric nucleic acid (e.g., mRNA) encoding a first T cell epitope, β2-microglobulin, and an MHC class I heavy chain sequence encapsulated in the lipid nanoparticles.

[0200] In one embodiment, the nanoparticles may comprise 40% to 60% ionizable lipids, cationic lipids, or any combination thereof; 1% to 2% polyethylene glycol-lipids; 8% to 12% helper lipids; 35% to 40% sterols; and a single-stranded trimeric nucleic acid (e.g., mRNA) encoding a first T cell epitope, β2-microglobulin, and an MHC class I heavy chain sequence encapsulated in the lipid nanoparticles.

[0201] In some embodiments, the single-stranded trimer nucleic acid may comprise a nucleic acid sequence selected from any one of the following: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 498, or SEQ ID NO: 500; and a nucleic acid sequence having the same sequence as SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, or SEQ ID NO: 20, SEQ ID NO: 498, or SEQ ID NO: 500. A nucleic acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8% or 99.9%) identical to the nucleic acid sequence of NO:500.

[0202] In some embodiments, the single-stranded trimer nucleic acid can encode an amino acid selected from any one of the following: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 497, or SEQ ID NO: 499; and a nucleic acid having a sequence similar to that of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 497, or SEQ ID NO: 499. An amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8% or 99.9%) identical to the amino acid sequence of NO:499.

[0203] In some embodiments, the first T cell epitope can include an epitope listed in Tables 1 to 3 or a homolog having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8% or 99.9%) identity to an epitope in Tables 1 to 3.

[0204] In some embodiments, the first T cell epitope can include an epitope listed in Table 2 or a homolog having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8% or 99.9%) identity to an epitope of Table 2.

[0205] In some embodiments, the single-chain trimer nucleic acid may comprise nucleic acids encoding a T cell epitope, β2 microglobulin, and an MHC heavy chain sequence in order from amino to carboxyl terminus.

[0206] In some embodiments, the single-stranded trimer nucleic acid may further comprise a second T cell epitope. In some embodiments, the second T cell epitope may be an MHC class II restricted epitope. In some embodiments, the second T cell epitope includes SEQ ID NO: 21 and SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 or an epitope listed in Table 5 or having a sequence selected from SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8% or 99.9%) homologues and the epitopes listed in Table 5.

[0207] In some embodiments, the single-chain trimer nucleic acid may further comprise a flexible first linker between the first T cell epitope and the β2 microglobulin and a second linker between the β2 microglobulin and the MHC class I heavy chain sequence.

[0208] Suitable MHC class I heavy chains can include, but are not limited to, HLA-A1 :01, HLA-A2:01, HLA-A2:03, HLA-A2:06, HLA-A2:07, HLA-A3:01, HLA-A9, HLA-A10, HLA-A11 :01, HLA-A23:01, HLA-A24:02, HLA-A25:01, HLA-A26:01, HLA-A29:02, HLA-A30:01, HLA-A30:02, HLA-A31 :01, HLA-A32:02, HLA-A33:03, HLA-A34:01, HLA-A34:02, HLA-A68:01, HLA-A68:02, HLA-A74:01, HLA-B5, HLA-B7:02, HLA-B8:01, HLA-B-12, HLA-B13:01, HLA-B14:02, HLA-B15:01, HLA-B15:02, HLA-B15:03, HLA-B15:18, HLA-B18:01, HLA-B35:01, HLA-B38:02, HLA-B40:01, HLA-B40:02, HLA-B42:01, HLA-B44:02, HLA-B44:03, HLA-B45:01, HLA-B46:01, HLA-B49:01, HLA-B51 :01, HLA-B52:01, HLA-B53:01, HLA-B54:01, HLA-B55:02, HLA-B57:01, HLA-B58:01, HLA-B67:01, HLA-C1 :02, HLA-C2:02, HLA-C3:02, HLA-C3:03, HLA-C3:04, HLA-C4:01, HLA-C5:01, HLA-C6:02, HLA-C7:01, HLA-C7:02, HLA-C8:01, HLA-C8:02, HLA-C12:03, HLA-C14:02, HLA-C16:01, HLA-C17:01, or HLA-C18:01 heavy chain.

[0209] Suitable MHC class II heavy chains can include, but are not limited to, HLA-DP, HLA-DQ, or HLA-DR.

[0210] In some embodiments, the cells can include dendritic cells, B cells, macrophages, or other cells derived from the subject that can be used for immunotherapy. In some embodiments, the T cell epitopes can include viral, bacterial, parasitic, or cancer T cell epitopes. In some embodiments, the T cell epitopes can include T cell epitopes of tumor-specific antigens (TSAs), tumor-associated antigens (TAAs), or virus-derived cancer antigens.

[0211] neutral lipid

[0212] In some embodiments, the lipid nanoparticle formulation comprises 0.5% to 15% (e.g., 3% to 12%, 5% to 10%, or 15%, 10%, or 7.5% by mole) of a neutral lipid. Examples of neutral lipids include, but are not limited to, DSPC, POPC, DPPC, DOPE, and SM. In some embodiments, the formulation comprises 5% to 50% (e.g., 15% to 45%, 20% to 40%, 40%, 38.5%, 35%, or 31% by mole) of a sterol. A non-limiting example of a sterol is cholesterol. In some embodiments, the lipid nanoparticle formulation comprises 0.5% to 20% (e.g., 0.5% to 10%, 0.5% to 5%, 1.5%, 0.5%, 1.5%, 3.5%, or 5% by mole) of a PEG or PEG-modified lipid. In some embodiments, the PEG or PEG-modified lipid comprises PEG molecules having an average molecular weight of 2,000 Da. In some embodiments, the PEG or PEG-modified lipid comprises PEG molecules having an average molecular weight of less than 2,000, e.g., about 1,500 Da, about 1,000 Da, or about 500 Da. Non-limiting examples of PEG-modified lipids include PEG-distearyl glycerol (PEG-DMG) (also referred to herein as PEG-C14 or C14-PEG), PEG-cDMA (further discussed in Reyes et al. J. Controlled Release, 107, 276-287 (2005), the contents of which are incorporated by reference herein in their entirety).

[0213] zwitterionic lipid

[0214] In some embodiments, the composition can be encapsulated in, linked to, and / or associated with a zwitterionic lipid. Non-limiting examples of zwitterionic lipids and methods of using zwitterionic lipids are described in U.S. Patent Publication No. US20130216607, the contents of which are incorporated by reference herein in their entirety. In some aspects, zwitterionic lipids can be used in the liposomes and lipid nanoparticles described herein.

[0215] cationic lipid

[0216] Suitable cationic lipids may include, but are not limited to, 2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,2Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol (compound 1 in US20130150625); 2-amino-3-[(9Z)-octadeca-9-en-1-yloxy]-2-{[(9Z)-octadeca-9-en-1-yloxy]methyl}propan-1-ol (compound 2 in US20130150625); Amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-[(octyloxy)methyl]propan-1-ol (compound 3 in US20130150625); and 2-(dimethylamino)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol (compound 4 in US20130150625); or any pharmaceutically acceptable salt or stereoisomer thereof. As non-limiting examples, the cationic lipids may be selected from (20Z,23Z)-N,N-dimethylnonacosa-20,23-dien-10-amine, (17Z,20Z)-N,N-dimethylhexacosa-17,20-dien-9-amine, (1Z,19Z)-N5N-dimethylpentacosa-16,19-dien-8-amine, (13Z,16Z)-N,N-dimethyldocosa-1 3,16-dien-5-amine, (12Z,15Z)-N,N-dimethylhexene-12,15-dien-4-amine, (14Z,17Z)-N,N-dimethyltricosan-14,17-dien-6-amine, (15Z,18Z)-N,N-dimethyltetracosa-15,18-dien-7-amine, (18Z,21Z)-N,N-dimethylheptacosa-18,21-dien- -10-amine, (15Z,18Z)-N,N-dimethyltetracosa-15,18-dien-5-amine, (14Z,17Z)-N,N-dimethyltricosac-14,17-dien-4-amine, (19Z,22Z)-N,N-dimethyloctacosa-19,22-dien-9-amine, (18Z,21Z)-N,N-dimethylheptacosac-18,21-dien-8-amine, ( (17Z,20Z)-N,N-dimethylhexacosa-17,20-dien-7-amine, (16Z,19Z)-N,N-dimethylpentacosa-16,19-dien-6-amine, (22Z,25Z)-N,N-dimethyltriaconta-22,25-dien-10-amine, (21Z,24Z)-N,N-dimethyltriaconta-21,24-dien-9-amine, (18Z)-N,N-dimethylheptacos-18-en-10-amine, (17Z)-N,N-dimethylhexacosa-17-en-9-amine, (19Z,22Z)-N,N-dimethyloctacosa-19,22-dien-7-amine, N,N-dimethylheptacosan-10-amine, (20Z,23Z)-N-ethyl-N-methylnonacosan-20,23-dien-10-amine, 1-[(11Z,14Z)-1-nonyleicosa-11,14-dien-1-yl]pyrrolidine, (20Z)-N,N-dimethylheptacosan-20-en-10-amine, (15Z)-N,N-dimethylheptacosan-15-en-10-amine, (14Z)-N,N-dimethyl N,N-dimethylnonacosa-14-en-10-amine, (17Z)-N,N-dimethylnonacosa-17-en-10-amine, (24Z)-N,N-dimethyltricaracon-24-en-10-amine, (20Z)-N,N-dimethylnonacosa-20-en-10-amine, (22Z)-N,N-dimethyltricaracon-22-en-10-amine, (16Z)-N,N-dimethylpentacosa-16-en-8-amine, (12Z,15Z)-N,N-dimethyl-2-nonylhexicosa-12,15-dien-1-amine, (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine, N,N-dimethyl-1 -[(1S,2R)-2-octylcyclopropyl]heptadecan-8-amine, 1-[(1S,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadecan-10-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]nonadecan-10-amine, N,N-dimethyl-21-[(1S,2R)-2-octylcyclopropyl]heneconyl-10-amine, N,N-dimethyl-1-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]nonadecan-10-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexadecane-8-amine, N,N-dimethyl-[(1R,2S)- 2-undecylcyclopropyl]tetradec-5-amine, N,N-dimethyl-3-{7-[(1S,2R)-2-octylcyclopropyl]heptyl}dodec-1-amine, 1-[(1R,2S)-2-heptylcyclopropyl]-N,N-dimethyloctadec-9-amine, 1-[(1S,2R)-2-decylcyclopropyl]-N,N-dimethylpentadecan-6-amine, N,N-dimethyl-1-R1S,2R)-2-octylcyclopropylpentadecan-8-amine, R—N,N-dimethyl-1-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine, S—N,N-dimethyl-1-[(9Z,12Z)-octadec-9,1-{2-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine, 1-{2-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}pyrrolidine, (2S)—N,N-dimethyl-1-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]-3-[(5Z)-oct-5-en-1-yloxy]propan-2-amine, 1-{2-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}azetidine, (2S)-1-(hexyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy] 1-(heptyloxy)-N,N-dimethyl-3-(R9Z,12Z)-octadeca-9,12-dien-1-yloxy)propan-2-amine, N,N-dimethyl-1-(nonyloxy)-3-(R9Z,12Z)-octadeca-9,12-dien-1-yloxy)propan-2-amine, N,N-dimethyl-1-[(9Z)-octadeca-9-en-1-yloxy]-3-(octyloxy)propan-2-amine; (2S)-N,N-dimethyl-1-[(6Z,9Z,12Z)-octadeca-6,9,12-trien-1-yloxy]-3-(octyloxy)propan-2-amine, (2S)-1-[(11Z,14Z)-eicosyl-11 ,14-dien-1-yloxy]-N,N-dimethyl-3-(pentyloxy)propan-2-amine, (2S)-1-(hexyloxy)-3-[(11Z,14Z)-eicos-11,14-dien-1-yloxy]-N,N-dimethylpropan-2-amine, 1-[(11Z,14Z)-eicos-11,14-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, 1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, (2S)-1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-3-(hexyloxy)propan-2-amine N,N-dimethylpropan-2-amine, (2S)-1-[(13Z)-docosa-13-en-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine, 1-[(13Z)-docosa-13-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, 1-[(9Z)-hexadec-9-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, (2R)-N,N-dimethyl-H(1-methyloctyl)oxy]-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propan-2-amine, (2R)-1-[(3,7-dimethyloctyl)oxy]-N,N-dimethyl-3-R9Z,12Z)-octadec-9,12-dien-1-yloxypropan-2-amine, N,N-dimethyl-1-(octyloxy)-3-({8-R1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]octyl}oxy)propan-2-amine, N,N-dimethyl-1-1[8-(2-octylcyclopropyl)octyl]oxy}-3-(octyloxy)propan-2-amine and (11E,20Z,23Z)-N,N-dimethylnonacosa-11,20,2-trien-10-amine or a pharmaceutically acceptable salt or stereoisomer thereof. In some embodiments, the lipid can be a cationic lipid such as, but not limited to, DLin-DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA, DODMA and amino alcohol lipids. The amino alcohol cationic lipid can be a lipid described in U.S. Patent Publication No. US20130150625, which is incorporated herein by reference in its entirety, and / or a lipid produced by the method described in said U.S. Patent Publication No. WO20130150625. In some embodiments, the nanoparticles described herein may include an amine cationic lipid, such as an amine cationic lipid described in International Patent Application No. WO2013059496, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the cationic lipid may have an amino-amine or amino-amide moiety.

[0217] In some embodiments, the cationic lipid may be a low molecular weight cationic lipid, such as those described in U.S. Patent Application No. 20130090372, the contents of which are herein incorporated by reference in their entirety.

[0218] Ionizable lipids

[0219] Exemplary ionizable lipids are described in U.S. Patent Publication Nos. US2016 / 0311759, US2015 / 0376115, US2016 / 0151284, US2017 / 0210697, US2015 / 0140070, US2013 / 0178541, US2013 / 0303587, US2015 / 0141678, US2015 / 0239926, US2016 / 0376224, US2017 / 0119904, US2012 / 0149894, US2015 / 0057373, US2013 / 0090372, US2013 / 0274523, US2013 / 0274504, US2013 / 0274504, US2009 / 0023673, US2012 / 01 No. 28760, No. US2010 / 03241240, No. US2014 / 0200257, No. US2015 / 0203446, No. US2018 / 0005363, No. US2014 / 0308304, No. US2013 / 0338210, No. No. US2012 / 0101148, No. US2012 / 0027796, No. US2012 / 0058144, No. US2013 / 0323269, No. US2011 / 0117125, No. US2011 / 0256175, No. US2012 / 0202871, No. US2011 / 0076335, No. US2006 / 0083780, No. US2013 / 0123338, No. US2015 / 0064242, No. US2006 / 0051405, No. US2013 / 0065939, No. U No. S2006 / 0008910, No. US2003 / 0022649, No. US2010 / 0130588, No. US2013 / 0116307, No. US2010 / 0062967, No. US2013 / 0202684, No. US2014 / 0141070, No. US2014 / 0255472, No. US2014 / 0039032, No. US2018 / 0028664, No. US2016 / 0317458, No. US2013 / 0195920, No. US2022 / 0062175, No. USU.S. 2022 / 0009878, U.S. 2022 / 0040325, U.S. 2012 / 61657480, U.S. 2016 / 0074514, U.S. 2013 / 0330401, U.S. 2019 / 0185410, U.S. 2012 / 61617468, U.S. 2019 / 0032087, U.S. 2015 / 62184188, U.S. 2019 / 0127318, U.S. 2021 / 0002813, U.S. 2020 / 0345641, U.S. 2014 / 61944336, U.S. 2012 / 61657480, U.S. 2021 / 0059953, U.S. 2022 / 0162521, U.S. 2022 / 0235377, U.S. 2018 / 0085474, U.S. 2018 / 0000953, U.S. 2020 / 0129445, U.S. 2021 / 0145982, U.S. 2021 / 0378980, U.S. 2020 / 0254086, U.S. 2021 / 0346306, and U.S. 2018 / 0000953, the disclosures of all of which are incorporated by reference herein in their entireties.

[0220] In some embodiments, the nanoparticle comprises ionizable lipids at a molar ratio of 0% to 80%. In some embodiments, the ionizable lipids can be present at a molar ratio of at least 0% (e.g., at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80%). In some embodiments, the ionizable lipids can be present at a molar ratio of 80% or less (e.g., 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, 1% or less, or 0.5% or less).

[0221] Ionizable lipids can be present in a molar ratio ranging from any of the above minimum values ​​to any of the above maximum values. For example, in some embodiments, ionizable lipids can be present in a molar ratio of 0% to 80% (e.g., greater than 0% to 80%, greater than 0% to 70%, greater than 0% to 60%, greater than 0% to 50%, greater than 0% to 40%, greater than 0% to 30%, greater than 0% to 20%, greater than 0% to 10%, greater than 0% to 5%, greater than 0% to 1%, greater than 0% to 0.5%, 1% to 30%, 1% to 20%, 1% to 10%, 1% to 5%, 5% to 30%, 5% to 20%, 5% to 10%, 10% to 30%, 10% to 20%, 20% to 30%, 20% to 40%, or 30% to 40%). Helper lipids

[0222] In some embodiments, the nanoparticle comprises a helper lipid. In some embodiments, the helper lipid can be a non-cationic lipid. In some embodiments, the non-cationic lipids may include, but are not limited to, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), 1,2-dioleyl-sn-glycero-3-phosphatidylcholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dioleoyl-5 / 7-glycero-3-phospho-(1'-rac-glycerol) (DOPG), or a combination thereof. In one embodiment, the non-cationic lipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). In one embodiment, the non-cationic lipid is 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), and in one embodiment, the non-cationic lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In one embodiment, the non-cationic lipid is 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE). Although several non-cationic lipids are described herein, additional non-cationic lipids can be used in combination with the compounds disclosed herein.

[0223] In some embodiments, the nanoparticles comprise a helper lipid at a molar ratio of 0% to 20%. In some embodiments, the nanoparticles comprise a polyethylene glycol-lipid at a molar ratio of about 0%, 0.25%, 0.5%, 0.75%, 1%, 1.5%, 2%, 3%, 4%, 5%, 10%, 15%, or 20%.

[0224] polyethylene glycol-lipid

[0225] In certain embodiments, the nanoparticles include polyethylene glycol-lipids (PEG-lipids). PEG-lipids are incorporated to form a hydrophilic outer layer and stabilize the particles. Non-limiting examples of polyethylene glycol-lipids include lipids modified by PEG-, such as PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols and PEG-modified dialkylglycerols. Representative polyethylene glycol-lipids include DMG-PEG, DLPE-PEG, DMPE-PEG, DPPC-PEG and DSPE-PEG. In one embodiment, polyethylene glycol-lipids are 1,2-dimyristoyl-sn-glycerol, methoxy polyethylene glycol (DMG-PEG). In one embodiment, polyethylene glycol-lipids are 1,2-dimyristoyl-sn-glycerol, methoxy polyethylene glycol-2000 (DMG-PEG2000). DMG-PEGXXXX means 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol-XXXX, where XXXX represents the molecular weight of the polyethylene glycol portion, for example, DMG-PEG2000 or DMG-PEG5000.

[0226] In some embodiments, the nanoparticles comprise a molar ratio of 0% to 5% polyethylene glycol-lipid. In some embodiments, the nanoparticles comprise a molar ratio of about 0%, 0.25%, 0.5%, 0.75%, 1%, 1.5%, 2%, 3%, 4%, or 5% polyethylene glycol-lipid. In one embodiment, the nanoparticles comprise a molar ratio of 0.75% polyethylene glycol-lipid.

[0227] In some embodiments, the ratio of PEG in the lipid nanoparticle formulation can be increased or decreased and / or the carbon chain length of the PEG lipid can be modified from C14 to C18 to alter the pharmacokinetics and / or biodistribution of the lipid nanoparticle formulation. As a non-limiting example, the lipid nanoparticle formulation can contain a lipid molar ratio of 0.5% to 3.0%, 1.0% to 3.5%, 1.5% to 4.0%, 2.0% to 4.5%, 2.5% to 5.0% and / or 3.0% to 5.0% PEG-c-DOMG (R-3-[(ω-methoxy-poly(ethylene glycol)2000)carbamoyl)]-1,2-dimyristyloxypropyl-3-amine) (also referred to herein as PEG-DOMG) compared to the cationic lipid, DSPC and cholesterol. In some embodiments, PEG-c-DOMG can be replaced by a PEG lipid such as, but not limited to, PEG-DSG (1,2-distearoyl-sn-glycerol, methoxypolyethylene glycol), PEG-DMG (1,2-dimyristoyl-sn-glycerol), and / or PEG-DPG (1,2-dipalmitoyl-sn-glycerol, methoxypolyethylene glycol).

[0228] In some embodiments, the LNP formulation may contain 3% PEG-c-DOMG at a lipid molar ratio. In some embodiments, the LNP formulation may contain 1.5% PEG-c-DOMG at a lipid molar ratio.

[0229] In some embodiments, the pharmaceutical composition may comprise at least one of the PEGylated lipids described in International Publication No. WO2012099755, the contents of which are herein incorporated by reference in their entirety.

[0230] In some embodiments, the LNP formulation may contain PEG-DMG 2000 (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000). In some embodiments, the LNP formulation may contain PEG-DMG 2000 (a cationic lipid known in the art) and at least one other component. In some embodiments, the LNP formulation may contain PEG-DMG 2000 (a cationic lipid known in the art), DSPC, and cholesterol. As a non-limiting example, the LNP formulation may contain PEG-DMG 2000, DLin-DMA, DSPC, and cholesterol. As another non-limiting example, the LNP formulation can contain PEG-DMG 2000, DLin-DMA, DSPC, and cholesterol in a molar ratio of 2:40:10:48 (see, e.g., Geall et al., Nonviral delivery of self-amplifying RNA (e.g., mRNA) vaccines, PNAS 2012; PMID: 22908294, the contents of each of which are herein incorporated by reference in their entirety).

[0231] sterols

[0232] In some embodiments, the nanoparticles comprise sterols. Sterols are well known to those skilled in the art and generally refer to compounds having a cyclopentane polyhydrophenanthrene ring system and having one or more OH substituents. Examples of sterols include, but are not limited to, cholesterol, campesterol, ergosterol, sitosterol, etc.

[0233] In some embodiments, the sterol is selected from a cholesterol-based lipid. In some embodiments, the one or more cholesterol-based lipids are selected from cholesterol, pegylated cholesterol, DC-Choi (N,N-dimethyl-N-ethylcarboxamide cholesterol), 1,4-bis(3-N-oleamido-propyl)piperazine, or a combination thereof.

[0234] Sterols can tune the permeability and fluidity of particles based on their function in cell membranes. In one embodiment, the sterol is cholesterol.

[0235] In some embodiments, the nanoparticles comprise sterols at a molar ratio of 20% to 80%. In some embodiments, the nanoparticles comprise sterols at a molar ratio of 25%, 30%, 35%, 40%, 45%, or 50%. In one embodiment, the nanoparticles comprise sterols at a molar ratio of 40%.

[0236] The nanoparticle formulation can be a carbohydrate nanoparticle comprising a carbohydrate carrier and a nucleic acid sequence described herein (e.g., mRNA). As non-limiting examples, the carbohydrate carrier can include, but is not limited to, anhydride-modified phytoglycogen or glycogen-like materials, octenylsuccinate phytoglycogen, phytoglycogen β-dextrin, and anhydride-modified phytoglycogen β-dextrin. (See, for example, International Publication No. WO2012109121; the contents of which are incorporated herein by reference in their entirety).

[0237] Nanoparticle formulations of the present disclosure can be coated with surfactants or polymers to improve the delivery of particles. In certain embodiments, nanoparticles can be coated with hydrophilic coatings, such as, but not limited to, PEG coatings and / or coatings with neutral surface charge. Hydrophilic coatings may contribute to the delivery of nanoparticles with larger payloads, such as, but not limited to, RNA (e.g., mRNA), in the central nervous system. As non-limiting examples, nanoparticles comprising hydrophilic coatings and methods for preparing such nanoparticles are described in U.S. Patent Publication No. US20130183244, the contents of which are incorporated herein by reference in their entirety.

[0238] In some embodiments, the lipid nanoparticles of the present disclosure may be hydrophilic polymer particles. Non-limiting examples of hydrophilic polymer particles and methods for preparing hydrophilic polymer particles are described in U.S. Patent Publication No. US20130210991, the contents of which are incorporated herein by reference in their entirety.

[0239] In some embodiments, the lipid nanoparticles of the present disclosure may be hydrophobic polymer particles.

[0240] In some embodiments, an immune response can be elicited by delivering lipid nanoparticles that can comprise a nanoparticle, a polymer, and an immunogen. (U.S. Publication No. 20120189700 and International Publication No. WO2012099805; each of which is incorporated herein by reference in its entirety). The polymer can encapsulate the nanoparticle or partially encapsulate the nanoparticle. The immunogen can be a recombinant protein, a modified RNA, and / or a polynucleotide as described herein. In some embodiments, the lipid nanoparticles can be formulated for use in vaccines, such as, but not limited to, vaccines against pathogens.

[0241] Lipid nanoparticles can be engineered to change the surface properties of particles so that lipid nanoparticles can penetrate mucosal barriers.Mucus is located on mucosal tissue, such as but not limited to oral cavity (for example, cheek and esophageal membrane and tonsil tissue), eye, gastrointestinal (for example, stomach, small intestine, large intestine, colon, rectum), nose, respiratory tract (for example, nose, pharynx, trachea and bronchial membrane), genitals (for example, vagina, cervix and urethra membrane).Being greater than the nanoparticle of 10-200nm (because of its higher drug encapsulation efficiency and the ability that multiple drugs are continuously delivered is preferred), it is considered that it is too large and can not be rapidly diffused by mucosal barriers.Mucus is constantly secreted, falls off, discarded or digested and circulates, so most of captured particles can be removed from mucosal tissue in a few seconds or a few hours. Large polymer nanoparticles (200 nm-500 nm in diameter) densely coated with low molecular weight polyethylene glycol (PEG) diffuse through mucus only 4 to 6 times slower than the same particles diffusing in water (Lai et al. PNAS 2007 104(5):1482-487; Lai et al. Adv Drug Deliv Rev. 2009 61(2):158-171; each of which is incorporated herein by reference in its entirety). Transport of nanoparticles can be determined by permeation rate and / or fluorescence microscopy techniques, including but not limited to fluorescence recovery after photobleaching (FRAP) and high-resolution multi-particle tracking (MPT). As non-limiting examples, compositions that can penetrate mucosal barriers can be made as described in U.S. Patent No. 8,241,670 or International Patent Publication No. WO2013110028, the contents of each of which are incorporated herein by reference in their entirety.

[0242] The lipid nanoparticles engineered to penetrate mucus can include polymeric materials (i.e., polymer cores) and / or polymer-vitamin conjugates and / or triblock copolymers. Polymeric materials can include, but are not limited to, polyamines, polyethers, polyamides, polyesters, polyurethanes, polyureas, polycarbonates, poly(styrene), polyimides, polysulfones, polyurethanes, polyacetylene, polyethylene, polyethyleneimine, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitrile, and polyarylates. Polymeric materials can be biodegradable and / or biocompatible. Non-limiting examples of biocompatible polymers are described in International Patent Publication No. WO2013116804, the contents of which are incorporated herein by reference in their entirety. Polymeric materials can be irradiated in addition. As a non-limiting example, polymeric materials can be irradiated with gamma rays (see, for example, International Application No. WO201282165, which is incorporated herein by reference in its entirety).Non-limiting examples of specific polymers include poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid) (PLGA), poly(L-lactic acid-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone), poly(D,L-lactide-co-caprolactone-co-glycolide), poly(D,L-lactide-co-PEO- co-D,L-lactide), poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacrylates, polyurethanes, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA), polyethylene glycol, poly-L-glutamic acid, poly(hydroxy acids), polyanhydrides, polyorthoesters, poly(esteramides), polyamides, poly(etheresters), polycarbonates, polyalkylenes such as polyethylene and polypropylene, polyalkylene glycols such as poly(ethylene glycol) (PEG), polyalkylene oxides (PEO), polyalkylene terephthalates such as poly(ethylene terephthalate) , polyvinyl alcohol (PVA), polyvinyl ethers, polyvinyl esters such as poly(vinyl acetate)), polyvinyl halides such as poly(vinyl chloride) (PVC)), polyvinyl pyrrolidone, polysiloxanes, polystyrene (PS), polyurethanes, derivatized celluloses such as alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitrocellulose, hydroxypropyl cellulose, and carboxymethyl cellulose, acrylic polymers such as poly(methyl (meth)acrylate) (PMMA), poly(ethyl (meth)acrylate), poly(butyl (meth)acrylate), poly(isobutyl (meth)acrylate), poly(methyl meth ... (meth)acrylate), poly(isodecyl (meth)acrylate), poly(lauryl (meth)acrylate), poly(phenyl (meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate) and copolymers and mixtures thereof), polydioxanone and its copolymers, polyhydroxyalkanoates, polypropylene fumarate, polyoxymethylene, poloxamer, poly(o-)esters, poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone), PEG-PLGA-PEG and trimethylene carbonate, polyvinyl pyrrolidone.The lipid nanoparticles can be coated with or combined with copolymers, such as, but not limited to, block copolymers (such as the branched polyether-polyamide block copolymers described in International Publication No. WO2013012476, which is incorporated herein by reference in its entirety) and (polyethylene glycol)-(polypropylene oxide)-(polyethylene glycol) triblock copolymers (see, for example, U.S. Publication No. 20120121718 and U.S. Publication No. 20100003337 and U.S. Patent No. 8,263,665, the contents of each of which are incorporated herein by reference in their entirety). The copolymer can be a generally recognized as safe (GRAS) polymer, and the formation of the lipid nanoparticles can be in a manner that does not create new chemical entities. For example, lipid nanoparticles can comprise poloxamer-coated PLGA nanoparticles without forming new chemical entities that are still able to rapidly penetrate human mucus (Yang et al., Angew. Chem. Int. Ed. 2011 50: 2597-2600; the contents of which are incorporated herein by reference in their entirety). Xu et al. describe a non-limiting, scalable method for producing nanoparticles that can penetrate human mucus (see, e.g., J Control Release 2013, 170(2): 279-86; the contents of which are incorporated herein by reference in their entirety).

[0243] The vitamin of the polymer-vitamin conjugate can be vitamin E. The vitamin portion of the conjugate can be substituted with other suitable components, such as, but not limited to, vitamin A, vitamin E, other vitamins, cholesterol, hydrophobic moieties, or hydrophobic components of other surfactants (e.g., sterol chains, fatty acids, hydrocarbon chains, and alkylene oxide chains).

[0244] Lipid nanoparticles engineered to penetrate mucus can include surface-altering agents such as, but not limited to, polynucleotides, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as, for example, dimethyldioctadecyl ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrins), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamer), mucolytics (e.g., N-acetylcysteine, artemisia, bromelain, papain, clerodendrum, acetylcysteine, bromhexine, carbocysteine, eprazone, mesna, ambroxol, sobrexol, domiol, letosteine, sitoronine, tiopronin, gelsolin, thymosin β4, dornase alfa, netexin, erdosteine), and various DNA enzymes including rhDNase. The surface-altering agent can be embedded or trapped in the surface of the particle, or placed (e.g., by coating, adsorption, covalent bonding, or other processes) on the surface of the lipid nanoparticle. (See, e.g., U.S. Publication No. 20100215580 and U.S. Publication Nos. 20080166414 and US20130164343; the contents of each of which are herein incorporated by reference in their entirety).

[0245] In some embodiments, the mucus-penetrating lipid nanoparticles may comprise at least one polynucleotide described herein. The polynucleotide may be encapsulated within the lipid nanoparticle and / or disposed on the surface of the particle. The polynucleotide may be covalently coupled to the lipid nanoparticle. The formulation of the mucus-penetrating lipid nanoparticles may comprise a plurality of nanoparticles.

[0246] In some embodiments, the composition can be formulated as solid lipid nanoparticles. Solid lipid nanoparticles (SLN) can be spherical with an average diameter between 10 and 1000 nm. SLN has a solid lipid core matrix that can dissolve lipophilic molecules and can be stabilized with surfactants and / or emulsifiers. In some embodiments, lipid nanoparticles can be self-assembling lipid-polymer nanoparticles (see Zhang et al., ACS Nano, 2008, 2 (8), pp 1696-1702; the content of this document is incorporated herein by reference in its entirety). As a non-limiting example, SLN can be the SLN described in International Patent Publication No. WO2013105101, the content of which is incorporated herein by reference in its entirety. As another non-limiting example, SLN can be manufactured by the method or process described in International Patent Publication No. WO2013105101, the content of which is incorporated herein by reference in its entirety.

[0247] In some embodiments, the compositions of the present disclosure may be encapsulated in nanoparticles. Nanoparticles may be prepared by methods described herein and known in the art, such as, but not limited to, International Publication Nos. WO2010005740, WO2010030763, WO2010005721, WO2010005723, WO2012054923, U.S. Publication Nos. US20110262491, US20100104645, US20100087337, US20100068285, US20110274759, US2010 Nos. 0068286, 0068286, 20120288541, 20130123351, and 20130230567, and U.S. Patent Nos. 8,206,747, 8,293,276, 8,318,208, and 8,318,211; the contents of each of which are incorporated herein by reference in their entirety. In some embodiments, polymer nanoparticles can be identified by the methods described in U.S. Publication No. US20120140790, the contents of which are incorporated herein by reference in their entirety.

[0248] In certain embodiments, nanoparticles can be formulated for sustained release. As used herein, "slow release" refers to a pharmaceutical composition or compound that complies with a release rate within a specific time period. This time period may include, but is not limited to, hours, days, weeks, months, and years. As a non-limiting example, sustained-release nanoparticles may include polymers and nucleic acid sequences disclosed herein (see International Publication No. 2010075072 and U.S. Publication No. US20100216804, No. US20110217377, and No. US20120201859, the contents of each of which are incorporated herein by reference in their entirety). In another non-limiting example, sustained-release formulations may include medicaments that allow sustained bioavailability, such as, but not limited to, crystals, macromolecular gels, and / or particle suspensions (see U.S. Patent Publication No. US20130150295, the contents of each of which are incorporated herein by reference in their entirety).

[0249] In some embodiments, the nanoparticles of the present disclosure can comprise a polymer matrix. As non-limiting examples, the nanoparticles can comprise two or more polymers, such as, but not limited to, polyethylene, polycarbonate, polyanhydride, polyhydroxy acid, polypropylene fumarate, polycaprolactone, polyamide, polyacetal, polyether, polyester, poly(orthoester), polycyanoacrylate, polyvinyl alcohol, polyurethane, polyphosphazene, polyacrylate, polymethacrylate, polycyanoacrylate, polyurea, polystyrene, polyamine, polylysine, poly(ethyleneimine), poly(serine ester), poly(L-lactide-to-L-lysine), poly(4-hydroxy-L-proline ester), or a combination thereof.

[0250] In some embodiments, the nanoparticles include diblock copolymers. In some embodiments, the diblock copolymers can include a combination of PEG and a polymer, such as, but not limited to, polyethylene, polycarbonate, polyanhydride, polyhydroxy acid, polypropylene fumarate, polycaprolactone, polyamide, polyacetal, polyether, polyester, poly(orthoester), polycyanoacrylate, polyvinyl alcohol, polyurethane, polyphosphazene, polyacrylate, polymethacrylate, polycyanoacrylate, polyurea, polystyrene, polyamine, polylysine, poly(ethyleneimine), poly(serine ester), poly(L-lactide-to-L-lysine), poly(4-hydroxy-L-proline ester), or a combination thereof. In yet another embodiment, the diblock copolymer can be a high x diblock copolymer, such as those described in International Patent Publication No. WO2013120052, the contents of which are incorporated herein by reference in their entirety.

[0251] As a non-limiting example, the nanoparticles comprise PLGA-PEG block copolymers (see U.S. Publication No. US20120004293 and U.S. Patent No. 8,236,330, each of which is incorporated herein by reference in its entirety). In another non-limiting example, the therapeutic nanoparticles are stealth nanoparticles comprising diblock copolymers of PEG and PLA or PEG and PLGA (see U.S. Patent No. 8,246,968 and International Publication No. WO2012166923, the contents of each of which are incorporated herein by reference in their entirety). In yet another non-limiting example, the nanoparticles are stealth nanoparticles or target-specific stealth nanoparticles, as described in U.S. Patent Publication No. US20130172406, the contents of which are incorporated herein by reference in their entirety.

[0252] In some embodiments, the nanoparticles may comprise multi-block copolymers (see, for example, U.S. Patent Nos. 8,263,665 and 8,287,910, and U.S. Patent Publication No. US20130195987, the contents of each of which are herein incorporated by reference in their entirety).

[0253] In yet another non-limiting example, the lipid nanoparticle comprises a block copolymer PEG-PLGA-PEG (see, e.g., Lee et al. Thermosensitive Hydrogel as a Tgf-β1 Gene Delivery Vehicle Enhances Diabetic Wound Healing. Pharmaceutical Research, 2003 20(12):1995-2000 Use of thermosensitive hydrogel (PEG-PLGA-PEG) as a TGF-β1 gene delivery vehicle; Li et al. Controlled Gene Delivery System Based on Thermosensitive Biodegradable Hydrogel. Pharmaceutical Research 2003 20(6):884-888 as a controlled gene delivery system; and Chang et al. Non-ionic amphiphilic biodegradable PEG-PLGA-PEG copolymer enhances gene delivery efficiency in rat skeletal muscle. J Controlled Release. 2007 118:245-253, the contents of each of which are herein incorporated by reference in their entirety). The nucleic acid (e.g., mRNA) vaccines of the present disclosure can be formulated into lipid nanoparticles comprising PEG-PLGA-PEG block copolymers.

[0254] In some embodiments, the nanoparticles may comprise multi-block copolymers (see, for example, U.S. Patent Nos. 8,263,665 and 8,287,910, and U.S. Patent Publication No. US20130195987, the contents of each of which are herein incorporated by reference in their entirety).

[0255] In some embodiments, the block copolymers described herein can be contained in a polyionic complex comprising a non-polymeric micelle and a block copolymer (see, for example, U.S. Publication No. 20120076836, the contents of which are incorporated herein by reference in their entirety).

[0256] In some embodiments, the nanoparticles may comprise at least one acrylic polymer. Acrylic polymers include, but are not limited to, acrylic acid, methacrylic acid, acrylic acid and methacrylic acid copolymers, methyl methacrylate copolymers, ethoxyethyl methacrylate, cyanoethyl methacrylate, aminoalkyl methacrylate copolymers, poly(acrylic acid), poly(methacrylic acid), polycyanoacrylates, and combinations thereof.

[0257] In some embodiments, the nanoparticle can include at least one poly (vinyl ester) polymer. The poly (vinyl ester) polymer can be a copolymer, such as a random copolymer. As a non-limiting example, the random copolymer can have a structure such as those described in International Application No. WO2013032829 or U.S. Patent Publication No. US20130121954, the contents of each of which are incorporated herein by reference in their entirety. In some embodiments, the poly (vinyl ester) polymer can be conjugated to a polynucleotide as described herein.

[0258] In some embodiments, the nanoparticles may comprise at least one diblock copolymer. The diblock copolymer may be, but is not limited to, a poly(lactic acid)-poly(ethylene glycol) copolymer (see, for example, International Patent Publication No. WO2013044219, the contents of which are incorporated herein by reference in their entirety). As a non-limiting example, the nanoparticles may be used to treat cancer (see International Patent Publication No. WO2013044219, the contents of which are incorporated herein by reference in their entirety).

[0259] In some embodiments, the nanoparticles may include at least one cationic polymer described herein and / or known in the art.

[0260] In some embodiments, the nanoparticles may include at least one amine-containing polymer, such as, but not limited to, polylysine, polyethyleneimine, poly(amidoamine) dendrimers, poly(β-amino esters) (see, e.g., U.S. Pat. No. 8,287,849, the contents of which are incorporated herein by reference in their entirety), and combinations thereof.

[0261] In some embodiments, the nanoparticles may comprise at least one degradable polyester, which may contain polycationic side chains. Degradable polyesters include, but are not limited to, poly(serine esters), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline esters), and combinations thereof. In some embodiments, the degradable polyesters may be conjugated with PEG to form PEGylated polymers.

[0262] In some embodiments, the composition may be formulated in a colloidal nanocarrier as described in U.S. Patent Publication No. US20130197100, the contents of which are incorporated herein by reference in their entirety.

[0263] In some embodiments, nanoparticles can be optimized for oral administration. Nanoparticles can comprise at least one cationic biopolymer, such as, but not limited to, chitosan or a derivative thereof. By way of non-limiting example, nanoparticles can be formulated by the methods described in U.S. Publication No. 20120282343, the contents of which are incorporated by reference in their entirety herein.

[0264] In some embodiments, LNP comprises lipid KL52 (an amino-lipid disclosed in U.S. Application Publication No. 2012 / 0295832, the contents of which are incorporated by reference in their entirety herein). By incorporating such lipids, the activity and / or safety of LNP administration can be improved (e.g., as measured by one or more of ALT / AST, white blood cell count, and cytokine induction). LNP comprising KL52 can be administered intravenously and / or in one or more doses. In some embodiments, administration of LNP comprising KL52 results in the same or improved mRNA and / or protein expression compared to LNP comprising MC3.

[0265] In some embodiments, the lipid nanoparticles described herein can be limit-size lipid nanoparticles described in International Patent Publication No. WO2013059922, the contents of which are incorporated by reference in their entirety herein. Limit-size lipid nanoparticles can comprise a lipid bilayer encapsulating an aqueous core or a hydrophobic core; wherein the lipid bilayer can comprise a phospholipid, such as, but not limited to, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, sphingomyelin, cerebrosides, C8-C20 fatty acid diacylphosphatidylcholine, and l-palmitoyl-2-oleoyl phosphatidylcholine (POPC). In some embodiments, limit-size lipid nanoparticles can comprise a polyethylene glycol-lipid, such as, but not limited to, DLPE-PEG, DMPE-PEG, DPPC-PEG, and DSPE-PEG.

[0266] In some embodiments, compositions can be formulated into nanoparticles comprising an inner core comprising non-cellular material and an outer surface comprising a cell membrane. The cell membrane can be derived from a cell or a membrane derived from a virus. By way of non-limiting example, nanoparticles can be manufactured by the methods described in International Patent Publication No. WO2013052167, the contents of which are incorporated by reference in their entirety herein. By way of another non-limiting example, nanoparticles described in International Patent Publication No. WO2013052167, the contents of which are incorporated by reference in their entirety herein, can be used to deliver the compositions described herein.

[0267] In some embodiments, the composition can be formulated as a porous nanoparticle-supported lipid bilayer (protocell). Protocells are described in International Patent Publication No. WO2013056132, the contents of which are incorporated herein by reference in their entirety.

[0268] In some embodiments, the compositions described herein can be formulated into polymer nanoparticles as described in U.S. Patent Nos. 8,420,123 and 8,518,963 and European Patent No. EP2073848B1 or manufactured by the methods described therein, the contents of each of which are incorporated herein by reference in their entirety. As a non-limiting example, the polymer nanoparticles can have a high glass transition temperature, such as the nanoparticles described in U.S. Patent No. 8,518,963 or nanoparticles manufactured by the methods described therein, the contents of which are incorporated herein by reference in their entirety. As another non-limiting example, polymer nanoparticles for oral and parenteral formulations can be manufactured by the methods described in European Patent No. EP2073848B1, the contents of which are incorporated herein by reference in their entirety.

[0269] In some embodiments, the compositions described herein can be formulated into nanoparticles for imaging. The nanoparticles can be liposome nanoparticles, such as those described in U.S. Patent Publication No. US20130129636, the contents of which are incorporated herein by reference in their entirety. As a non-limiting example, liposomes can include gadolinium (III) 2-{4,7-bis-carboxymethyl-10-[(N,N-distearoylamidomethyl-N′-amido-methyl]-1,4,7,10-tetraazacyclododecan-1-yl}-acetic acid and a neutral, fully saturated phospholipid component (see, for example, U.S. Patent Publication No. US20130129636, the contents of which are incorporated herein by reference in their entirety).

[0270] In some embodiments, nanoparticles useful in the present disclosure may be formed by the methods described in U.S. Patent Application No. US20130130348, the contents of which are incorporated herein by reference in their entirety.

[0271] In some embodiments, the compositions of the present disclosure can be formulated as expandable nanoparticles. The expandable nanoparticles can be, but are not limited to, those described in U.S. Patent No. 8,440,231, the contents of which are incorporated herein by reference in their entirety.

[0272] The compositions of the present disclosure may be formulated as polyanhydride nanoparticles such as, but not limited to, those described in US Patent No. 8,449,916, the contents of which are incorporated herein by reference in their entirety.

[0273] Nanoparticles and microparticles of the present disclosure can be geometrically engineered to regulate macrophages and / or immune responses. In certain embodiments, the particles of geometric engineering can have different shapes, sizes and / or surface charges to incorporate the polynucleotides of the present disclosure for targeted delivery. Other physical features that the particles of geometric engineering can have include, but are not limited to, perforations, tilted arms, asymmetry and surface roughness, and charges that can change interactions with cells and tissues. As a non-limiting example, nanoparticles of the present disclosure can be manufactured by the method described in International Publication No. WO2013082111, the contents of which are incorporated herein by reference in their entirety.

[0274] In some embodiments, the nanoparticles of the present disclosure can be water-soluble nanoparticles, such as, but not limited to, those described in International Publication No. WO2013090601, the contents of which are incorporated herein by reference in their entirety. The nanoparticles can be inorganic nanoparticles with dense zwitterionic ligands to exhibit good water solubility. The nanoparticles can also have a smaller hydrodynamic diameter (HD), stability to time, pH, and salinity, and low levels of nonspecific protein binding.

[0275] In some embodiments, the nanoparticles of the present disclosure may be developed by the methods described in U.S. Patent Publication No. US20130172406, the contents of which are incorporated herein by reference in their entirety.

[0276] In some embodiments, the nanoparticles of the present disclosure are stealth nanoparticles or target-specific stealth nanoparticles, such as, but not limited to, those described in U.S. Patent Publication No. US20130172406, the contents of which are incorporated herein by reference in their entirety. The nanoparticles of the present disclosure can be made by the methods described in U.S. Patent Publication No. US20130172406, the contents of which are incorporated herein by reference in their entirety.

[0277] In some embodiments, the stealth nanoparticles or target-specific stealth nanoparticles can comprise a polymer matrix. The polymer matrix can comprise two or more polymers, such as, but not limited to, polyethylene, polycarbonate, polyanhydride, polyhydroxy acid, polypropylene fumarate, polycaprolactone, polyamide, polyacetal, polyether, polyester, poly(orthoester), polycyanoacrylate, polyvinyl alcohol, polyurethane, polyphosphazene, polyacrylate, polymethacrylate, polycyanoacrylate, polyurea, polystyrene, polyamine, polyester, polyanhydride, polyether, polyurethane, polymethacrylate, polyacrylate, polycyanoacrylate, or a combination thereof.

[0278] In some embodiments, the nanoparticle can be a nanoparticle-nucleic acid hybrid structure having a high-density nucleic acid layer. As a non-limiting example, the nanoparticle-nucleic acid hybrid structure can be manufactured by the method described in U.S. Patent Publication No. US20130171646, the contents of which are incorporated herein by reference in their entirety. The nanoparticle can contain nucleic acids, such as, but not limited to, polynucleotides described herein and / or known in the art.

[0279] At least one of the nanoparticles of the present disclosure can be embedded in the core of the nanostructure or coated with a low-density porous 3-D structure or coating that can carry or bind at least one payload inside or on the surface of the nanostructure. Non-limiting examples of nanostructures comprising at least one nanoparticle are described in International Patent Publication No. WO2013123523, the contents of which are incorporated herein by reference in their entirety.

[0280] In some embodiments, smaller LNPs can be used to deliver the composition. Such particles can include particles less than 0.1 μm up to 100 nm, such as, but not limited to, less than 0.1 μm, less than 1.0 μm, less than 5 μm, less than 10 μm, less than 15 μm, less than 20 μm, less than 25 μm, less than 30 μm, less than 35 μm, less than 40 μm, less than 50 μm, less than 55 μm, less than 60 μm, less than 65 μm, less than 70 μm, less than 75 μm, less than 80 μm, less than 85 μm, less than 90 μm, less than 95 μm, less than 100 μm, less than 125 μm, less than 150 μm, less than 175 μm, less than 200 μm, less than 225 μm, less than 250 μm, less than 275 μm, less than 300 μm, less than 350 μm, less than 400 μm, less than 500 μm, less than 550 μm, less than 600 μm, less than 65 μm, less than 700 μm, less than 750 μm, less than 800 μm, less than 85 μm, less than 900 μm, less than 95 μm, less than 100 μm, less than 125 μm, less than 150 μm, less than 175 μm, less than 200 μm, less than 225 μm, less than 250 μm, less than 275 μm, less than 300 μm, less than 350 μm, less than 350 μm, less than 350 μm, less than 350 μm, less than less than 300 μm, less than 325 μm, less than 350 μm, less than 375 μm, less than 400 μm, less than 425 μm, less than 450 μm, less than 475 μm, less than 500 μm, less than 525 μm, less than 550 μm, less than 575 μm, less than 600 μm, less than 625 μm, less than 650 μm, less than 675 μm, less than 700 μm, less than 725 μm, less than 750 μm, less than 775 μm, less than 800 μm, less than 825 μm, less than 850 μm, less than 875 μm, less than 900 μm, less than 925 μm, less than 950 μm, less than 975 μm or less than 1000 μm in diameter.

[0281] In some embodiments, smaller LNP delivery compositions may be used, which may include 1 nm to 100 nm, 1 nm to 10 nm, 1 nm to 20 nm, 1 nm to 30 nm, 1 nm to 40 nm, 1 nm to 50 nm, 1 nm to 60 nm, 1 nm to 70 nm, 1 nm to 80 nm, 1 nm to 90 nm, 5 nm to 100 nm, 5 nm to 10 nm, 5 nm to 20 nm, 5 nm to 30 nm, 5 nm to 40 nm, 5 nm to 50 nm, 5 nm to 60 nm, 5 nm to 70 nm, 5 nm to 80 nm, 5 nm to 90 nm, 10 nm to 50 nm, 20 nm or a diameter of 50 nm, 30 to 50 nm, 40 to 50 nm, 20 to 60 nm, 30 to 60 nm, 40 to 60 nm, 20 to 70 nm, 30 to 70 nm, 40 to 70 nm, 50 to 70 nm, 60 to 70 nm, 20 to 80 nm, 30 to 80 nm, 40 to 80 nm, 50 to 80 nm, 60 to 80 nm, 20 to 90 nm, 30 to 90 nm, 40 to 90 nm, 50 to 90 nm, 60 to 90 nm, and / or 70 to 90 nm.

[0282] In some embodiments, such LNPs are synthesized using methods comprising a microfluidic mixer. Examples of microfluidic mixers can include, but are not limited to, slit interdigital micromixers, including but not limited to those manufactured by Microinnova (Allerheiligen bei Wildon, Austria) and / or staggered herringbone micromixers (SHM) (Zhigaltsev, IV et al., Bottom-up design and synthesis of limit size lipid nanoparticle systems with aqueous and triglyceride cores using millisecond microfluidic mixing. Langmuir. 2012. 28: 3633-40; Belliveau, NM et al., Microfluidic synthesis of highly potent limit-size lipid nanoparticles for in vivo delivery of siRNA. Molecular Therapy-Nucleic Acids. 2012. 1: e37; Chen, D. et al., Rapid discovery of potent siRNA-containing lipid nanoparticles enabled by controlled microfluidic formulation. J Am Chem Soc. 2012.134(16):6948-51, the contents of each of which are incorporated herein by reference in their entirety). In some embodiments, the LNP generation method comprising SHM further comprises mixing at least two input streams, wherein the mixing occurs by microstructure-induced chaotic convection (MICA). According to the method, the fluid stream flows through a channel in a herringbone pattern, thereby generating a rotational flow and causing the fluids to fold over each other. The method may also include a surface for fluid mixing, wherein the surface changes direction during fluid circulation. Methods for generating LNPs using SHM include those disclosed in U.S. Application Publication Nos. 2004 / 0262223 and 2012 / 0276209, the contents of each of which are incorporated herein by reference in their entirety.

[0283] In some embodiments, the compositions of the present disclosure can be formulated into lipid nanoparticles produced using a micromixer such as, but not limited to, a Notched Interdigital Micromixer (SIMM-V2) or a Standard Notched Interdigital Micromixer (SSIMM) or a Caterpillar (CPMM) or Impact Jet (IJMM) from the Institut für Mikrotechnik Mainz GmbH, Mainz, Germany.

[0284] In some embodiments, the compositions of the present disclosure can be formulated into lipid nanoparticles produced using microfluidics technology (see, e.g., Whitesides, George M. The Origins and the Future of Microfluidics. Nature, 2006 442: 368-373; and Abraham et al. Chaotic Mixer for Microchannels. Science, 2002 295: 647-651; each of which is incorporated herein by reference in its entirety). As a non-limiting example, controlled microfluidic formulations include passive methods for mixing stable pressure-driven flows in microchannels at low Reynolds numbers (see, e.g., Abraham et al. Chaotic Mixer for Microchannels. Science, 2002 295: 647-651, the contents of which are incorporated herein by reference in their entirety).

[0285] In some embodiments, the compositions of the present disclosure can be formulated into lipid nanoparticles produced using micromixer chips, such as, but not limited to, those from Harvard Apparatus (Holliston, Mass.) or Dolomite Microfluidics (Royston, UK). Micromixer chips can be used to rapidly mix two or more fluid streams through a split and recombinant mechanism.

[0286] In some embodiments, the compositions of the present disclosure may be formulated to have a diameter of 10 to 100 nm, such as, but not limited to, 10 to 20 nm, 10 to 30 nm, 10 to 40 nm, 10 to 50 nm, 10 to 60 nm, 10 to 70 nm, 10 to 80 nm, 10 to 90 nm, 20 to 30 nm, 20 to 40 nm, 20 to 50 nm, 20 to 60 nm, 20 to 70 nm, 20 to 80 nm, 20 to 90 nm, 20 to 100 nm, 30 to 40 nm, 30 to 50 nm, 30 to 60 nm, 30 to 70 nm, 30 to 80 nm, In some embodiments, the present invention relates to lipid nanoparticles having a diameter of 30 to 90 nm, 30 to 100 nm, 40 to 50 nm, 40 to 60 nm, 40 to 70 nm, 40 to 80 nm, about 40 to about 90 nm, about 40 to 100 nm, 50 to 60 nm, 50 to 70 nm, 50 to 80 nm, 50 to 90 nm, 50 to 100 nm, 60 to 70 nm, 60 to 80 nm, 60 to 90 nm, 60 to 100 nm, 70 to 80 nm, 70 to 90 nm, 70 to 100 nm, 80 to 90 nm, 80 to 100 nm, and / or 90 to 100 nm.

[0287] In some embodiments, the lipid nanoparticles may have a diameter of 10 to 500 nm.

[0288] In some embodiments, the lipid nanoparticles may have a diameter greater than 100 nm, greater than 150 nm, greater than 200 nm, greater than 250 nm, greater than 300 nm, greater than 350 nm, greater than 400 nm, greater than 450 nm, greater than 500 nm, greater than 550 nm, greater than 600 nm, greater than 650 nm, greater than 700 nm, greater than 750 nm, greater than 800 nm, greater than 850 nm, greater than 900 nm, greater than 950 nm, or greater than 1000 nm.

[0289] Also described herein are compositions comprising effective doses of nanoparticles as herein described and pharmaceutically acceptable carriers. In some respects, pharmaceutical compositions can include pharmaceutically acceptable carriers and nanoparticles, which include single-stranded trimer nucleic acids (e.g., mRNA) encoding the first T cell epitope, β2-microglobulin, and the first MHC heavy chain sequence encapsulated in the nanoparticles. In some respects, pharmaceutical compositions can include pharmaceutically acceptable carriers and lipid nanoparticles, which include single-stranded trimer nucleic acids (e.g., mRNA) encoding the first T cell epitope, β2-microglobulin, and the first MHC heavy chain sequence encapsulated in the lipid nanoparticles.

[0290] Also described herein are compositions comprising effective doses of nano-particles as herein described and pharmaceutically acceptable carriers. In some respects, pharmaceutical compositions can include pharmaceutically acceptable carriers and nano-particles, which comprise single-stranded nucleic acid dimers (e.g., mRNA) that are encapsulated in the coding T cell epitopes and one-level MHC heavy chain sequences in the nano-particles. In some respects, pharmaceutical compositions can include pharmaceutically acceptable carriers and lipid nanoparticles, which comprise single-stranded nucleic acid dimers (e.g., mRNA) that are encapsulated in the coding T cell epitopes and one-level MHC heavy chain sequences in the lipid nanoparticles.

[0291] Liposomes, lipoplexes or lipid nanoparticles can be used to improve the efficacy of polynucleotide-directed protein production because these formulations can be able to increase cell transfection of RNA (e.g., mRNA) polynucleotides; and / or increase translation of encoded proteins. One such example involves the use of lipid encapsulation to achieve efficient systemic delivery of polyplex plasmid DNA (Heyes et al., Mol Ther. 2007 15: 713-720; the contents of which are incorporated herein by reference in their entirety). Liposomes, lipoplexes or lipid nanoparticles can also be used to increase the stability of polynucleotides.

[0292] Liposomes are artificially prepared vesicles that are primarily composed of a lipid bilayer and can be used as delivery vehicles for nutrients and drug formulations. Liposomes can be of different sizes, such as, but not limited to, multilamellar vesicles (MLVs), which can be hundreds of nanometers in diameter and can contain a series of concentric bilayers separated by narrow aqueous compartments; small unicellular vesicles (SUVs), which can be less than 50 nm in diameter; and large unilamellar vesicles (LUVs), which can be between 50 and 500 nm in diameter. Liposome designs can include, but are not limited to, opsonins or ligands to improve the attachment of liposomes to unhealthy tissues or to activate events such as, but not limited to, endocytosis. Liposomes can have a low or high pH to improve the delivery of drug formulations.

[0293] The formation of liposomes may depend on physicochemical properties such as, but not limited to, the entrapped drug formulation and liposome components, the nature of the medium in which the lipid vesicles are dispersed, the effective concentration of the entrapped substance and its potential toxicity, any additional processing involved during vesicle application and / or delivery, the optimal size, polydispersity and shelf life of the vesicles for the intended use, as well as batch-to-batch reproducibility and the possibility of large-scale production of safe and effective liposome products.

[0294] In some embodiments, the pharmaceutical compositions described herein may include, but are not limited to, liposomes such as those formed from 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA) liposomes, DiLa2 liposomes from Marina Biotech (Bothell, WA), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), and MC3 (US20100324120; incorporated herein by reference in its entirety), as well as small molecule drug deliverables such as, but not limited to, liposomes from Janssen Biotech, Inc. (Horsham, Pa.). ) liposomes.

[0295] In some embodiments, the pharmaceutical compositions described herein may include, but are not limited to, liposomes, such as those formed by the synthesis of stable plasmid-lipid particles (SPLPs) or stable nucleic acid lipid particles (SNALPs), which have been previously described and shown to be suitable for oligonucleotide delivery in vitro and in vivo (see Wheeler et al. Gene Therapy. 1999 6:271-281; ​​Zhang et al. Gene Therapy. 1999 6:1438-1447; Jeffs et al. Pharm Res. 2005 22:362-372; Morrissey et al. Nat Biotechnol. 2005 2:1002-1007; Zimmermann et al. Nature. 2006 441:111-114; Heyes et al. J Contr Rel. 2005 107:276-287; Semple et al. Nature Biotech. 2010). (Wheeler et al., 2001; 28:172-176; Judge et al., J Clin Invest. 2009 119:661-673; deFougerolles Hum Gene Ther. 2008 19:125-132; U.S. Patent Publication No. US20130122104; all of which are incorporated herein in their entirety). The original manufacturing method by Wheeler et al. was a detergent dialysis method, which was later improved by Jeffs et al. and referred to as the spontaneous vesicle formation method. Liposome formulations are composed of three to four lipid components in addition to the polynucleotide. As an example, liposomes can contain, but are not limited to, 55% cholesterol, 20% distearoylphosphatidylcholine (DSPC), 10% PEG-S-DSG, and 15% 1,2-dioleyl-N,N-dimethylaminopropane (DODMA), as described by Jeffs et al. As another example, certain liposome formulations may contain, but are not limited to, 48% cholesterol, 20% DSPC, 2% PEG-c-DMA, and 30% cationic lipid, where the cationic lipid may be 1,2-distearyloxy-N,N-dimethylaminopropane (DSDMA), DODMA, DLin-DMA, or 1,2-dilinolenyl-3-dimethylaminopropane (DLenDMA), as described by Heyes et al.

[0296] In some embodiments, the liposome formulations can comprise from about 25.0% cholesterol to about 40.0% cholesterol, from about 30.0% cholesterol to about 45.0% cholesterol, from about 35.0% cholesterol to about 50.0% cholesterol, and / or from about 48.5% cholesterol to about 60% cholesterol. In some embodiments, the formulations can comprise a cholesterol percentage selected from the group consisting of 28.5%, 31.5%, 33.5%, 36.5%, 37.0%, 38.5%, 39.0%, and 43.5%. In some embodiments, the formulations can comprise from about 5.0% to about 10.0% DSPC and / or from about 7.0% to about 15.0% DSPC.

[0297] In some embodiments, the composition can be formulated into liposomes, such as, but not limited to, DiLa2 liposomes (Marina Biotech, Bothell, Wash.), (Marina Biotech, Bothell, Wash.), neutral DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine)-based liposomes (e.g., for siRNA delivery in ovarian cancer (Landen et al. Cancer Biology & Therapy 2006 5(12)1708-1713); incorporated herein by reference in its entirety), and hyaluronic acid-coated liposomes (Quiet Therapeutics, Israel).

[0298] In some embodiments, the composition can be formulated as lipid vesicles, which can have crosslinks between functionalized lipid bilayers.

[0299] In some embodiments, the composition can be formulated in a lipid-polycation complex. The formation of the lipid-polycation complex can be accomplished by methods known in the art and / or as described in U.S. Publication No. 20120178702, which is incorporated herein by reference in its entirety. As non-limiting examples, the polycation can include a cationic peptide or polypeptide such as, but not limited to, polylysine, polyornithine, and / or polyarginine. In some embodiments, the composition can be formulated in a lipid-polycation complex, which can further include a non-cationic lipid such as, but not limited to, cholesterol or dioleoylphosphatidylethanolamine (DOPE).

[0300] In some embodiments, the composition may be formulated as a liposome complex, such as, but not limited to, ATUPLEX® from Silence Therapeutics (London, United Kingdom). TM System, DACC system, DBTC system and other siRNA-liposome complex technologies, from STEMFECT (Cambridge, Mass.) TM , and targeted and nontargeted delivery of nucleic acids based on polyethyleneimine (PEI) or protamine (Aleku et al. Cancer Res. 2008 68:9788-9798; Strumberg et al. Int J Clin Pharmacol Ther 2012 50:76-78; Santel et al. Gene Ther 2006 13:1222-1234; Santel et al. Gene Ther 2006 13:1360-1370; Gutbier et al. Pulm Pharmacol. Ther. 2010 23:334-344; Kaufmann et al. Microvasc Res 2010 80:286-293; Weide et al. J Immunother. 2009 32:498-507; Weide et al. J Immunother. 2008 31:180-188; Pascolo Expert Opin. Biol. Ther. 4: 1285-1294; Fotin-Mleczek et al., 2011 J. Immunother. 34: 1-15; Song et al., Nature Biotechnol. 2005, 23: 709-717; Peer et al., Proc Natl Acad Sci USA. 2007 6; 104: 4095-4100; deFougerolles Hum Gene Ther. 2008 19: 125-132, the contents of each of which are herein incorporated by reference in their entirety).

[0301] In some embodiments, such formulations can also be constructed or the compositions modified so that they are passively or actively targeted to different types of cells in the body, including but not limited to hepatocytes, immune cells, tumor cells, endothelial cells, antigen presenting cells, and leukocytes (Akinc et al. Mol Ther. 2010 18:1357-1364; Song et al., Nat Biotechnol. 2005 23:709-717; Judge et al., J Clin Invest. 2009 119:661-673; Kaufmann et al., Microvasc Res 2010 80:286-293; Santel et al., Gene Ther 2006 13:1222-1234; Santel et al., Gene Ther 2006 13:1360-1370; Gutbier et al., Pulm Pharmacol. Ther. 2010 23:334-344; Basha et al., Mol. Ther. 2011 19:2186-2200; Fenske and Cullis, Expert Opin Drug Deliv. 2008 5:25-44; Peer et al., Science. 2008 319:627-630; Peer and Lieberman, Gene Ther. 2011 18:1127-1133, the contents of each of which are incorporated herein by reference in their entirety). One example of passive targeting of formulations to hepatocytes includes lipid nanoparticle formulations based on DLin-DMA, DLin-KC2-DMA, and DLin-MC3-DMA, which have been shown to bind to apolipoprotein E and promote binding and uptake of these formulations to hepatocytes in vivo (Akinc et al., Mol Ther. 2010 18:1357-1364, the contents of which are incorporated herein by reference in their entirety).The formulations can also be selectively targeted by expressing different ligands on their surface, as exemplified by, but not limited to, folic acid, transferrin, N-acetylgalactosamine (GalNAc), and antibody targeting approaches (Kolhatkar et al., Curr Drug Discov Technol. 2011 8:197-206; Musacchio and Torchilin, Front Biosci. 2011 16:1388-1412; Yu et al., Mol Membr Biol. 2010 27:286-298; Patil et al., Crit Rev Ther Drug Carrier Syst. 2008 25:1-61; Benoit et al., Biomacromolecules. 2011 12:2708-2714; Zhao et al., Expert Opin Drug Deliv. 2008 5:309-319; Akinc et al., Mol Ther. 2010 18:1357-1364; Srinivasan et al., Methods Mol Biol. 2012 820:105-116; Ben-Arie et al., Methods Mol Biol. 2012 757:497-507; Peer 2010 J Control Release. 20:63-68; Peer et al., Proc Natl Acad Sci USA. 2007 104:4095-4100; Kim et al., Methods Mol Biol. 2011 721:339-353; Subramanya et al., Mol Ther. 2010 18:2028-2037; Song et al., Nat Biotechnol. 2005 23:709-717; Peer et al., Science. 2008 319:627-630; Peer and Lieberman, Gene Ther. 2011 18:1127-1133, the contents of each of which are herein incorporated by reference in their entirety).

[0302] In some embodiments, the compositions of the present disclosure can be formulated for controlled release and / or targeted delivery. As used herein, "controlled release" refers to a pharmaceutical composition or compound release profile that adheres to a specific release pattern to achieve a therapeutic outcome. In some embodiments, the composition can be encapsulated into a delivery agent described herein and / or known in the art for controlled release and / or targeted delivery. As used herein, the term "encapsulate" means to enclose, surround, or wrap around. When it comes to formulations of compounds of the present disclosure, encapsulation can be substantial, complete, or partial. The term "substantially encapsulate" means that at least greater than 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.9% or greater than 99.999% of the pharmaceutical composition or compound of the present disclosure can be enclosed, surrounded, or wrapped in a delivery agent. "Partially encapsulated" means that less than 10, 10, 20, 30, 40, 50 or less of the pharmaceutical composition or compound of the present disclosure can be enclosed, surrounded or wrapped in the delivery agent. Advantageously, encapsulation can be determined by measuring the escape or activity of the pharmaceutical composition or compound of the present disclosure using fluorescence and / or electron micrographs. For example, at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or more of the pharmaceutical composition or compound of the present disclosure is encapsulated in the delivery agent.

[0303] In some embodiments, the controlled release formulation may include, but is not limited to, triblock copolymers. As a non-limiting example, the formulation may include two different types of triblock copolymers (U.S. Pat. Nos. 9,901,554 and 9,795,679, the contents of each of which are incorporated herein by reference in their entirety).

[0304] In some embodiments, the composition can be encapsulated into lipid nanoparticles or rapidly eliminated lipid nanoparticles, and the lipid nanoparticles or rapidly eliminated lipid nanoparticles are encapsulated into polymers, hydrogels and / or surgical sealants described herein and / or known in the art. As non-limiting examples, the polymer, hydrogel or surgical sealant can be PLGA, ethylene vinyl acetate copolymer (EVAc), poloxamer, (Nanotherapeutics, Inc. Alachua, Fla.), (Halozyme Therapeutics, San Diego Calif.), surgical sealants such as fibrinogen polymers (Ethicon Inc. Cornelia, Ga.), THERACOL® (Baxter International, Inc Deerfield, III.), PEG-based sealants and THERACOL® (Baxter International, Inc Deerfield, III.).

[0305] In some embodiments, the compositions described herein formulated for controlled release and / or targeted delivery can further comprise at least one degradable polyester, which can contain a polycationic side chain. Degradable polyesters include, but are not limited to, poly(fermionic ester), poly(L-lactide-L-lysine), poly(4-hydroxy-L-proline ester), and combinations thereof. In some embodiments, the degradable polyester can comprise PEG conjugation to form a pegylated polymer.

[0306] In some embodiments, the compositions described herein formulated for controlled release and / or targeted delivery can further comprise at least one PEG and / or PEG-related polymer derivative, as described in U.S. Patent No. 8,404,222, the contents of which are incorporated by reference in their entirety herein.

[0307] In some embodiments, the compositions described herein formulated for controlled release delivery can be the controlled release polymer system described in US20130130348, the contents of which are incorporated by reference in their entirety herein.

[0308] In some embodiments, the synthetic nanocarriers can be formulated for targeted release. In some embodiments, the synthetic nanocarriers are formulated to release the polynucleotide at a specified pH and / or after a desired time interval. As a non-limiting example, the synthetic nanoparticles can be formulated to release an RNA (e.g., mRNA) vaccine after 24 hours and / or at a pH of 4.5 (see International Publication Nos. WO2010138193 and WO2010138194 and U.S. Publication Nos. US20110020388 and US20110027217, each of which is incorporated by reference in its entirety herein).

[0309] In some embodiments, the synthetic nanocarriers can be formulated for controlled release and / or sustained release of the nucleic acids described herein. As a non-limiting example, the synthetic nanocarriers for sustained release can be formulated by methods known in the art, which are described herein and / or as described in International Publication No. WO2010138192 and U.S. Publication No. 20100303850, each of which is incorporated by reference in its entirety herein.

[0310] In certain embodiments, synthetic nanocarriers can be formulated for vaccine applications. As non-limiting examples, synthetic nanocarriers can include, but are not limited to, the nanocarriers described in International Publication No. WO2011150264, No. WO2011150249, No. WO2012024621, No. WO201202629, No. WO2012024632, and U.S. Publication No. US20110293701, U.S. Publication No. US20110293723, No. US20120064110, No. US20120058153, and No. US20120058154, the contents of each of which are incorporated herein by reference in their entirety. The vaccine dosage form may be selected by the methods described herein, known in the art, and / or described in International Publication No. WO2011150258 and US Publication No. US20120027806, the contents of each of which are herein incorporated by reference in their entirety.

[0311] In some embodiments, the composition may be formulated in a colloidal nanocarrier as described in U.S. Patent Publication No. US20130197100, the contents of which are incorporated herein by reference in their entirety.

[0312] In certain embodiments, the compositions of the present disclosure can be formulated for delivery using drug-encapsulated microspheres described in International Patent Publication No. WO2013063468 or U.S. Patent No. 8,440,614, the contents of each of which are incorporated herein by reference in their entirety. The microspheres may comprise compounds of formula (I), (II), (III), (IV), (V) or (VI) as described in International Patent Publication No. WO2013063468, the contents of which are incorporated herein by reference in their entirety. In certain embodiments, amino acids, peptides, polypeptides, lipids (APPL) can be used to deliver RNA (e.g., mRNA) polynucleotides of the present disclosure to cells (see International Patent Publication No. WO2013063468, the contents of which are incorporated herein by reference in their entirety).

[0313] In some embodiments, the composition can be delivered, positioned, and / or concentrated to a specific location using the delivery methods described in International Patent Publication No. WO2013063530, the contents of which are incorporated herein by reference in their entirety. As a non-limiting example, the empty polymer particles can be administered to the subject before, simultaneously with, or after the composition described herein is delivered to the subject. Once the empty polymer particles come into contact with the subject, they change in volume and become lodged, embedded, fixed, or embedded in a specific location within the subject's body.

[0314] In some embodiments, the compositions can be formulated in an active substance release system (see, e.g., U.S. Patent Publication No. US20130102545, the contents of which are incorporated by reference herein in their entirety). The active substance release system can comprise: 1) at least one nanoparticle bound to an oligonucleotide inhibitor strand that hybridizes to a catalytically active nucleic acid; 2) a compound bound to at least one substrate molecule bound to a therapeutically active substance (e.g., a polynucleotide described herein), wherein the therapeutically active substance is released by cleavage of the substrate molecule by the catalytically active nucleic acid.

[0315] In some embodiments, the compositions can be combined with cationic or polycationic compounds including protamine, nucleolin, spermine or spermidine, or other cationic peptides or proteins, such as poly-L-lysine (PLL), polyarginine, basic polypeptides, cell-penetrating peptides (CPPs), (including HIV-binding peptides, HIV-1 Tat (HIV), Tat-derived peptides, penetratin, VP 22 derived peptides or similar peptides), pestivirus Ems, HSV, VP 22(herpes simplex), MAP, KALA or protein transduction domain (PTD), PpT620, proline-rich peptide, arginine-rich peptide, lysine-rich peptide, MPG-peptide, Pep-1, L-oligomer, calcitonin peptide, Antennapedia-derived peptide (particularly from Drosophila antennapedia), pAntp, plsl, FGF, lactoferrin, Transportan, Buforin-2, Bac715-24, SynB, SynB(1), pVEC, hCT-derived peptide, SAP, histone, cationic polysaccharide (e.g. chitosan, polybrene), cationic polymer (e.g. polyethyleneimine (PEI)) , cationic lipids (e.g., DOTMA: [1-(2,3-dioleoyloxy)propyl)]-N,N,N-trimethylammonium chloride, DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOPC, DODAP, DOPE: dioleylphosphatidylethanolamine, DOSPA, DODAB, DOIC, DMEPC, DOGS: dioctadecylamidoglycylspermine, DIMRI: dimyristyloxypropyldimethylhydroxyethylammonium bromide, DOTAP: dioleoyloxy-3-(trimethylammonium)propane, DC-6-14: O,O-ditetradecanoyl-N-α-trimethylammoniumacetyl)diethanolamine chloride, CLIP 1: rac-[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammonium chloride, CLIP6: rac-[2(2,3-dihexadecyloxypropyloxymethoxy)ethyl]-trimethylammonium, CLIP9: rac-[2(2,3-dihexadecyloxypropyloxysuccinyloxy)ethyl]-trimethylammonium, oligofectamine; or cationic or polycationic polymers such as modified polyamino acids, such as β-amino acid-polymers or reverse polyamides, etc., modified polyethylene such as PVP (poly(N-ethyl-4-vinylpyridinium bromide)), etc., modified acrylates such as pDMAEMA (poly(dimethylaminoethyl methacrylate)), etc., modified amidoamines such as pAM AM (poly(amidoamine)), etc., modified poly-β-amino esters (PBAE) such as diamine-terminated 1,4-butanediol diacrylate-co-5-amino-1-pentanol polymers, etc., dendritic polymers such as polypropylamine dendritic polymers or pAMAM-based dendritic polymers, etc., polyimines such as PEI: poly(ethyleneimine), poly(propyleneimine), etc., polyallylamine, sugar-based polymers such as cyclodextrin-based polymers, dextran-based polymers, chitosan, etc., silane-based polymers such as PMOXA-PDMS copolymers, etc., block polymers composed of a combination of one or more cationic blocks (for example, selected from the cationic polymers mentioned above) and one or more hydrophilic or hydrophobic blocks (for example, polyethylene glycol), etc.

[0316] In other embodiments, the composition is not combined with a cationic or polycationic compound.

[0317] Vaccine

[0318] Described herein are vaccines comprising the compositions described herein. In some aspects, described herein are vaccines comprising a composition comprising a single chain trimeric nucleic acid (e.g., mRNA) encoding a first T cell epitope, beta 2-microglobulin, and a primary MHC heavy chain sequence.

[0319] In some aspects, also described herein are vaccines comprising a composition comprising a single chain nucleic acid dimer (e.g., mRNA) encoding a T cell epitope and a primary MHC heavy chain sequence.

[0320] In some embodiments, the vaccine can be formulated as a nanoparticle described herein. In some embodiments, the vaccine can be formulated as a lipid nanoparticle described herein. In some embodiments, the vaccine can be formulated in a lipid-polycation complex described herein (referred to as a cationic lipid nanoparticle). The vaccines of the present disclosure can be formulated using one or more liposomes, lipid complexes, or lipid nanoparticles described herein. In some embodiments, the pharmaceutical composition of the vaccine can comprise a liposome described herein. In some embodiments, the vaccine can be formulated as a lipid vesicle, which can have a crosslinking between the functionalized lipid bilayers. In some embodiments, the vaccine can be formulated in a lipid-polycation complex.

[0321] In some embodiments, described herein are vaccines comprising a lipid nanoparticle comprising a single chain trimeric nucleic acid (e.g., mRNA) encoding a first T cell epitope, beta 2-microglobulin, and a primary MHC heavy chain sequence. In some embodiments, also described herein are vaccines comprising a lipid nanoparticle comprising a single chain nucleic acid dimer (e.g., mRNA) encoding a T cell epitope and a primary MHC heavy chain sequence.

[0322] Methods of administration

[0323] The compositions used in the methods described herein can be administered by any suitable methods and techniques presently or prospectively known to those skilled in the art. For example, the active ingredients described herein can be formulated in a physiologically or pharmaceutically acceptable form and administered by any suitable route known in the art, including, for example, oral and parenteral routes of administration. As used herein, the term “parenteral” includes subcutaneous, intradermal, intravenous, intramuscular, intraperitoneal, and intrasternal administration, such as by injection. Administration of the active ingredients of its composition can be a single administration, or administered at successive and distinct time intervals that can be readily determined by one skilled in the art.

[0324] "Excipients" include any and all solvents, diluents or other liquid vehicles, dispersing or suspending aids, surfactants, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, etc., suitable for the desired particular dosage form. General considerations in formulation and / or manufacturing can be found, for example, in Remington's Pharmaceutical Sciences, 16th ed., E.W. Martin (Mark Publishing Company, Easton, Pennsylvania, 1980) and Remington: The Science and Practice of Pharmacy, 21st ed. (Lippincott Williams & Wilkins, 2005).

[0325] Exemplary excipients include, but are not limited to, any non-toxic, inert solid, semisolid, or liquid filler, diluent, encapsulating material, or formulation auxiliary of any type. Some examples of materials that can be used as excipients include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil, and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; detergents such as Tween 80; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; and phosphate buffered solutions, as well as other nontoxic compatible lubricants (such as sodium lauryl sulfate and magnesium stearate), as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives, and antioxidants can also be present in the composition (at the discretion of the formulator). As will be understood by those skilled in the art, the excipient can be selected based on the purpose of the composition. For example, for pharmaceutical compositions, the choice of excipient will depend on the route of administration, the agent being delivered, the time course of agent delivery, etc., and can be administered to humans and / or animals orally, rectally, parenterally, intracisternal, intravaginal, intranasal, intraperitoneally, topically (such as by powders, creams, ointments or drops), orally, or as an oral or nasal spray. In some embodiments, the active compounds disclosed herein are administered topically.

[0326] Exemplary diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, dibasic calcium phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar, and the like, and combinations thereof.

[0327] Exemplary granulating and / or dispersing agents include potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponges, cation exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methyl cellulose, pregelatinized starch (starch 1500), microcrystalline starch, water-insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, and the like, and combinations thereof.

[0328] Exemplary surfactants and / or emulsifiers include natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan gum, pectin, gelatin, egg yolk, casein, lanolin, cholesterols, waxes, and lecithin), colloidal clays (e.g., bentonite [aluminum silicate] and Veegum [magnesium aluminum silicate]), long-chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carbopol, polyacrylic acid, acrylic acid polymers, and carboxyvinyl polymers), carrageenan, cellulose derivatives (e.g., sodium carboxymethylcellulose, powdered cellulose, hydroxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, methylcellulose), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate [Tween 20], polyoxyethylene sorbitan [Tween 60], polyoxyethylene sorbitan monooleate [Tween 80], sorbitan monopalmitate [Span 40], sorbitan monostearate [Span 60], sorbitan tristearate [Span 65], glyceryl monooleate, sorbitan monooleate [Span 80]), polyoxyethylene esters (e.g., polyoxyethylene monostearate [Myrj 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and Solutol), sucrose fatty acid esters, fatty acid polyethylene glycol esters (e.g., Cremophor), polyoxyethylene ethers (e.g., polyoxyethylene lauryl ether [Brij 30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic F 68, Poloxamer 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, sodium docusate, and the like, and / or combinations thereof.Exemplary binders include starches (e.g., corn starch and starch paste), gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol, etc.), natural and synthetic gums (e.g., gum arabic, sodium alginate, chondrus crispus extract, panwar gum, ghattigum, mucilage of isapol husks, carboxymethyl cellulose, methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum), and larch arabinogalactan), alginates, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylates, waxes, water, alcohols, etc., and / or combinations thereof.

[0329] Exemplary preservatives include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and other preservatives.

[0330] Exemplary antioxidants include alpha tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite.

[0331] Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA) and its salts and hydrates (e.g., sodium edetate, disodium edetate, trisodium edetate, calcium disodium edetate, dipotassium edetate, etc.), citric acid and its salts and hydrates (e.g., citric acid monohydrate), fumaric acid and its salts and hydrates, malic acid and its salts and hydrates, phosphoric acid and its salts and hydrates, and tartaric acid and its salts and hydrates. Exemplary antimicrobial preservatives include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethanol, glycerin, hexetidine, imidazolidinyl urea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.

[0332] Exemplary antifungal preservatives include butylparaben, methylparaben, ethylparaben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid.

[0333] Exemplary alcohol preservatives include ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoates, and phenylethanol.

[0334] Exemplary acidic preservatives include vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid. Other preservatives include tocopherol, tocopherol acetate, deteroxime mesylate, cetylpyridinium bromide, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, Glydant Plus, Phenonip, methyl paraben, Germall 115, Germaben II, Neolone, Kathon, and Euxyl. In certain embodiments, the preservative is an antioxidant. In other embodiments, the preservative is a chelating agent.

[0335] Exemplary buffering agents include citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glucoheptonate, calcium gluconate, D-gluconic acid, glycerylphosphocholine, calcium lactate, propionic acid, calcium levulinate, valeric acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethanol, and the like, and combinations thereof.

[0336] Exemplary lubricants include magnesium stearate, calcium stearate, stearic acid, silicon dioxide, talc, malt, glyceryl behenate, hydrogenated vegetable oil, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and the like, and combinations thereof.

[0337] Exemplary natural oils include almond oil, apricot kernel oil, avocado oil, babassu oil, bergamot oil, black currant seed oil, borage oil, juniper oil, chamomile oil, canola oil, caraway oil, palm wax oil, castor oil, cinnamon oil, cocoa butter oil, coconut oil, cod liver oil, coffee oil, corn oil, cottonseed oil, emu oil, eucalyptus oil, evening primrose oil, fish oil, linseed oil, geraniol oil, gourd oil, grapeseed oil, hazelnut oil, hyssop oil, isopropyl myristate oil, jojoba oil, macadamia nut oil, lavender oil, lavender

[0014] Examples of the present invention include, but are not limited to, citrus oil, lemon oil, litsea cubeba oil, macadamia nut oil, mallow oil, mango seed oil, meadowfoam seed oil, mink oil, nutmeg oil, olive oil, orange oil, orange snapper oil, palm oil, palm kernel oil, peach kernel oil, peanut oil, poppy seed oil, pumpkin seed oil, rapeseed oil, rice bran oil, rosemary oil, safflower oil, sandalwood oil, camellia oil, savory oil, sea buckthorn oil, sesame oil, shea butter, silicone oil, soybean oil, sunflower oil, tea tree oil, thistle oil, tsubaki oil, vetiver oil, walnut oil, and wheat germ oil. Exemplary synthetic oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and combinations thereof.

[0338] In addition, the composition may also include a polymer. Exemplary polymers encompassed herein include, but are not limited to, cellulosic polymers and copolymers, such as cellulose ethers, such as methylcellulose (MC), hydroxyethylcellulose (HEC), hydroxypropylcellulose (HPC), hydroxypropylmethylcellulose (HPMC), methylhydroxyethylcellulose (MHEC), methylhydroxypropylcellulose (MHPC), carboxymethylcellulose (CMC) and its various salts (including, for example, sodium salt), hydroxyethylcarboxymethylcellulose (HECMC) and its various salts, carboxymethylhydroxyethylcellulose (CMHEC) and its various salts; other polysaccharides and polysaccharide derivatives, such as starch, polydextrose, polydextrose derivatives, chitosan, and alginic acid and its various salts; carrageenan; various gums, including xanthan gum, guar gum, gum arabic, gum karaya, gum ghatti, konjac gum, and gum tragacanth; glycosaminoglycans and proteoglycans, such as hyaluronic acid and its salts; proteins, such as gelatin, collagen, albumin, and fibrin; other polymers, such as polyhydroxy acids, such as polylactide. , polyglycolide, poly(lactide-co-glycolide) and poly(ε-caprolactone-co-glycolide), carboxyvinyl polymers and their salts (e.g., carbomer), polyvinylpyrrolidone (PVP), polyacrylic acid and its salts, polyacrylamide, polyacrylic acid / acrylamide copolymers; polyalkylene oxides such as polyethylene oxide, polypropylene oxide, poly(ethylene oxide-propylene oxide) and Pluronic polymers; polyoxyethylene (polyethylene glycol), polyanhydrides, polyvinyl alcohol, polyvinylamine and polypyridine; poly Ethylene glycol (PEG) polymers, such as PEGylated lipids (e.g., PEG stearate, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000], 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000]), copolymers and salts thereof.

[0339] In addition, the composition may also include an emulsifier. Exemplary emulsifiers include, but are not limited to, polyethylene glycol (PEG), polypropylene glycol, polyvinyl alcohol, poly-N-vinyl pyrrolidone, and copolymers thereof, poloxamer nonionic surfactants, neutral water-soluble polysaccharides (e.g., polydextrose, Ficoll, cellulose), non-cationic poly(meth)acrylates, non-cationic polyacrylates (e.g., poly(meth)acrylic acid) and their esteramides and hydroxyalkylamides, natural emulsifiers (e.g., gum arabic, agar, alginic acid, sodium alginate, tragacanth, carrageenan, cholesterol, xanthan gum, pectin, gelatin, egg yolk, casein, lanolin, cholesterol, wax, and lecithin), colloidal clays (e.g., bentonite [aluminum silicate] and Vee gum [magnesium aluminum silicate]), long-chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxypolymethylene, polyacrylic acid, acrylic acid polymers, and carboxyvinyl polymers), carrageenan, cellulose derivatives (e.g., sodium carboxymethylcellulose, powdered cellulose, hydroxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, methylcellulose), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate [Tween 20], polyoxyethylene sorbitan [Tween 60], polyoxyethylene sorbitan monooleate [Tween 80], sorbitan monopalmitate [Span 40], sorbitan monostearate [Span 60], sorbitan tristearate [Span 65], glyceryl monooleate, sorbitan monooleate [Span 80]), polyoxyethylene esters (e.g., polyoxyethylene monostearate [Myrj 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and Solutol), sucrose fatty acid esters, fatty acid polyethylene glycol esters (e.g., Cremophor), polyoxyethylene ethers (e.g., polyoxyethylene lauryl ether [Brij 30]), poly (vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic F 68, poloxamer 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, etc. and / or combinations thereof. In certain embodiments, the emulsifier is cholesterol.

[0340] Liquid compositions include emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compound, the liquid composition can contain inert diluents commonly used in the art, such as water or other solvents, solubilizers, and emulsifiers, such as ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (e.g., cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and sorbitan fatty acid esters, and mixtures thereof. In addition to inert diluents, oral compositions can also include adjuvants, such as wetting agents, emulsifiers, and suspending agents, sweeteners, flavorings, and aromatics.

[0341] Injectable compositions (e.g., injectable aqueous or oily suspensions) can be prepared using suitable dispersants or wetting agents and suspending agents according to known techniques. Sterile injectable formulations can also be injectable solutions, suspensions, or emulsions in non-toxic parenteral acceptable diluents or solvents, for example, in the form of solutions in 1,3-butanediol. Acceptable vehicles and solvents for pharmaceutical or cosmetic compositions that can be used are water, Ringer's solution, USP, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally used as solvents or suspending media. Any mild, non-volatile oil can be used, including synthetic monoglycerides or diglycerides. In addition, fatty acids, such as oleic acid, are used in the preparation of injectables. In certain embodiments, the particles are suspended in a carrier fluid comprising 1% (w / v) sodium carboxymethylcellulose and 0.1% (v / v) Tween 80. The injectable compositions can be sterilized, for example, by filtration through a bacteria-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0342] Compositions for rectal or vaginal administration may be in the form of suppositories which can be prepared by mixing the particles with a suitable non-irritating excipient or carrier such as cocoa butter, polyethylene glycol or a suppository wax which is solid at ambient temperature but liquid at body temperature and therefore melts in the rectum or vaginal cavity and releases the particles.

[0343] Solid compositions include capsules, tablets, pills, powders, and granules. In these solid compositions, the particles are mixed with at least one excipient and / or the following: a) fillers or extenders such as starch, lactose, sucrose, glucose, mannitol, and silicic acid; b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; c) humectants such as glycerol; d) disintegrants such as agar-agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain silicates, and sodium carbonate; e) solution retardants such as paraffin; f) absorption accelerators such as quaternary ammonium compounds; g) wetting agents such as cetyl alcohol and glyceryl monostearate; h) adsorbents such as kaolin and bentonite; and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also include a buffer. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using excipients such as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

[0344] Tablet, capsule, pill and granule can be prepared with coating and shell, as other coating known in the field of enteric coating and pharmaceutical preparation.It can optionally contain opacifying agent, and can also have the composition that makes it optionally only or preferentially release one or more active components in a delayed manner in a certain part of intestinal tract.The example of operable embedding composition comprises polymeric substance and wax.Can also use excipient such as lactose or milk sugar and high molecular weight polyethylene glycol and its analogue with similar type solid composition as the filler in soft fill and hard fill gelatin capsule.

[0345] Compositions for topical or transdermal administration include ointments, pastes, creams, emulsions, gels, powders, solutions, sprays, inhalants or patches.The active compound is mixed with excipients and any needed preservatives or buffers as may be required.

[0346] Ointments, pastes, creams and gels may contain, in addition to the active compounds, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

[0347] In addition to the active compounds, powders and sprays may contain excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate and polyamide powder, or mixtures of these substances. Sprays may additionally contain customary propellants such as chlorofluorohydrocarbons.

[0348] Transdermal patches have the additional advantage of providing controlled delivery of the compound to health. These dosage forms can be manufactured by dissolving or distributing the nanoparticles in a suitable medium. Absorption enhancers can also be used to increase the flow of the compound through the skin. Rate can be controlled by providing a rate-controlling membrane or by dispersing the particles in a polymer matrix or gel.

[0349] The compound can be incorporated into microparticles, nanoparticles, or combinations thereof that provide controlled release of the compound and / or additional active agents. For example, the compound can be incorporated into polymer microparticles that provide controlled release of the drug. The release of the drug is controlled by diffusion of the drug from the microparticles and / or degradation of the polymer particles by hydrolysis and / or enzymatic degradation. Suitable polymers include ethylcellulose and other natural or synthetic cellulose derivatives.

[0350] Polymers that slowly dissolve and form gels in aqueous environments, such as hydroxypropyl methylcellulose or polyethylene oxide, may also be suitable as materials for drug-containing microparticles. Other polymers include, but are not limited to, polyanhydrides, poly(ester anhydrides), polyhydroxy acids, such as polylactide (PLA), polyglycolide (PGA), poly(lactide-co-glycolide) (PLGA), poly-3-hydroxybutyrate (PHB) and copolymers thereof, poly-4-hydroxybutyrate (P4HB) and copolymers thereof, polycaprolactone and copolymers thereof, and combinations thereof.

[0351] Alternatively, the compound can be incorporated into microparticles made of a material that is insoluble in aqueous solution or slowly soluble in aqueous solution but can be degraded in the gastrointestinal tract by means including enzymatic degradation, the action of surfactants such as bile acids, and / or mechanical erosion. As used herein, the term "slowly soluble in water" refers to a material that is insoluble in water within a period of 30 minutes. Preferred examples include fats, fatty substances, waxes, waxy substances, and mixtures thereof. Suitable fats and fatty substances include fatty alcohols (such as lauryl alcohol, myristyl alcohol, stearyl alcohol, cetyl alcohol, or 1864 alcohol), fatty acids and derivatives, but are not limited to fatty acid esters, fatty acid glycerides (mono-, di-, and triglycerides), and hydrogenated fats. Specific examples include, but are not limited to, hydrogenated vegetable oils, hydrogenated cottonseed oil, hydrogenated castor oil, and products sold under the trade name Available hydrogenated oils, stearic acid, cocoa butter, and stearyl alcohol. Suitable waxes and waxy materials include natural or synthetic waxes, hydrocarbons, and conventional waxes. Specific examples of waxes include beeswax, glycowax, castor wax, carnauba wax, paraffin wax, and candelilla wax. As used herein, a waxy material is defined as any material that is generally solid at room temperature and has a melting point of about 30°C to 300°C.

[0352] In some cases, it may be necessary to change the rate at which water penetrates into the microparticles. To this end, a rate-controlling formulation (wicking agent) can be formulated with the fats or waxes listed above. Examples of rate-controlling materials include certain starch derivatives (e.g., waxy maltodextrin and drum-dried corn starch), cellulose derivatives (e.g., hydroxypropyl methylcellulose, hydroxypropyl cellulose, methylcellulose, and carboxymethylcellulose), alginic acid, lactose, and talc. In addition, a pharmaceutically acceptable surfactant (e.g., lecithin) can also be added to promote the degradation of such microparticles.

[0353] Water-insoluble proteins, such as zein, can also be used as materials for forming drug-containing microparticles. Furthermore, water-soluble proteins, polysaccharides, and combinations thereof can be formulated into microparticles with the drug and subsequently cross-linked to form an insoluble network. For example, cyclodextrin can be complexed with individual drug molecules and subsequently cross-linked.

[0354] The drug can be encapsulated or incorporated into a carrier material by known pharmaceutical formulation technology to produce drug-containing microparticles. In the case of a formulation in fat, wax or waxy material, the carrier material is usually heated to more than its melting point temperature, and medicine is added to form a mixture comprising drug particles suspended in the carrier material, the medicine dissolved in the carrier material or its mixture. Microparticles can be prepared subsequently by a variety of methods, including but not limited to solidification, extrusion, spray cooling or water dispersion methods. In a preferred method, wax is heated to more than its melting point temperature, medicine is added, and while the mixture is cooling, the molten wax-drug mixture is solidified under continuous stirring. Alternatively, the molten wax-drug mixture can be extruded and spheronized to form a pill or pearl. These methods are known in the art.

[0355] For some carrier materials, it may be necessary to use solvent evaporation techniques to produce drug-containing microparticles. In this case, the drug and carrier material are co-dissolved in a mutual solvent, and the microparticles can then be produced by a variety of techniques, including but not limited to forming an emulsion in water or other suitable medium, spray drying, or evaporating the solvent from the bulk solution and grinding the resulting material.

[0356] In certain embodiments, the drug in granular form is uniformly dispersed in a water-insoluble or slowly water-soluble material. In order to minimize the size of the drug particles in the composition, the drug powder itself can be ground to generate fine particles before preparation. The jet milling process known in the pharmaceutical field can be used for this purpose. In certain embodiments, the drug in granular form is uniformly dispersed in the wax or waxy substance by heating the wax or waxy substance to above its melting point and adding the drug particles while stirring the mixture. In this case, a pharmaceutically acceptable surfactant can be added to the mixture to promote the dispersion of the drug particles.

[0357] The particles can also be coated with one or more modified release coatings. Solid fatty acid esters that are hydrolyzed by lipases can be sprayed onto the microparticles or drug particles. Zein is an example of a naturally water insoluble protein. Zein can be coated onto drug-containing microparticles or drug particles by either spray coating or by wet granulation techniques. In addition to naturally water insoluble materials, some substrates of digestive enzymes can also be treated by a cross-linking procedure, resulting in the formation of an insoluble network. Numerous methods of cross-linking proteins have been reported, both by chemical and physical means. One of the most common methods of obtaining cross-linking is the use of chemical cross-linking agents. Examples of chemical cross-linking agents include aldehydes (glutaraldehyde and formaldehyde), epoxides, carbodiimides, and genipin. In addition to these cross-linking agents, oxidized sugars and natural sugars have also been used to cross-link gelatin. Cross-linking can also be accomplished using enzymatic means; for example, transglutaminase has been approved as a GRAS substance for cross-linking seafood products. Finally, cross-linking can be initiated by physical means, such as heat treatment, UV irradiation, and gamma irradiation.

[0358] To create a coating layer of cross-linked proteins surrounding the drug-containing microparticles or drug particles, water-soluble proteins can be sprayed onto the microparticles and subsequently cross-linked by one of the methods described above. Alternatively, the drug-containing microparticles can be microencapsulated within the proteins by coacervation phase separation (e.g., by the addition of salt) and subsequently cross-linked. Some proteins suitable for this purpose include gelatin, albumin, casein, and gluten.

[0359] Polysaccharides can also be cross-linked to form water insoluble networks. For many polysaccharides, this can be achieved by reaction with calcium salts or multivalent cations, which cross-link the main polymer chains. Pectin, alginate, dextran, amylose, and guar gum are subjected to cross-linking in the presence of multivalent cations. Complexes can also form between oppositely charged polysaccharides; for example, pectin and chitosan can complex via electrostatic interactions.

[0360] In certain embodiments, it can be desirable to provide sustained delivery of one or more compounds to a patient in need thereof. For intravenous or arterial routes, this can be accomplished using an infusion system, such as by intravenous administration. For topical applications, repeated applications can be made, or a patch can be used to provide continuous compound administration over an extended period of time.

[0361] The compounds described herein can be incorporated into injectable / implantable solid or semi-solid implants, such as polymeric implants. In one embodiment, the compound is incorporated into a polymer that is a liquid or paste at room temperature, but exhibits an increase in viscosity upon contact with an aqueous medium, such as a physiological fluid, to form a semi-solid or solid material. Exemplary polymers include, but are not limited to, hydroxyalkanoic acid polyesters derived from at least one unsaturated hydroxy fatty acid copolymerized with a hydroxyalkanoic acid. The polymer can be melted, mixed with the active, and cast or injection molded into a device. Such melt fabrication requires a polymer with a melting point below the temperature at which the substance to be delivered and the polymer degrade or react. The device can also be prepared by solvent casting, in which the polymer is dissolved in a solvent and the drug is dissolved or dispersed in the polymer solution, and the solvent is evaporated. The solvent method requires that the polymer be soluble in an organic solvent. Another method is compression molding of a mixed powder of the polymer and drug loaded with active agent or polymer particles.

[0362] Alternatively, the compound can be incorporated into a polymer matrix and molded, compressed, or extruded into a device that is solid at room temperature. For example, the compound can be incorporated into a biodegradable polymer, such as a polyanhydride, a polyhydroalkanoate (PHA), PLA, PGA, PLGA, polycaprolactone, a polyester, a polyamide, a polyorthoester, a polyphosphazene, a protein, and a polysaccharide, such as collagen, hyaluronic acid, albumin, and gelatin, and combinations thereof, and compressed into a solid device, such as a disc, wafer, or extruded into a device, such as a rod.

[0363] The release of the compound from the implant can be varied by the choice of polymer, the molecular weight of the polymer, and / or modification of the polymer to increase degradation, such as the incorporation of pores and / or hydrolyzable linkages. Methods for altering the properties of biodegradable polymers to modify the release profile of a compound from an implant are well known in the art.

[0364] In some embodiments, the compound or pharmaceutical composition can be administered topically. In some embodiments, the compound is incorporated into a delivery system, such as a gel, nanoparticle, microparticle, or implant, such as a rod, disc, wafer, orthopedic implant, for sustained release. In some embodiments, the compound can be administered using a local delivery implantable system comprising a compound incorporated within a gel, nanoparticle, microparticle, or implant. In some embodiments, the pharmaceutical composition comprises a delivery system, such as a gel, nanoparticle, microparticle, or implant, such as a rod, disc, wafer, orthopedic implant, for sustained release of paroxetine, or a pharmaceutically acceptable salt or derivative thereof.

[0365] The active ingredient can be administered in the amount, time, and route thought to be necessary to achieve the desired result. The exact amount of the active ingredient will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the infection, the specific active ingredient, its mode of administration, its mode of activity, etc. The active ingredient (whether the active compound itself or a combination of the active compound and the medicament) is preferably formulated in a unit dosage form for ease of administration and dosage uniformity. However, it should be understood that the total daily dose of the active ingredient will be determined by the attending physician within the scope of reasonable medical judgment. For any particular subject, the specific therapeutically effective dose level will depend on a variety of factors, including the severity of the disorder and the disorder being treated; the activity of the active ingredient used; the specific composition used; the patient's age, weight, overall health, sex, and diet; the time of administration, route of administration, and excretion rate of the specific active ingredient used; the duration of treatment; the drugs used in combination with or simultaneously with the specific active ingredient used; and similar factors well-known in the medical field.

[0366] The active ingredient can be administered by any route. In some embodiments, the active ingredient is administered via a variety of routes, including oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, intradermal, rectal, intravaginal, intraperitoneal, topical (e.g., by powders, ointments, creams, and / or drops), mucosal, nasal, buccal, intestinal, sublingual; by intratracheal instillation, bronchial instillation, and / or inhalation; and / or in the form of oral sprays, nasal sprays, and / or aerosols. Typically, the most appropriate route of administration will depend on a variety of factors, including the properties of the active ingredient (e.g., its stability in the gastrointestinal environment), the condition of the individual (e.g., whether the individual can tolerate oral administration), etc.

[0367] The exact amount of active ingredient required to achieve a therapeutically or prophylactically effective amount will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of side effects or symptoms, the nature of the specific compound or compounds, the mode of administration, etc. The amount administered to, for example, a child or adolescent can be determined by a licensed physician or person skilled in the art and may be less than or equal to the amount administered to an adult.

[0368] Useful dosages of the compositions disclosed herein can be determined by comparing their in vitro activity with their in vivo activity in animal models. Methods for extrapolating effective dosages in mice and other animals to humans are known in the art.

[0369] The dosage ranges for composition administration are those that are large enough to produce the desired effect affecting the symptoms or conditions. The dosage should not be large enough to cause adverse side effects, such as unwanted cross-reactions, allergic reactions, etc. Generally, the dosage will vary with the patient's age, condition, sex, and degree of disease, and can be determined by those skilled in the art. In the case of any contraindications, the dosage can be adjusted by an individual physician. The dosage can vary and can be administered in one or more doses per day, for one or several days.

[0370] The compositions described herein are typically formulated in dosage unit form for ease of administration and uniformity of dosage. However, it will be understood that the total daily usage of the composition can be determined by the attending physician within the scope of sound medical judgment. For any particular patient, the specific therapeutically effective dose, prophylactically effective dose, or appropriate imaging dose level will depend on a variety of factors, including the condition being treated and the severity of the condition; the activity of the specific compound used; the specific composition used; the patient's age, weight, general health, sex, and diet; the time of administration, route of administration, and excretion rate of the specific compound used; the duration of treatment; drugs used in combination with or concurrently with the specific compound used; and similar factors well known in the medical arts.

[0371] In some embodiments, the composition may be administered at a dosage level sufficient to deliver 0.0001 mg / kg to 100 mg / kg, 0.001 mg / kg to 0.05 mg / kg, 0.005 mg / kg to 0.05 mg / kg, 0.001 mg / kg to 0.005 mg / kg, 0.05 mg / kg to 0.5 mg / kg, 0.01 mg / kg to 50 mg / kg, 0.1 mg / kg to 40 mg / kg, 0.5 mg / kg to 30 mg / kg, 0.01 mg / kg to 10 mg / kg, 0.1 mg / kg to 10 mg / kg, or 1 mg / kg to 25 mg / kg of the subject's body weight daily, once or multiple times a day, weekly, monthly, etc., to achieve the desired therapeutic, diagnostic, prophylactic or imaging effect (see, e.g., the unit dose ranges described in International Publication No. WO2013078199, the contents of which are herein incorporated by reference in their entirety). The desired dosage can be delivered three times a day, twice a day, once a day, every other day, every three days, once a week, every two weeks, every three weeks, every four weeks, every two months, every three months, every six months. In certain embodiments, it is possible to use multiple administrations (for example, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, eleven times, twelve times, thirteen times, fourteen times or more administrations) to deliver the desired dosage. When adopting multiple administrations, it is possible to use a split dose regimen, such as those described herein. In an exemplary embodiment, compositions can be used to be enough to deliver 0.0005mg / kg to 0.01mg / kg (for example, about 0.0005mg / kg to about 0.0075mg / kg, for example, about 0.0005mg / kg, about 0.001mg / kg, about 0.002mg / kg, about 0.003mg / kg, about 0.004mg / kg or about 0.005mg / kg) dosage levels.

[0372] In some embodiments, the composition may be administered once or twice (or more) at a dose level sufficient to deliver 0.025 mg / kg to 0.250 mg / kg, 0.025 mg / kg to 0.500 mg / kg, 0.025 mg / kg to 0.750 mg / kg, or 0.025 mg / kg to 1.0 mg / kg.

[0373] In some embodiments, the composition may be administered at a total dose of 0.0100 mg, 0.025 mg, 0.050 mg, 0.075 mg, 0.100 mg, 0.125 mg, 0.150 mg, 0.175 mg, 0.200 mg, 0.225 mg, 0.250 mg, 0.275 mg, 0.300 mg, 0.325 mg, 0.350 mg, 0.375 mg, 0.400 mg, 0.425 mg, 0.450 mg, 0.475 mg, 0.500 mg, 0.525 mg, 0.550 mg, 0.575 mg, 0.600 mg, 0.625 mg, 0.650 mg, 0.675 mg, 0.700 mg, 0.725 mg, 0.750 mg, 0.760 mg, 0.775 mg, 0.780 mg, 0.790 mg, 0.800 mg, 0.810 mg, 0.825 mg, 0.830 mg, 0.840 mg, 0.850 mg, 0.860 mg, 0.875 mg, 0.880 mg, 0.890 mg, 0.900 mg, 0.910 mg, 0.925 mg, In some embodiments, the present invention provides an oral administration of 50 mg, 0.775 mg, 0.800 mg, 0.825 mg, 0.850 mg, 0.875 mg, 0.900 mg, 0.925 mg, 0.950 mg, 0.975 mg or 1.0 mg, or a dose level sufficient to deliver the above total dose, administered twice (e.g., day 0 and day 7, day 0 and day 14, day 0 and day 21, day 0 and day 28, day 0 and day 60, day 0 and day 90, day 0 and day 120, day 0 and day 150, day 0 and day 180, day 0 and 3 months later, day 0 and 6 months later, day 0 and 9 months later, day 0 and 12 months later, day 0 and 18 months later, day 0 and 2 years later, day 0 and 5 years later, or day 0 and 10 years later). The present disclosure encompasses higher and lower doses and administration frequencies.For example, the compositions described herein can be administered three or four times.

[0374] In some embodiments, the composition can be administered twice (e.g., day 0 and day 7, day 0 and day 14, day 0 and day 21, day 0 and day 28, day 0 and day 60, day 0 and day 90, day 0 and day 120, day 0 and day 150, day 0 and day 180, day 0 and 3 months later, day 0 and 6 months later, day 0 and 9 months later, day 0 and 12 months later, day 0 and 18 months later, day 0 and 2 years later, day 0 and 5 years later, or day 0 and 10 years later) at a total dose of 0.010 mg, 0.025 mg, 0.100 mg, or 0.400 mg, or a dose level sufficient to deliver the above total dose.

[0375] In some embodiments, the composition (e.g., vaccine) used in the method of vaccinating a subject is administered to the subject with a single dose of a nucleic acid vaccine between 10 μg / kg and 400 μg / kg (an effective amount to vaccinate the subject). In some embodiments, the composition (e.g., vaccine) used in the method of vaccinating a subject is administered to the subject with a single dose of a nucleic acid vaccine between 10 μg and 400 μg (an effective amount to vaccinate the subject). In some embodiments, the composition (e.g., vaccine) used in the method of vaccinating a subject is administered to the subject with a single dose of 25-1000 μg (e.g., a single dose of mRNA encoding an infectious agent and an mRNA encoding a T cell epitope, or an mRNA encoding an infectious agent and at least one universal T cell epitope). In some embodiments, the composition (e.g., vaccine) is administered to a subject in a single dose of 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 μg. For example, the composition (e.g., vaccine) is administered to a subject in a single dose of 25-100, 25-500, 50-100, 50-500, 50-1000, 100-500, 100-1000, 250-500, 250-1000, or 500-1000 μg. In some embodiments, the composition (e.g., vaccine) used in the method for vaccinating a subject is administered to a subject in two doses, the combination of the two doses being equal to a composition (e.g., vaccine) of 25-1000 μg.

[0376] The compositions described herein (e.g., vaccine pharmaceutical compositions) can be formulated into dosage forms described herein, such as intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intradermal, intracardiac, intraperitoneal, and subcutaneous).

[0377] How to use

[0378] Described herein are methods for activating and / or amplifying antigen-presenting cells, comprising co-culturing antigen-presenting cells and cells comprising single-chain trimer nucleic acids, compositions, nanoparticles, vaccines, or pharmaceutical compositions as described herein. In some embodiments, methods for activating and / or amplifying antigen-presenting cells can comprise co-culturing antigen-presenting cells and cells comprising lipid nanoparticles as described herein.

[0379] Also described herein are methods for treating subjects with viral infections, bacterial infections, parasitic infections, and / or cancers, comprising administering to the subject a therapeutically effective amount of a single-stranded trimer nucleic acid as described herein, a composition as described herein, a nanoparticle as described herein, a vaccine as described herein, a cell as described herein, or a pharmaceutical composition as described herein. In some embodiments, methods for treating subjects with viral infections, bacterial infections, parasitic infections, and / or cancers are described, comprising administering to the subject a therapeutically effective amount of a lipid nanoparticle as described herein.

[0380] In some embodiments, described herein are methods for treating a subject with cancer, comprising administering to the subject a therapeutically effective amount of a single-stranded trimer nucleic acid as described herein, a composition as described herein, a nanoparticle as described herein, a vaccine as described herein, a cell as described herein, or a pharmaceutical composition as described herein. In some embodiments, described herein are methods for treating a subject with cancer, comprising administering to the subject a therapeutically effective amount of a lipid nanoparticle as described herein.

[0381] Also described herein are methods of treating a subject having a viral infection, a bacterial infection, a parasitic infection, and / or cancer, comprising obtaining antigen-presenting cells from the subject; contacting the antigen-presenting cells from the subject with a single-chain trimer nucleic acid described herein, a composition described herein, a nanoparticle described herein, a vaccine described herein, or a pharmaceutical composition described herein, thereby activating the antigen-presenting cells, and administering the activated antigen-presenting cells to the subject.

[0382] In some embodiments, described herein are methods of treating a subject having a viral infection, a bacterial infection, a parasitic infection, and / or cancer, the methods comprising obtaining antigen-presenting cells from the subject; contacting the antigen-presenting cells from the subject with a lipid nanoparticle described herein, thereby activating the antigen-presenting cells, and administering the activated antigen-presenting cells to the subject.

[0383] In some embodiments, described herein are methods of treating a subject having cancer, comprising obtaining antigen-presenting cells from the subject; contacting the antigen-presenting cells from the subject with a single-chain trimer nucleic acid described herein, a composition described herein, a nanoparticle described herein, a vaccine described herein, a cell described herein, or a pharmaceutical composition described herein, thereby activating the antigen-presenting cells, and administering the activated antigen-presenting cells to the subject.

[0384] In some embodiments, a method of treating a subject having cancer is described, the method comprising obtaining antigen-presenting cells from the subject; contacting the antigen-presenting cells from the subject with a lipid nanoparticle described herein, thereby activating the antigen-presenting cells, and administering the activated antigen-presenting cells to the subject.

[0385] Also described herein are methods for preventing a subject from developing a viral infection, a bacterial infection, a parasitic infection, and / or cancer, comprising administering to the subject a therapeutically effective amount of a single-stranded trimer nucleic acid, a composition, a nanoparticle, a vaccine, a cell, or a pharmaceutical composition as described herein. In some embodiments, methods for preventing a subject from developing a viral infection, a bacterial infection, a parasitic infection, and / or cancer are described, comprising administering to the subject a therapeutically effective amount of a lipid nanoparticle as described herein.

[0386] In some embodiments, described herein are methods for preventing a subject from developing cancer, comprising administering to the subject a therapeutically effective amount of a single-stranded trimer nucleic acid as described herein, a composition as described herein, a nanoparticle as described herein, a vaccine as described herein, a cell as described herein, or a pharmaceutical composition as described herein. In some embodiments, described herein are methods for preventing a subject from developing cancer, comprising administering to the subject a therapeutically effective amount of a lipid nanoparticle as described herein.

[0387] Also described herein are methods of preventing a subject from developing a viral infection, a bacterial infection, a parasitic infection, and / or cancer, the methods comprising obtaining antigen-presenting cells from the subject; contacting the antigen-presenting cells from the subject with a single-chain trimer nucleic acid described herein, a composition described herein, a nanoparticle described herein, a vaccine described herein, a cell described herein, or a pharmaceutical composition described herein, thereby activating the antigen-presenting cells; and administering the activated antigen-presenting cells to the subject.

[0388] In some embodiments, a method of preventing a subject from developing a viral infection, a bacterial infection, a parasitic infection, and / or cancer is described, the method comprising obtaining antigen-presenting cells from the subject; contacting the antigen-presenting cells from the subject with a lipid nanoparticle described herein, thereby activating the antigen-presenting cells, and administering the activated antigen-presenting cells to the subject.

[0389] In some embodiments, a method of preventing cancer in a subject is described, comprising obtaining antigen-presenting cells from the subject; contacting the antigen-presenting cells from the subject with a single-chain trimer nucleic acid described herein, a composition described herein, a nanoparticle described herein, a vaccine described herein, a cell described herein, or a pharmaceutical composition described herein, thereby activating the antigen-presenting cells, and administering the activated antigen-presenting cells to the subject.

[0390] In some embodiments, methods of preventing a subject from developing a cancer are described, the methods comprising obtaining an antigen presenting cell from the subject; contacting the antigen presenting cell from the subject with a lipid nanoparticle described herein, thereby activating the antigen presenting cell, and administering the activated antigen presenting cell to the subject.

[0391] In some embodiments, the antigen presenting cell can be a dendritic cell, a B cell, a macrophage, or other cell derived from the subject that can be used for immunotherapy.

[0392] In some cases, the cancer is a circulating cancer cell (circulating tumor cell). In some cases, the cancer is a metastatic cancer cell. In some embodiments, the compositions and methods described herein are used to treat both localized tumors and metastatic tumors. In some embodiments, the compositions and methods described herein can be used to treat or prevent metastasis or recurrence of a cancer. In some embodiments, the compositions and methods described herein can be used to prevent recurrence of a resected solid tumor. In some embodiments, the compositions and methods described herein can be used to prevent metastasis of a resected solid tumor.

[0393] In one aspect, the methods described herein are used to treat cancers such as melanoma, lung cancer (including lung adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, large cell carcinoma, bronchioalveolar carcinoma, bronchogenic carcinoma, non-small cell carcinoma, small cell carcinoma, mesothelioma); breast cancer (including ductal carcinoma, lobular carcinoma, inflammatory breast cancer, clear cell carcinoma, mucinous carcinoma, serous cavity breast cancer); colorectal cancer (colon cancer, rectal cancer, colorectal adenocarcinoma); anal cancer; pancreatic cancer (including pancreatic adenocarcinoma, islet cell carcinoma, neuroendocrine tumors); prostate cancer; prostate adenocarcinoma; ovarian cancer (ovarian epithelial carcinoma or surface epithelial stromal tumors, including serous tumors, endometrioid tumors and mucinous cystadenocarcinomas, sex cord stromal tumors); hepatobiliary cancer (including hepatocellular carcinoma, bile duct cancer, hemangioma); esophageal cancer (including esophageal adenocarcinoma and squamous cell carcinoma); oral and oropharyngeal squamous cell carcinoma; salivary gland adenoid cystic carcinoma; bladder cancer; bladder cancer. carcinoma); uterine cancer (including endometrial adenocarcinoma, eye cancer, uterine papillary serous carcinoma, uterine clear cell carcinoma, uterine sarcoma, leiomyosarcoma, and mixed Müllerian tumor); glioma, glioblastoma, medulloblastoma, and other brain tumors; kidney cancer (including renal cell carcinoma, clear cell carcinoma, and Wilms' tumor); head and neck cancer (including squamous cell carcinoma); stomach cancer (antral cancer, gastric adenocarcinoma, gastrointestinal stromal tumor); testicular cancer; germ cell tumors; neuroendocrine tumors; cervical cancer; carcinoid tumors of the gastrointestinal tract, breast, and other organs; signet ring cell carcinoma; stromal tumors, including sarcoma, fibrosarcoma, cervical hemangioma, angiomatosis, hemangiopericytoma, pseudoangiomatous stromal proliferation, myofibroblastic tumor, fibromatosis, inflammatory myofibroblastic tumor, lipoma, angiolipoma, granular cell tumor, neurofibroma, neurilemoma, angiosarcoma, liposarcoma, rhabdomyosarcoma, osteosarcoma, leiomyoma, leiomyosarcoma, skin cancer (including melanoma), cervical cancer, retinoblastoma, head and neck cancer, pancreatic cancer, brain cancer, thyroid cancer, and testicular cancer , kidney cancer, bladder cancer, soft tissue cancer, adrenal cancer, urethral cancer, penile cancer, myxosarcoma, chondrosarcoma, osteosarcoma, chordoma, malignant fibrous histiocytoma, lymphangiosarcoma, mesothelioma, squamous cell carcinoma; epidermoid carcinoma, malignant skin adnexal tumor, adenocarcinoma, liver cancer, hepatocellular carcinoma, renal cell carcinoma, adrenal tumor, hepatobiliary type hepatocellular carcinoma, transitional cell carcinoma, choriocarcinoma, seminoma, embryonal cell carcinoma, asexual glioma; glioblastoma multiforme, neuroblastoma, medulloblastoma, malignant meningioma, malignant neurilemoma, neurofibrosarcoma, parathyroid cancer, medullary thyroid cancer, bronchial carcinoid, pheochromocytoma, pancreatic islet cell carcinoma, malignant carcinoid, malignant paraganglioma, melanoma, Merkel cell tumor (Merkelcell neoplasm), phyllodes cystosarcoma, salivary gland cancer, thymic cancer and vaginal cancer, etc.

[0394] In one embodiment, bacterial organisms that can cause bacterial infections can include, but are not limited to, Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Streptococcus pyogenes, Corynebacterium diphtheriae, Listeria monocytogenes, Bacillus anthracis, Clostridium tetani, Clostridium botulinum, Clostridium perfringens, Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus mutans, Pseudomonas aeruginosa, Salmonella typhi, Haemophilus parainfluenzae, Bordetella pertussis, Francisella tularensis, Yersinia pestis, Vibrio cholerae, Legionella pneumophila, Mycobacterium tuberculosis, Mycobacterium leprae, Treponema pallidum, Leptospira interrogans, Borrelia burgdorferi, Campylobacter jejuni, and the like. Viruses that can cause viral infections include, but are not limited to, metapneumoviruses such as human metapneumovirus (hMPV), parainfluenza viruses such as human parainfluenza virus (hPIV) types 1, 2, and 3 (hPIV1, hPIV2, and hPIV3, respectively), respiratory syncytial virus (RSV), measles virus (MeV), coronaviruses (e.g., MERS-CoV, SARS-CoV, SARS-CoV2, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV- NL, HCoV-NH, HCoV-HKU1), poxviruses (e.g., smallpox, monkeypox), influenza A and B, human immunodeficiency virus (HIV), varicella-zoster, herpes simplex 1 and 2, cytomegalovirus, Epstein-Barr virus, rotavirus, rhinovirus, adenovirus, papillomavirus, poliovirus, mumps, rabies, rubella, coxsackievirus, equine encephalitis, Japanese encephalitis, yellow fever, Rift Valley fever, hepatitis A, B, C, D, and E viruses, etc. Other organisms that can cause infection include fungi, protozoa, and parasitic organisms such as Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia rickettsii, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydia psittaci, Chlamydia trachomatis, Plasmodium falciparum, Trypanosoma brucei, Entamoeba histolytica, Toxoplasma gondii, Trichomonas vaginalis, Schistosoma mansoni, and the like.

[0395] In some embodiments, the method can further comprise administering an additional active agent. In some embodiments, the additional active agent.

[0396] As used herein, "active agent" refers to a therapeutic agent, a diagnostic agent, or a prophylactic agent. As discussed herein, the therapeutic agent can be released from the disclosed compounds, compositions, and systems in a biologically active form.

[0397] It will also be understood that, as used herein, the term "therapeutic agent" refers to one or more therapeutic agents, active ingredients, or substances that can be used to treat a medical condition. Therapeutic agents include any synthetic or naturally occurring biologically active compound or composition of matter that, when administered to an organism (human or non-human animal), induces a desired pharmacological, immunogenic, and / or physiological effect by local and / or systemic action. The term thus encompasses those compounds or chemicals that are traditionally considered to include molecules such as proteins, peptides, hormones, nucleic acids, genetic constructs, drugs, vaccines, and biopharmaceuticals. Examples of therapeutic agents are described in well-known literature references, such as the Merck Index (14th edition), Physicians' Desk Reference (64th edition), and The Pharmacological Basis of Therapeutics (12th edition), and include, but are not limited to: drugs; vitamins and minerals, such as essential amino acids, calcium, iron, potassium, zinc, vitamin B12, and the like; substances used in the treatment, prevention, diagnosis, cure, or alleviation of disease or disorder; substances that affect the structure or function of the body, or prodrugs that become biologically active or more active when placed in a physiological environment. For example, the term "therapeutic agent" includes compounds or compositions used in all major therapeutic areas, including but not limited to adjuvants; antimicrobial agents (including antibiotics, antivirals, antiparasitics and antifungals), anti-inflammatory agents (including steroidal and non-steroidal anti-inflammatory agents), anticoagulants, ophthalmic agents, gastrointestinal agents, antiplatelet agents and antiseptics, steroid agents, anti-tumor agents, anti-cancer agents, antigens, antibodies (e.g., cetuximab, anti-CD24 antibodies, panitumumab and bevacizumab), contraceptives, progestins, anticholinergics, Nutritional agents, analgesics and analgesic combinations such as acetaminophen, acetylsalicylic acid, etc.; anesthetics such as lidocaine, xylocaine, etc., anorectic drugs such as dextrothyroxine, phendimetrazine tartrate, etc.; antiepileptics, local and general anesthetics, hypnotics, sedatives, antipsychotics, neuroleptics, antidepressants, such as isocarboxazid, amoxapine, etc.; anxiolytics, antagonists, neuroleptics, anticholinergics and cholinergic agents, antimuscarinics and muscarinics, anti-Parkinson's disease drugs, anti-Alzheimer's disease drugs (anti-Alzheimer's agent), antiadrenergic drugs, antiarrhythmic drugs, antihypertensive drugs, hormones such as insulin, progesterone, estrogen, corticosteroids, glucocorticoids, and androgens; as well as nutrients, gout drugs such as methylprednisolone and ibuprofen; antiasthma drugs such as terbutaline sulfate, theophylline, ephedrine; anticonvulsants such as phenytoin sodium and diazepam;Antiallergics, antihistamines such as diphenhydramine HCl, chlorpheniramine maleate, etc.; antinauseants, antitumor drugs, antipruritics, antipyretics; antispasmodics such as belladonna alkaloids, dicyclomine hydrochloride, etc.; cardiovascular drugs such as prazosin HCl, nitroglycerin, propranolol HCl, hydralazine HCl, pancreatic lipase, succinate dehydrogenase, etc.; vasoactive agents, cardiovascular preparations (including calcium channel blockers, beta blockers, beta agonists and antiarrhythmic drugs), antihypertensive drugs, diuretics such as furosemide, spironolactone, etc.; vasodilators; central nervous system stimulants; cough and cold preparations; decongestants; diagnostic drugs; bone growth stimulants and bone resorption inhibitors; muscle relaxants; psychostimulants; sedatives; tranquilizers such as thorazine, diazepam, chlorpromazine HCl, reserpine, chlordiazepoxide HCl, etc.; anti-peptic ulcer drugs such as ranilazole HCl HCl cimetidine, etc.; anti-asthma drugs, antidiarrheal drugs, slimming agents, antithrombotic agents, antitussive drugs, anti-uremic drugs, anti-anginal drugs, appetite suppressants, expectorants, hyperglycemic drugs, hypoglycemic drugs, thyroid and antithyroid drugs, tissue growth agents, uterine relaxants, immunomodulators, including cytokines, interleukins, interferons, colony stimulating factors, tumor necrosis factors, etc.; immunosuppressants, such as rapamycin, tacrolimus, etc.; immunopharmaceuticals; antigens, factors, growth factors, amino acids, peptides and proteins and fragments thereof (whether naturally occurring, chemically synthesized or recombinantly produced) such as LHRH, somatostatin, calcitonin, growth hormone, glucagon-like peptide, growth-releasing factor, angiotensin, FSH, EGF, bone morphogenetic protein (BMP), erythropoietin (EPO), interferon, interleukin, collagen, fibrinogen, insulin, factor VIII, factor IX, etc.; α-glucosidase, Vasopressin, ACTH, human serum albumin, gamma globulin, structural proteins, blood product proteins, complex proteins, antigens or antigenic polypeptides, enzymes, antibodies, monoclonal antibodies, etc.; and nucleic acid molecules (polymeric forms of two or more nucleotides, polynucleotides, ribonucleotides (RNA) or deoxyribonucleotides (DNA), including both double-stranded and single-stranded molecules, gene constructs, expression vectors, antisense molecules, etc.), small molecules (e.g., doxorubicin) and other biologically active macromolecules such as, for example, proteins and enzymes. The pharmaceutical agent can be a bioactive agent for medical (including veterinary) applications and agriculture (such as plants) and other fields. In certain embodiments of the present disclosure, the pharmaceutical agent to be delivered can be a mixture of active agents.

[0398] Representative examples of antibiotics include amikacin, amoxicillin, ampicillin, atovaquone, azithromycin, aztreonam, bacitracin, carbenicillin, cefadroxil, cefazolin, cefdinir, cefditoren, cefepime, cefdirox, cefoperazone, cefotetan, cefoxitin, cefotaxime, cefpodoxime, cefprozil, ceftaroline, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, chloramphenicol, polymyxin E mesylate, cefuroxime, cephalexin, cephradine, cilastatin, cinoxacin, ciprofloxacin, clarithromycin, clindamycin, dalbavancin, dalfopristin, daptomycin, demeclocycline, dicloxacillin, doripenem, doxycycline, eracycline, ertacycline, lincomycin, erythromycin, fidaxomicin, fosfomycin, gatifloxacin, gemifloxacin, gentamicin, imipenem, lefamulin, lincomycin, linezolid, lomefloxacin, loracarbef, meropenem, metronidazole, minocycline, moxifloxacin, nafcillin, nalidixic acid, neomycin, norfloxacin, ofloxacin, omadacycline, oritavancin, oxacillin, oxytetracycline, paromomycin, penicillin, pentamidine, piperacillin, prazomicin, quinupristin, rifaximin, sarecycline, secnidazole, sparfloxacin, spectinomycin, sulfamethoxazole, sulfisoxazole, tedizolid, telocycline, telithromycin, ticarcillin, tigecycline, tobramycin, trimethoprim, trovafloxacin, and vancomycin.

[0399] Representative examples of antiviral agents include, but are not limited to, abacavir, acyclovir, adefovir, amantadine, amprenavir, atazanavir, balavir, baloxavir, boceprevir, cidofovir, cobicistat, daclatasvir, darunavir, delavirdine, didanosine, docasanol, dolutegravir, doravirine, ecoliever, edoxuridine, efavirenz, elvitegravir, emtricitabine, enfuvirtide, entecavir, etravirine, famciclovir, fomivirsen, fosamprenavir, forscarnet, fosnonet, famciclovir, favipiravir, fomivirsen, foscavir, ganciclovir, ibacitabine, idoxuridine, indinavir, inosine, isoprenosine, type I interferon, type II interferon, type III interferon, lamivudine, lemovir , lemovir, lopinavir, loviride, maraviroc, methylthiazone, morphine, nelfinavir, nevirapine, nitazoxanide, oseltamivir, peginterferon alfa-2a, peginterferon alfa-2b, penciclovir, peramivir, pleconaril, podophyllotoxin, pyramidine, raltegravir, remdesevir, ribavirin, rilpivirine, rimantadine, Retamod, monaprevir, ritonavir, saquinavir, simeprevir, sofosbuvir, stavudine, tarabivirin, telaprevir, telbivudine, tenofovir alafenamide, tenofovir disoproxil, tenofovir, tipravir, trifluridine, triamcinolone, tromantanamide, arbidol hydrochloride, valacyclovir, valganciclovir, vidarabine, zalcitabine, zanamivir, and zidovudine.

[0400] Representative examples of anticoagulants include, but are not limited to, heparin, warfarin, rivaroxaban, dabigatran, apixaban, edoxaban, enoxaparin, and fondaparinux.

[0401] Representative examples of antiplatelet agents include, but are not limited to, clopidogrel, ticagrelor, prasugrel, dipyridamole, dipyridamole / aspirin, ticlopidine, and eptifibatide.

[0402] Representative examples of antifungal agents include, but are not limited to, voriconazole, itraconazole, posaconazole, fluconazole, ketoconazole, clotrimazole, isavuconazole, miconazole, caspofungin, anidulafungin, micafungin, griseofulvin, terbinafine, flucytosine, terbinafine, nystatin, and amphotericin B.

[0403] Representative examples of steroidal anti-inflammatory agents include, but are not limited to, hydrocortisone, dexamethasone, prednisolone, prednisone, triamcinolone, methylprednisolone, budesonide, betamethasone, cortisone, and deflazacort. Representative examples of nonsteroidal anti-inflammatory drugs include ibuprofen, naproxen, ketoprofen, tolmetin, etodolac, fenoprofen, flurbiprofen, diclofenac, piroxicam, indomethacin, sulindax, meloxicam, nabumetone, oxaprozin, mefenamic acid, and diflunisal.

[0404] Other examples of active agents include chloroquine, hydrochloroquine, pyridoxal phosphate, vitamin D, and vitamin C.

[0405] Representative examples of anti-cytokine or immunomodulatory agents include, but are not limited to, tocilizumab, salinomycin, bevacizumab, fingolimod, imiquimod, and eculizumab.

[0406] Immunotherapeutic agents may include, but are not limited to, anti-CD40 antibodies, anti-PDL1 antibodies (e.g., atezolizumab, durvalumab, or avelumab), anti-PD1 antibodies, anti-CTLA4 antibodies, programmed death protein 1 (PD-1) inhibitors, or programmed death protein ligand 1 or 2 inhibitors including (e.g., nivolumab (BMS), pembrolizumab (Merck), pidilizumab (CureTech / Teva), AMP-244 (Amplimmune / GSK), BMS-936559 (BMS), and MEDI4736 (Roche / Genentech)), or combinations thereof.

[0407] Representative examples of contraceptives include, but are not limited to, progestogens, estrogens, or any combination thereof. For example, suitable progestogens include, but are not limited to, natural and synthetic compounds with progestational activity, such as, for example, progesterone, chlormadinone acetate, norethisterone, cyproterone acetate, norethisterone acetate, desogestrel, levonorgestrel, drospirenone, trimegestone, norgestrel, norgestimate, methylprogesterone, etonogestrel, gestodene, and other natural and / or synthetic progestogens. For example, suitable estrogens include, but are not limited to, natural and synthetic compounds with estrogenic activity, such as, for example, estradiol (17β-estradiol), 17α-estradiol, estriol, estrone, and esters thereof, such as acetates, sulfates, valerates, or benzoates of these compounds, including, for example, estradiol 17β-cypionate, estradiol 17-propionate, estradiol 3-benzoate, and piperazine estrone sulfate; ethinyl estradiol; conjugated estrogens (natural and synthetic); ethinyl estradiol methyl ether; agonist antiestrogens; and selective estrogen receptor modulators. Other examples of contraceptives include gonadotropin-releasing hormone (GnRh) or its analogs, such as deslorelin, avorelin, leuprorelin, triptorelin, nafarelin, goserelin, buserelin, and fetirelin.

[0408] The term "steroid" refers to compounds belonging to or related to the following exemplary families of compounds: corticosteroids, mineral steroids, and sex steroids (including, for example, potential androgens or estrogens or anti-androgens and anti-estrogens). These include, for example, prednisone, prednisolone, methylprednisolone, triamcinolone, fluocinolone, aldosterone, spironolactone, danazol (also known as OPTINA), and others. In some embodiments, the therapeutic agent may comprise a steroid.

[0409] Exemplary cancer drugs or anticancer agents can include, but are not limited to, antimetabolite anticancer agents and antimitotic anticancer agents and combinations thereof. Various antimetabolite and antimitotic anticancer agents (including single such agents or combinations of such agents) can be used in the methods and compositions described herein.

[0410] Antimetabolite anticancer agents are usually similar in structure to natural metabolites, which participate in the normal metabolic processes of cancer cells, such as the synthesis of nucleic acids and proteins. However, antimetabolites are very different from natural metabolites, so that they interfere with the metabolic processes of cancer cells. In cells, antimetabolites are mistaken for their similar metabolites and are processed by cells in a manner similar to normal compounds. The presence of "decoy" metabolites prevents cells from performing important functions, and cells cannot grow and survive. For example, antimetabolites can destroy cell division by replacing these fraudulent nucleotides into cellular DNA, or by inhibiting key cellular enzymes, preventing DNA replication, to exert cytotoxic activity.

[0411] Thus, in one aspect, the antimetabolite anticancer agent is a nucleotide or nucleotide analog. In certain aspects, for example, the antimetabolite can comprise a purine (e.g., guanine or adenosine) or an analog thereof, or a pyrimidine (cytidine or thymidine) or an analog thereof, with or without a sugar moiety attached.

[0412] Suitable antimetabolite anticancer agents for use in the present disclosure can generally be classified according to the metabolic processes they affect, and can include, but are not limited to, analogs and derivatives of folic acid, pyrimidine, purine, and cytidine. Thus, in one aspect, the antimetabolite is selected from the group consisting of cytidine analogs, folic acid analogs, purine analogs, pyrimidine analogs, and combinations thereof.

[0413] In one particular aspect, for example, the antimetabolite is a cytidine analog. According to this aspect, for example, the cytidine analog can be selected from cytarabine (cytosine arabinoside), azacytidine (5-azacytidine), and salts, analogs, and derivatives thereof.

[0414] In another specific aspect, for example, the antimetabolite is a folic acid analog. Folic acid analogs or antifolates generally work by inhibiting dihydrofolate reductase (DHFR), an enzyme involved in nucleotide formation; when this enzyme is blocked, nucleotides cannot be formed, thereby disrupting DNA replication and cell division. According to certain aspects, for example, the folic acid analog can be selected from the group consisting of normethopterin, methotrexate (methotrexate), pemetrexed, pteropterin, raltitrexed, trimesapatite, and salts, analogs, and derivatives thereof.

[0415] In another specific aspect, for example, the antimetabolite is a purine analog. Purine-based antimetabolites work by inhibiting DNA synthesis, for example, by interfering with the production of purines containing nucleotides, adenine and guanine, thereby stopping DNA synthesis and thus stopping cell division. Purine analogs can also be incorporated into the DNA molecule itself during DNA synthesis, which can interfere with cell division. According to certain aspects, for example, the purine analog can be selected from the group consisting of acyclovir, allopurinol, 2-aminoadenosine, arabinoadenine (ara-A), azacytidine, azathioprine, 8-aza-adenosine, 8-fluoro-adenosine, 8-methoxy-adenosine, 8-oxo-adenosine, cladribine, deoxycoformycin, fludarabine, ganciclovir, 8-aza-guanosine, 8-fluoro-guanosine, 8-methoxy-guanosine, 8-oxo-guanosine, guanosine diphosphate, guanosine diphosphate-β-L-2-aminofucose, guanosine diphosphate-D-arabinose, guanosine diphosphate-2-fluorofucose, guanosine diphosphate fucose, mercaptopurine (6-MP), pentostatin, thiopurine, thioguanine (6-TG), and salts, analogs, and derivatives thereof.

[0416] In another specific aspect, for example, antimetabolites are pyrimidine analogs. Similar to the purine analogs discussed above, pyrimidine-based antimetabolites block pyrimidine-containing nucleotides (cytosine and thymine in DNA; cytosine and uracil in RNA). By acting as "bait", pyrimidine-based compounds can prevent the production of nucleotides, and / or can be incorporated into growing DNA chains and cause them to terminate. According to certain aspects, for example, pyrimidine analogs can be selected from ancitabine, azacytidine, 6-azauridine, bromouracil (e.g., 5-bromouracil), capecitabine, carmofur, chlorouracil (e.g., 5-chlorouracil), cytarabine (cytosine arabinoside), cytosine, dideoxyuridine, 3'-azido-3'-deoxythymidine, 3'-dideoxycytidine-2'-ene, 3'-deoxy-3'-deoxythymidine In one embodiment, the pyrimidine analogue is a pyrimidine analogue comprising a 5-fluorouracil, a 2-fluorodeoxycytidine, a 3-fluoro-3'-deoxythymidine, a fluorouracil, such as a 5-fluorouracil (also known as 5-FU), a 5-methylcytosine, a 5-propynylcytosine, a 5-propynylthymidine, a 5-propynyluracil, a thymine, a uracil, a uridine, and salts, analogs, and derivatives thereof. In one aspect, the pyrimidine analogue is not 5-fluorouracil. In another aspect, the pyrimidine analogue is gemcitabine or a salt thereof.

[0417] In some aspects, the antimetabolite is selected from the group consisting of 5-fluorouracil, capecitabine, 6-mercaptopurine, methotrexate, gemcitabine, cytarabine, fludarabine, pemetrexed and its salts, analogs, derivatives and combinations. In other aspects, the antimetabolite is selected from the group consisting of capecitabine, 6-mercaptopurine, methotrexate, gemcitabine, cytarabine, fludarabine, pemetrexed and its salts, analogs, derivatives and combinations. In a specific aspect, the antimetabolite is not 5-fluorouracil. In particularly preferred aspects, the antimetabolite is gemcitabine or its salts (e.g., HCl gemcitabine). ).

[0418] Other antimetabolite anticancer agents may be selected from, but are not limited to, the group consisting of: acaciferic acid, aminothiadiazole, brequinar sodium, Ciba-Geigy CGP-30694, cyclopentylcytosine, cytarabine phosphate stearate, cytarabine conjugate, Lilly DATHF, MerrelDow DDFC, dezaguanine, dideoxycytidine, dideoxyguanosine, didox, Yoshitomi DMDC, Wellcome EHNA, Merck & Co. EX-015, fazarabine, fludarabine phosphate, N-(2′-furylalkyl)-5-fluorouracil, Daiichi Seiyaku FO-152, 5-FU-fibrinogen, isopropylpyrrolizine, Lilly LY-188011; Lilly LY-264618, meprobamate, Wellcome MZPES, norspermidine, NCI NSC-127716, NCI NSC-264880, NCI NSC-39661, NCI NSC-612567, Warner-Lambert PALA, pentostatin, pirtrexine, plicamycin, Asahi Chemical PL-AC, Takeda TAC-788, thiazofurine, Erbamont TIF, tyrosine kinase inhibitors, Taiho UFT, and uricytin, among others.

[0419] On the one hand, anti-mitotic anticancer agent is microtubule inhibitor or microtubule stabilizer.In general, microtubule stabilizer, such as taxane and epothilone, is combined with the inner surface of β-microtubule chain, and strengthens microtubule assembly by promoting the nucleation and extension phase of polymerization reaction and by reducing the critical tubulin subunit concentration required for microtubule assembly.Different from the microtubule inhibitor (such as vinca alkaloid) that hinders microtubule assembly, microtubule stabilizer (such as taxane) reduces lag time, and significantly makes the dynamic equilibrium between tubulin dimer and microtubule polymer shift to the polymerization direction.Therefore, in one aspect, microtubule stabilizer is taxane or epothilone.In another aspect, microtubule inhibitor is vinca alkaloid.

[0420] In some embodiments, the anticancer agent may comprise a taxane or a derivative or analog thereof. Taxanes may be naturally derived compounds or related forms, or may be chemically synthesized compounds or derivatives thereof that have antitumor properties. Taxanes are a family of terpenoids that include, but are not limited to, paclitaxel. and docetaxel They are primarily derived from the Pacific yew tree Taxus brevifolia and have activity against certain tumors, particularly breast and ovarian tumors. In one aspect, the taxane is docetaxel or paclitaxel. Paclitaxel is the preferred taxane and is considered an antimitotic agent that promotes the assembly of microtubules from tubulin dimers and stabilizes microtubules by preventing depolymerization. This stabilization results in inhibition of the normal dynamic reorganization of the microtubule network, which is essential for interphase and mitotic cell function.

[0421] Also included are various known taxane derivatives, including hydrophilic and hydrophobic derivatives. Taxane derivatives include, but are not limited to, galactose and mannose derivatives described in International Patent Application No. WO 99 / 18113; piperazine and other derivatives described in WO 99 / 14209; taxane derivatives described in WO 99 / 09021, WO 98 / 22451, and U.S. Pat. No. 5,869,680; 6-thio derivatives described in WO 98 / 28288; sulfenamide derivatives described in U.S. Pat. No. 5,821,263; deoxypaclitaxel compounds, such as those described in U.S. Pat. No. 5,440,056; and taxane derivatives described in U.S. Pat. No. 5,415,869. As described above, it further includes prodrugs of paclitaxel, including, but not limited to, those described in WO 98 / 58927; WO 98 / 13059; and U.S. Pat. No. 5,824,701. The taxane may also be a taxane conjugate, such as, for example, paclitaxel-PEG, paclitaxel-dextran, paclitaxel-xylose, docetaxel-PEG, docetaxel-dextran, docetaxel-xylose, etc. Other derivatives are mentioned in references such as "Synthesis and Anticancer Activity of Taxol Derivatives," DGI Kingston et al., Studies in Organic Chemistry, Vol. 26, entitled "New Trends in Natural Products Chemistry" (1986), Atta-ur-Rabman, PWle Quesne, eds. (Elsevier, Amsterdam 1986). Each of these references is hereby incorporated by reference in its entirety.

[0422] Various taxanes can be readily prepared using techniques known to those skilled in the art (see also WO 94 / 07882, WO 94 / 07881, WO 94 / 07880, WO 94 / 07876, WO 93 / 23555, WO 93 / 10076; U.S. Pat. Nos. 5,294,637; 5,283,253; 5,279,949; 5,274,137; 5,202,448; 5,200,534; 5,229,529; and EP 590,267) (each of which is hereby incorporated by reference in its entirety), or obtained from a variety of commercial sources, including, for example, Sigma-Aldrich Co., St. Louis, Mo.

[0423] Alternatively, the anti-mitotic anticancer agent can be a microtubule inhibitor; in a preferred aspect, the microtubule inhibitor is a vinca alkaloid. Generally speaking, vinca alkaloids are mitotic spindle poisons. Vinca alkaloid agents work during mitosis, when chromosomes divide and begin to migrate along the tubules of the mitotic spindle to one of its poles before cell separation. Under the action of these spindle poisons, the spindle becomes disordered due to the dispersion of chromosomes during mitosis, affecting cell reproduction. According to certain aspects, for example, the vinca alkaloid is selected from the group consisting of vinblastine, vincristine, vindesine, vinorelbine and its salts, analogs and derivatives.

[0424] Anti-mitotic anticancer agents can also be epothilones. In general, members of the epothilone class of compounds stabilize microtubule function according to a mechanism similar to that of the taxanes. Epothilones can also cause cell cycle arrest at the G2-M transition phase, leading to cytotoxicity and ultimately apoptosis. Suitable epithiones include epothilone A, epothilone B, epothilone C, epothilone D, epothilone E, and epothilone F, and their salts, analogs, and derivatives. A specific epothilone analog is the epothilone B analog ixabepilone (Ixempra TM ).

[0425] In certain aspects, the antimitotic anticancer agent is selected from the group consisting of a taxane, an epothilone, a vinca alkaloid, and salts and combinations thereof. Thus, for example, in one aspect, the antimitotic agent is a taxane. In this regard, more preferably, the antimitotic agent is paclitaxel or docetaxel, still more preferably paclitaxel. In another aspect, the antimitotic agent is an epothilone (e.g., an epothilone B analog). In another aspect, the antimitotic agent is a vinca alkaloid.

[0426] Examples of anticancer drugs that can be used in the present disclosure include, but are not limited to, thalidomide; platinum coordination complexes such as cisplatin (cis-DDP), oxaliplatin, and carboplatin; anthracenediones such as mitoxantrone; substituted ureas such as hydroxyurea; methylhydrazine derivatives such as procarbazine (N-methylhydrazine, MIH); adrenocortical suppressants such as mitotane (o,p′-DDD) and aminoglutethimide; RXR agonists such as bexarotene; and tyrosine kinase inhibitors such as sunitinib and imatinib.

[0427] Examples of additional anticancer drugs include alkylating agents, antimetabolites, natural products, hormones and antagonists, and other drugs. Alternative names are indicated in parentheses. Examples of alkylating agents include nitrogen mustards, such as mechlorethamine, cyclophosphamide, ifosfamide, melphalan (Alkeran®), and chlorambucil; ethylenimines and methylmelamines, such as hexamethylmelamine and thiotepa; alkyl sulfonates, such as busulfan; nitrosoureas, such as carmustine (BCNU), semustine (methyl-CCNU), lomustine (CCNU), and streptozocin (Zanosar®); DNA synthesis antagonists, such as estramustine phosphate; and triazines, such as dacarbazine (DTIC, dimethyl-triazenoimidazole carboxamide) and temozolomide. Examples of antimetabolites include folic acid analogs, such as methotrexate (amethopterin); pyrimidine analogs, such as fluorouracil (5-fluorouracil, 5-FU, SFU), floxuridine (fluorodeoxyuridine, FUdR), cytarabine (cytosine arabinoside), and gemcitabine; purine analogs, such as mercaptopurine (6-mercaptopurine, 6-MP), thioguanine (6-thioguanine, TG), and pentostatin (2'-deoxycoformycin, deoxycoformycin), cladribine, and fludarabine; and topoisomerase inhibitors, such as amsacrine. Examples of natural products include vinca alkaloids, such as vinblastine (VLB) and vincristine; taxoids, such as paclitaxel, protein-bound paclitaxel (Abraxane), and docetaxel (Taxotere); epipodophyllotoxins, such as etoposide and teniposide; camptothecins, such as topotecan and irinotecan; antibiotics, such as dactinomycin (actinomycin D), daunorubicin (daunomycin, rubidomycin), doxorubicin, histrelin, bleomycin, mitomycin (mitomycin C), idarubicin, epirubicin; enzymes, such as L-asparaginase; and biological response modifiers, such as interferon alpha and interleukin 2.Examples of hormones and antagonists include luteinizing hormone-releasing hormone agonists such as buserelin; adrenocortical steroids such as prednisone and related preparations; progesterone such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate; estrogen such as diethylstilbestrol and ethinyl estradiol and related preparations; estrogen antagonists such as tamoxifen and anastrozole; androgen such as testosterone propionate and fluoxymesterone and related preparations; androgen antagonists such as flutamide and bicalutamide; and gonadotropin-releasing hormone analogs such as leuprolide. Alternative names and trade names for these and additional examples of anticancer drugs, as well as methods of use thereof (including dosing and administration regimens) will be known to those skilled in the art.

[0428] In some aspects, the anticancer agent may comprise a chemotherapeutic agent. Suitable chemotherapeutic agents include, but are not limited to, alkylating agents, antibiotic agents, antimetabolites, hormone agents, plant-derived agents and synthetic derivatives thereof, anti-angiogenic agents, differentiation inducers, cell growth arrest inducers, apoptosis inducers, cytotoxic agents, agents that affect cell bioenergetics (i.e., affect cellular ATP levels and molecules / activities that regulate these levels), biological agents (e.g., monoclonal antibodies, kinase inhibitors, and inhibitors of growth factors and their receptors), gene therapy agents, cell therapies (e.g., stem cells), or any combination thereof.

[0429] According to these aspects, the chemotherapeutic agent is selected from the group consisting of cyclophosphamide, chlorambucil, melphalan, dichloromethyl diethylamine, ifosfamide, busulfan, lomustine, streptozotocin, temozolomide, dacarbazine, cisplatin, carboplatin, oxaliplatin, procarbazine, uracil, methotrexate, pemetrexed, fludarabine, cytarabine, fluorouracil, floxuridine, gemcitabine, capecitabine, vinblastine, vincristine, vinorelbine, etoposide, paclitaxel, docetaxel, doxorubicin, daunorubicin, epirubicin, idarubicin, mitoxantrone, bleomycin, mitomycin, hydroxyurea, topotecan, irinotecan, amsacrine, teniposide, erlotinib hydrochloride, and combinations thereof. Each possibility represents an independent aspect of the present invention.

[0430] The anti-tumor agent can be selected from the group consisting of: abiraterone acetate, Abitrexate (methotrexate), Abraxane (paclitaxel albumin-stabilized nanoparticle formulation), ABVD, ABVE, ABVE-PC, AC, AC-T, Adcetris (broutuximab), ADE, Ado-trastuzumab-emtansine, doxorubicin (doxorubicin hydrochloride), Adrucil (fluorouracil), afatinib bismaleate, Afinitor (everolimus), Akynzeo (netupitant and palonosetron hydrochloride), Aldara (imiquimod), aldesleukin, alemtuzumab, Alinda (pemetrexed disodium), Aloxi (palonosetron hydrochloride), ), Ambochlorin (chlorambucil), Amboclorin (chlorambucil), aminolevulinic acid, anastrozole, aprepitant, Acorda (pamidronate disodium), Arimidex (anastrozole), Aromasin (exemestane), Arranon (nelarabine), arsenic trioxide, Arzerra (ofatumumab), asparaginase Erwinia chrysanthemi, Avastin (bevacizumab), axitinib, azacitidine, BEACOPP, Becenum (carmustine), Beleodaq (belinostat), belinostat, bendamustine hydrochloride, BEP, bevacizumab, bexarotene, Bexxar (tositumomab and iodine I 131 tositumomab), bicalutamide, BiCNU (carmustine), bleomycin, blinatumomab, Blincyto (blinatumomab), bortezomib, Bosulif (bosutinib), bosutinib, velimar, busulfan, busulfan, cabazitaxel, cabozantinib-S-malate, CAF, Campath (alemtuzumab), Camptosar (irinotecan hydrochloride), capecitabine, CAPOX, carboplatin, carboplatin-paclitaxel, carfilzomib, Carmubris (carmustine), carmustine, carmustine implant, Casodex (bicalutamide), CeeNU (lomustine), ceritinib, Cerubidine (doxazosin hydrochloride), Cervarix (recombinant HPV bivalent vaccine), cetuximab, chlorambucil, chlorambucil-prednisone, CHOP, cisplatin, Clafen (cyclophosphamide), clofarabine, Clofarex (clofarabine), Clolar (clofarabine), CMF, Cometriq (cabozantinib-S-malate), COPP, COPP-ABV, Cosmegen (dactinomycin), crizotinib, CVP, cyclophosphamide, Cyfos (ifosfamide), cyamza (ramucirumab), cytarabine, cytarabine, liposomal, Cytosar-U (cytarabine), Cytoxan (cyclophosphamide), dabrafenib, dacarbazine,Dacogen (decitabine), dactinomycin, dasatinib, daunorubicin hydrochloride, decitabine, degarelix, dasatinib, denileukin, denosumab, DepoCyt (liposomal cytarabine), DepoFoam (liposomal cytarabine), dexrazoxane hydrochloride, denosumab, docetaxel, doxorubicin (liposomal doxorubicin hydrochloride), doxorubicin hydrochloride, doxorubicin hydrochloride liposomal, Dox-SL (liposomal doxorubicin hydrochloride), doxorubicin hydrochloride, doxorubicin hydrochloride liposomal, Dox-SL (liposomal doxorubicin hydrochloride), DTIC-Dome (dacarbazine), Efudex (fluorouracil), Elitek (rasburicase), Ellence (epirubicin hydrochloride), Eloxatin (oxaliplatin), eltrombopag, Emend (affinity). Enzalutamide, epirubicin hydrochloride, EPOCH, Erbitux (cetuximab), eribulin mesylate, Erivedge (vimodigib), erlotinib hydrochloride, Erwinaze (asparaginase), Etopophos (etoposide phosphate), etoposide, etoposide phosphate, Evacet (liposomal doxorubicin hydrochloride), everolimus, Evista (raloxifene hydrochloride), exemestane, Fareson (toremifene), Farydak (panobinostat), Faslodex (fulvestrant), FEC, Femara (letrozole), filgrastim, Fludara (fludarabine phosphate), fludarabine phosphate, Fluoroplex (fluorouracil), fluorouracil, Folex (methotrexate), Folex PFS (methotrexate), FOLFIRI, FOLFIRI-bevacizumab, FOLFIRI-cetuximab, FOLFIRINOX, FOLFOX, Folotyn (pralatrexate), FU-LV, fulvestrant, Gardasil (recombinant HPV quadrivalent vaccine), Gardasil 9 (recombinant HPV nine-valent vaccine), Gazyva (otuzumab), gefitinib, gemcitabine hydrochloride, gemcitabine-cisplatin, gemcitabine-oxaliplatin, gemtuzumab ozagribuzumab, Gemzar (gemcitabine hydrochloride), Gilotrif (afatinib bismaleate), Gleevec (imatinib mesylate), Gliadel (carmustine implant), Gliadel wafer (carmustine implant), glutarcapase, goserelin acetate, Halaven (eribulin mesylate), Herceptin (trastuzumab), HPV bivalent vaccine, recombinant, HPV 9-valent vaccine, recombinant, HPV quadrivalent vaccine, recombinant, Hycamtin (topotecan hydrochloride), Hyper-CVAD, Ibrance (palbociclib), ibritumomab tiuxetan, ibrutinib, ICE, Iclusig (ponatinib hydrochloride),Idamycin (idarubicin hydrochloride), idarubicin hydrochloride, idelalisib, Ifex (ifosfamide), ifosfamide, Ifosfamidum (ifosfamide), imatinib mesylate, Imbruvica (ibrutinib), imiquimod, Inlyta (axitinib), interferon alfa-2b, recombinant, intron A (recombinant interferon alfa-2b), iodine I 131 tositumomab and tositumomab, ipilimumab, Iressa (gefitinib), irinotecan hydrochloride, Istodax (romidep), ixabepilone, Ixempra (ixabepilone), Jakafi (ruxolitinib phosphate), Jevtana (cabazitaxel), Kadcyla (Ado-trastuzumab-emtansine), Keoxifene (raloxifene hydrochloride), Kepivance (palifermin), Keytruda (pembrolizumab), Kyp rolis (carfilzomib), lanreotide acetate, lapatinib ditosylate, lenalidomide, lenvatinib mesylate, Lenvima (lenvatinib mesylate), letrozole, folinate, Leukeran (chlorambucil), leuprorelin acetate, Levulan (aminolevulinic acid), Linfolizin (chlorambucil), LipoDox (liposomal doxorubicin hydrochloride), liposomal cytarabine, lomustine, Lupron (leuprorelin acetate), Lupron Depot (leuprolide acetate), Lupron Depot-Ped (leuprolide acetate), Lupron Depot-3Month (leuprolide acetate), Lupron Depot-4 Month (leuprolide acetate), Lynparza (olaparib), Marqibo (liposomal vincristine sulfate), Matulane (procarbazine hydrochloride), Nitrogen mustard hydrochloride, Megace (megestrol acetate), megestrol acetate, Mekinist (trametinib), Mercaptopurine, Mesna, Mesnex (mesna), Methazolastone (temozolomide), Methotrexate, Methotrexate LPF (methotrexate), Mexate (methotrexate), Mexate-AQ (methotrexate), Mitomycin C, Mitoxantrone hydrochloride, Mitozytrex (mitomycin C), MOPP, Mozobil (plerixafor), Mustargen (mechlorethamine hydrochloride), Mutamycin (mitomycin C), Myleran (busulfan), Mylosar (azacitidine), Mylotarg (gemtuzumab ozagamin), Nanoparticulate Paclitaxel (albumin-stabilized nanoparticle formulation of paclitaxel), Navelbine (vinorelbine tartrate), Nelarabine, Neosar (cyclophosphamide), Netupitant, and Palonosetron hydrochloride,Neupogen (filgrastim), Nexavar (sorafenib tosylate), nilotinib, nivolumab, Nolvadex (tamoxifen citrate), Nplate (romiplostim), obetuzumab, Odomzo (sonidegi), OEPA, ofatumumab, OFF, olaparib, homoharringtonine, Oncaspar (pegapagastatin), ondansetron hydrochloride, Ontak (denileukin), Opdivo (nivolumab), OPPA, oxaliplatin, paclitaxel, taxane Albumin-stabilized nanoparticle formulation, PAD, palbociclib, palifermin, palonosetron hydrochloride, palonosetron hydrochloride and netupitant, pamidronate disodium, panitumumab, panobinostat, Paraplat (carboplatin), Paraplatin (carboplatin), pazopanib hydrochloride, pegaptapagastat, peginterferon alfa-2b, PEG-Intron (pegylated interferon alfa-2b), pembrolizumab, pemetrexed disodium, Perjeta (pertuzumab), pertuzumab, Platinol (cisplatin) , Platinol-AQ (cisplatin), Plerixafor, Pomalidomide, Pomalyst (pomalidomide), Ponatinib hydrochloride, Pralatrexate, Prednisone, Procarbazine hydrochloride, Proleukin (aldesleukin), Prolia (denosumab), Promacta (eltrombopag), Provenge (Cipruset-T), Purinethol (mercaptopurine), Purixan (mercaptopurine), Radium-223 dichloride, Raloxifene hydrochloride, Ramucirumab, Rasburicase, R- CHOP, R-CVP, recombinant human papillomavirus (HPV) bivalent vaccine, recombinant human papillomavirus (HPV) 9-valent vaccine, recombinant human papillomavirus (HPV) quadrivalent vaccine, recombinant interferon alfa-2b, regorafenib, R-EPOCH, Revlimid (lenalidomide), Rheumatrex (methotrexate), Rituxan (rituximab), rituximab, romidepsin, romiplostim, Rubidomycin (daunomycin hydrochloride), ruxolitinib phosphate, Sclerosol Intrapleural Aerosol (talc), siltuximab, Siprucept-T, Somatuline Depot (lanreotide acetate), Sonidegi, sorafenib tosylate, Sprycel (dasatinib), STANFORD V, Sterile Talc (talc), Steritalc (talc), Stivarga (regorafenib), sunitinib malate, Sutent (sunitinib malate), Sylatron (pegylated interferon alfa-2b), Sylvant (siltuximab), Synovir (thalidomide), Synribo (homoharringtonine), TAC, Tafinlar (dabrafenib), talc,Tamoxifen citrate, Tarabine PFS (cytarabine), Tarceva (erlotinib hydrochloride), Targretin (bexarotene), Tasigna (nilotinib), Taxol (paclitaxel), Taxotere (docetaxel), Temodar (temozolomide), Temozolomide, Temsirolimus, Thalidomide, Thalomid (thalidomide), Thiotepa, Toposar (etoposide), Topotecan hydrochloride, Toremifene, Torisel (temsirolimus), Tositumomab and Iodine I131 Tositumomab, Totect (dextromethorphan hydrochloride), TPF, Trametinib, Trastuzumab, Treanda (bendamustine hydrochloride), Trisenox (arsenic trioxide), Tykerb (lapatinib ditosylate), Unituxin (denosumab), vandetanib, VAMP, Vectibix (panitumumab), VeIP, Velban (vinblastine sulfate), Velcade (bortezomib), Velsar (vinblastine sulfate), Vemurafenib, VePesid (etoposide), Viadur (leuprorelin acetate), Vidaza (azacitidine), vinblastine sulfate, Vincasar PFS (vincristine sulfate), vincristine sulfate, liposomal vincristine sulfate, vinorelbine tartrate, VIP, vismodegib, Voraxaze (glutarcapase), Vorinostat, Votrient (pazopanib hydrochloride), Wellcovorin (leucovorin), Xalkori (crizotinib), Xeloda (capecitabine), XELIRI, XELOX, Xgeva (denosumab), Xofigo (radium 223 dichloride), Xtandi (enzalutamide), Yervo y (ipilimumab), Zaltrap (ziv-aflibercept), Zelboraf (vemurafenib), Zevalin (ibritumomab tiuxetan), Zinecard (dexrazoxane hydrochloride), Ziv-aflibercept, Zofran (ondansetron hydrochloride), Zoladex (goserelin acetate), zoledronic acid, Zolinza (vorinostat), Zometa (zoledronic acid), Zydelig (idelalisib), Zykadia (ceretinib), and Zytiga (abiraterone acetate).

[0431] Growth factors that can be used as therapeutic agents include, but are not limited to, transforming growth factor-α ("TGF-α"), transforming growth factor ("TGF-β"), platelet-derived growth factor ("PDGF"), fibroblast growth factor ("FGF") (including FGF acidic isoforms 1 and 2), FGF basic form 2 and FGF 4, 8, 9 and 10, nerve growth factor ("NGF") (including NGF 2.5s, NGF7.0s and βNGF and neurotrophic factors), brain-derived neurotrophic factor, cartilage-derived factor, bone growth factor (BGF), basic fibroblast growth factor, insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), granulocyte colony-stimulating factor (G-CSF), insulin-like growth factor (IGF) I and II, hepatocyte growth factor, glial neurotrophic growth factor (GDNF), stem cell factor (SCF), keratinocyte growth factor (KGF), transforming growth factor (TGF) (including TGFα, β, β1, β2, β3) bone growth factor, bone matrix-derived growth factor and bone-derived growth factor, and mixtures thereof.

[0432] Cytokines used as anticancer agents may include, but are not limited to, cardiotrophin, stromal cell-derived factor, macrophage-derived chemokine (MDC), macrophage inflammatory protein 1α (MIP-1α), 2, 3α, 3β, 4, and 5, interleukin (IL)-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, and IL-18. TNF-α and TNF-β, interferons (IFNs) (e.g., interferon α, interferon αA, interferon β, interferon γ, interferon 1α), growth factors (e.g., colony stimulating factor (CSF), granulocyte colony stimulating factor (G-CSF) (e.g., filgrastim ( Amgen)) hematopoietic growth factor (HGF)), melanoma growth stimulating activity (MGSA), monoclonal antibody (mAb) - anti-CD40 antibody, anti-PDL1 antibody (e.g., atezolizumab, durvalumab or avelumab), anti-PD1 antibody, anti-CTLA4 antibody, programmed death protein 1 (PD-1) inhibitor or programmed death protein ligand 1 or 2 inhibitor including (e.g., nivolumab (BMS), pembrolizumab (Merck), pidilizumab (CureTech / Teva), AMP-244 (Amplimmune / GSK), BMS-936559 (BMS) and MEDI4736 (Roche / Genentesch)) or a combination thereof.

[0433] Immunoglobulins useful in the present disclosure include, but are not limited to, IgG, IgA, IgM, IgD, IgE, and mixtures thereof. Some preferred growth factors include VEGF (vascular endothelial growth factor), NGF (nerve growth factor), PDGF-AA, PDGF-BB, PDGF-AB, FGFb, FGFa, and BGF.

[0434] Other molecules that can be used as anticancer agents include, but are not limited to, growth hormone, leptin, leukemia inhibitory factor (LIF), tumor necrosis factor alpha and beta, endostatin, thrombospondin, osteogenic protein-1, bone morphogenetic proteins 2 and 7, osteonectin, somatomedin-like peptide, and osteocalcin.

[0435] Tumor antigens can be based on specific mutations (neo-epitopes) and those mutations expressed by cancer germline genes (antigens common to tumors found in multiple patients, referred to herein as "traditional cancer antigens" or "shared cancer antigens"). In some embodiments, traditional antigens are antigens known to be prevalent in cancers or tumors or in a specific type of cancer or tumor. In some embodiments, traditional cancer antigens are non-mutated tumor antigens. In some embodiments, traditional cancer antigens are mutated tumor antigens. Tumor antigens can include, but are not limited to, CD2, CD19, CD20, CD22, CD27, CD33, CD37, CD38, CD40, CD44, CD47, CD52, CD56, CD70, CD79, CD137, 4-IBB, 5T4, AGS-5, AGS-16, Angiopoietin 2, B2M, B7.1, B7.2, B7DC, B7H1, B7H2, B7H3, BT-062, BTLA, CAIX, C TLA4, Cripto, ED-B, ErbB1, ErbB2, ErbB3, ErbB4, EGFL7, EpCAM, EphA2, EphA3, EphB2, FAP, fibronectin, folate receptor, ganglioside GM3, GD2, glucocorticoid-induced tumor necrosis factor receptor (GITR), gp100, gpA33, GPNMB, ICOS, IGF1R, integrin av, integrin αvβ, LAG-3, Lewis Y, mesothelin, c-MET, MN carbonic anhydrase IX, MUC1, MUC16, nectin-4, NKGD2, NOTCH, OX40, OX40L, PD-1, PDL1, PSCA, PSMA, RANKL, ROR1, ROR2, SLC44A4, Syndecan-1, TACI, TAG-72, Tenascin, TIM3, TRAILR1, TRAILR2, VEGFR-1, VEGFR-2, VEGFR-3, cell tumor antigen p53, abnormal product of ras, cancer antigen 15-3 (CA 15-3), cancer antigen 19-9 (CA 19-9), cancer antigen 125 (CA-125), cancer antigen 195 (CA 195), C219, cancer antigen 549 (CA 549), HER-2 or novel oncoprotein (C-erb B-2), MHC Class II antigens (e.g., human leukocyte antigen (HLA)-A, HLA-B, HLA-C, HLA-A*01, HLA-A*02, or HLA-A*11), MHC class I antigens (e.g., human leukocyte antigen-DR, HLA-DQ, HLA-DP, HLA-DRB1*04, HLA-DRB1*07, or HLA-DRB1), carcinoembryonic antigens (e.g., alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA)),癌症-睾丸抗原(例如MAGEA1、MAGEA2、MAGEA3、MAGEA4、MAGEA5、MAGEA6、MAGEA8、MAGEA9、MAGEA10、MAGEA11、MAGEA12、BAGE、BAGE2、BAGE3、BAGE4、BAGE5、MAGEB1、MAGEB2、MAGEB5、MAGEB6、MAGEB3、MAGEB4、GAGE1、GAGE2A、GAGE3、GAGE4、GAGE5、GAGE6、GAGE7、GAGE8、SSX1、SSX2、SSX2b、SSX3、SSX4、CTAG1B、LAGE-1b、CTAG2、MAGEC1、MAGEC3、SYCP1、BRDT、MAGEC2、SPANXA1、SPANXB1、SPANXC、SPANXD、SPANXN1、SPANXN2、SPANXN3、SPANXN4、SPANXN5、XAGE1D、XAGE1C、XAGE1B、XAGE1、XAGE2、XAGE3、XAGE-3b、XAGE-4 / RP11-167P23.2、XAGE5、DDX43、SAGE1、ADAM2、PAGE5、CT16.2、PAGE1、PAGE2、PAGE2B、PAGE3、PAGE4、LIPI、VENTXP1、IL13RA2、TSP50、CTAGE1、CTAGE-2、CTAGE5、SPA17、ACRBP、CSAG1、CSAG2、DSCR8、MMA1b、DDX53、CTCFL、LUZP4、CASC5、TFDP3、JARID1B、LDHC、MORC1、DKKL1、SPO11、CRISP2、FMR1NB、FTHL17、NXF2、TAF7L、TDRD1、TDRD6、TDRD4、TEX15、FATE1、TPTE、CT45A1、CT45A2、CT45A3、CT45A4、CT45A5、CT45A6、HORMAD1、HORMAD2、CT47A1、CT47A2、CT47A3、CT47A4、CT47A5、CT47A6、CT47A7、CT47A8、CT47A9、CT47A10、CT47A11、CT47B1、SLCO6A1、TAG、LEMD1、HSPB9、CCDC110、ZNF165、SPACA3、CXorf48、THEG、ACTL8、NLRP4、COX6B2、LOC348120、CCDC33、LOC196993、PASD1、LOC647107、TULP2、CT66 / AA884595、PRSS54、RBM46, CT69 / BC040308, CT70 / BI818097, SPINLW1, TSSK6, ADAM29, CCDC36, LOC440934, SYCE1, CPXCR1, TSPY3, TSGA10, HIWI, MIWI, PIWI, PIWIL2, ARMC3, AKAP3, Cxorf61, PBK, C21orf99, OIP5, CEP290, CABYR, SPAG9, MPHOSPH1, ROPN1, PLAC1, CALR3, PRM1, PRM2, CAGE1, TTK, LY6K, IMP-3, AKAP4, DPPA2, KIAA0100, DCAF12, SEMG1, POTED, POTEE, POTEA, POTEG, POTEB, POTEC, POTEH, GOLGAGL2FA, CDCA1, PEPP2, OTOA, CCDC62, GPATCH2, CEP55, FAM46D, TEX14, CTNNA2, FAM133A, LOC130576, ANKRD45, ELOVL4, IGSF11, TMEFF1, TMEFF2, ARX, SPEF2, GPAT2, TMEM108, NOL4, PTPN20A, SPAG4, MAEL, RQCD1, PRAME, TEX101, SPATA19, ODF1, ODF2, ODF3, ODF4, ATAD2, ZNF645, MCAK, SPAG1, SPAG6, SPAG8, SPAG17, FBXO39, RGS22, cyclin Al, C15orf60, CCDC83, TEKT5, NR6A1, TMPRSS12, TPPP2, PRSS55, DMRT1, EDAG, NDR, DNAJB8, CSAG3B, CTAG1A, GAGE12B, GAGE12C, GAGE12D, GAGE12E, GAGE12F, GAGE12G, GAGE12H, GAGE12I, GAGE12J, GAGE13, LOC728137, MAGEA2B, MAGEA9B / LOC728269, NXF2B, SPANXA2, SPANXB2, SPANXE, SSX4B, SSX5, SSX6, SSX7, SSX9, TSPY1D, TSPY1E, TSPY1F, TSPY1G, TSPY1H, TSPY1I, TSPY2, XAGE1E, XAGE2B / CTD-2267G17.3, and / or variants thereof), oncoviral antigens (e.g., human papilloma virus (hpv) or hepatitis B), oncoviral antigens (e.g., Helicobacter pylori), and / or variants thereof.

[0436] Diagnostic agents include gases; metals; commercially available imaging agents for positron emission tomography (PET), computer-assisted tomography (CAT), single photon emission computed tomography, x-rays, fluoroscopy, and magnetic resonance imaging (MRI); and contrast agents. Examples of suitable materials for use as contrast agents in MRI include gadolinium chelates, as well as iron, magnesium, manganese, copper, and chromium. Examples of materials useful for CAT and x-ray imaging include iodine-based materials.

[0437] Vaccines can include isolated proteins or peptides, inactivated organisms and viruses, dead organisms and viruses, genetically altered organisms or viruses, cell extracts and RNA encoding at least one antigenic polypeptide or its immunogenic fragment (e.g., an immunogenic fragment capable of inducing an immune response to the antigenic polypeptide). Active agents can be combined with interleukins, interferons, cytokines and adjuvants such as cholera toxin, alum, Freund's adjuvant, etc. Prophylactics can include antigens of bacterial organisms such as Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Streptococcus pyogenes, Corynebacterium diphtheriae, Listeria monocytogenes, Bacillus anthracis, Clostridium tetani, Clostridium botulinum, Clostridium perfringens, Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus mutans, Pseudomonas aeruginosa, Salmonella typhi, Haemophilus parainfluenzae, Bordetella pertussis, Francisella tularensis, Yersinia pestis, Vibrio cholerae, Legionella pneumophila, tuberculosis, Mycobacterium, Mycobacterium leprae, Treponema pallidum, Leptospira interrogans, Borrelia burgdorferi, Campylobacter jejuni, etc.; Antigens of viruses, such as human metapneumovirus (hMPV), human parainfluenza virus (hPIV) types 1, 2 and 3 (hPIV1, hPIV2 and hPIV3, respectively), respiratory syncytial virus (RSV), measles virus (MeV), coronaviruses (e.g., MERS-CoV, SARS-CoV, SARS-CoV2, HCoV-OC43, HCoV-229E, HCoV-NL63, HCoV-NL, HCoV-NH, HCoV-HKU1), poxviruses (e.g., smallpox, monkeypox), influenza A and B, HIV, varicella-zoster, herpes simplex 1 and 2, cytomegalovirus, Epstein-Barr virus, rotavirus, rhinovirus, adenovirus, papillomavirus, poliovirus, mumps, rabies, rubella, coxsackievirus , equine encephalitis, Japanese encephalitis, yellow fever, Rift Valley fever, hepatitis A, B, C, D and E viruses, etc.; antigens of fungi, protozoa and parasitic organisms, such as Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia rickettsii, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydia psittaci, Chlamydia trachomatis, Plasmodium falciparum, Trypanosoma brucei, Entamoeba histolytica, Toxoplasma gondii, Trichomonas vaginalis, Schistosoma mansoni, etc. These antigens can be in the form of whole killed organisms, peptides, proteins, glycoproteins, carbohydrates, or a combination thereof.

[0438] Nucleic acids that can be introduced according to the methods herein include all types of DNA, cDNA, and RNA sequences. For example, the polynucleotide can be double-stranded DNA, single-stranded DNA, complexed DNA, encapsulated DNA, naked RNA, encapsulated RNA, plasmid RNA, mini RNA, circular RNA, messenger RNA (mRNA), tRNA, short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), antisense RNA (asRNA), self-amplifying mRNA (saRNA), guide RNA (gRNA), cRNA, and combinations thereof. In some embodiments, the nucleic acid can include plasmid DNA, mini DNA, mRNA, self-amplifying RNA, or circular RNA. The nucleic acid can also be a DNA construct, such as an expression vector, an expression vector encoding a desired gene product (e.g., a gene product that is homologous or heterologous to the subject into which it is to be introduced), and the like.

[0439] In some embodiments, nucleic acids (e.g., mRNA) can be used to induce a balanced immune response against an infectious agent. In some embodiments, nucleic acids (e.g., mRNA) can be used to induce a balanced immune response against a virus such as a metapneumovirus such as human metapneumovirus (hMPV), a parainfluenza virus such as human parainfluenza virus (hPIV) 1, 2, and 3 (hPIV1, hPIV2, and hPIV3, respectively), respiratory syncytial virus (RSV), measles virus (MeV), coronaviruses (e.g., MERS-CoV, SARS-CoV, SARS-CoV2, HCoV-OC43, HCoV-229E, HCoV-NL63, etc.), or a combination thereof. In some embodiments, the present invention provides a balanced immune response to respiratory viruses, such as HCoV-NL, HCoV-NH, HCoV-HKU1), poxviruses (e.g., smallpox, monkeypox), influenza A and B, HIV, varicella-zoster, herpes simplex 1 and 2, cytomegalovirus, Epstein-Barr virus, rotavirus, rhinovirus, adenovirus, papillomavirus, poliovirus, mumps, rabies, rubella, coxsackievirus, equine encephalitis, Japanese encephalitis, yellow fever, Rift Valley fever, hepatitis A, B, C, D and E, etc. In some embodiments, nucleic acids (e.g., mRNA) can be used to induce a balanced immune response to respiratory viruses. The term "respiratory virus" refers to a virus that causes respiratory disease in this article. For example, negative sense single stranded RNA viruses of the family Paramyxoviridae, such as human metapneumovirus (hMPV), human parainfluenza virus (hPIV) types 1, 2 and 3 (hPIV1, hPIV2 and hPIV3 respectively), RSV and measles virus (MeV). Another example of a respiratory virus is the coronavirus. Metapneumovirus is an enveloped virus with a positive sense single stranded RNA genome and a helical symmetric nucleocapsid. Coronavirus is a virus species belonging to the order Nidovirale, family Coronaviridae, subfamily Orthocoronavirinae.

[0440] Representative examples of betacoronaviruses include, but are not limited to, Urbani coronavirus 1 (e.g., Betacoronavirus 1, Human coronavirus OC43, Murine coronavirus HKU24, Human coronavirus HKU1, Murine coronavirus), Hubei virus (e.g., Bat Hp-βcoronavirus Zhejiang2013), Merbein virus (e.g., Hedgehog coronavirus 1, Middle East respiratory syndrome-related coronavirus (MERS-CoV), Pipistrelle bat coronavirus HKU5, Flat-faced bat coronavirus HKU4), Nobecovirus (e.g., Fruit bat coronavirus GCCDC1, Fruit bat coronavirus HKU9), BeCoV (e.g., Severe acute respiratory syndrome coronavirus (SARS-CoV), Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0441] Representative examples of gammacoronaviruses include, but are not limited to, cegacovirus (e.g., Beluga coronavirus SQ1) and Igacovirus (e.g., Avian coronavirus (IBV)).

[0442] Representative examples of deltacoronaviruses include, but are not limited to, andecovirus (e.g., Wild duck coronavirus HKU20), buldecovirus (e.g., Nightingale coronavirus HKU11, Porcine coronavirus HKU15 (PorCoV HKU15), Finch subcoronavirus HKU13, Finch coronavirus HKU16), herdecovirus (e.g., Night heron coronavirus HKU19), and moordecovirus (e.g., Common moorhen coronavirus HKU21).

[0443] In some embodiments, the coronavirus is a human coronavirus. Representative examples of human coronaviruses include, but are not limited to, Human coronavirus 229E (HCoV-229E), Human coronavirus OC43 (HCoV-OC43), Human coronavirus HKU1 (HCoV-HKU1), Human coronavirus NL63 (HCoV-NL63), Severe acute respiratory syndrome coronavirus (SARS-CoV), Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and Middle East respiratory syndrome-related coronavirus (MERS-CoV).

[0444] In one embodiment, the coronavirus infection is an infection of the upper and / or lower respiratory tract. The “upper respiratory tract” includes the mouth, nose, sinuses, middle ear, throat, larynx, and trachea. The “lower respiratory tract” includes the bronchi (bronchial tubes) and lungs (bronchioles and alveoli) and the interstitial tissue of the lungs.

[0445] In another embodiment, the coronavirus infection is a gastrointestinal infection. The“gastrointestinal tract” can include any region of the tube from mouth to anus, including the mouth, esophagus, stomach, and intestines. In yet another embodiment, the coronavirus infection is a kidney infection.

[0446] Other indications associated with coronavirus infection are described in Gralinski and Baric, 2015, J. Pathol. 235:185-195 and Cavanagh, 2005, “Coronaviridae: a review of coronavirus and toroviruses”, Coronaviruses with Special Emphasis on First Insights Concerning SARS 1, A. Schmidt, M. H. Wolff, and O. Weber, Birkhauser Verlag Baser, Switzerland, each of which is incorporated by reference herein in its entirety.

[0447] In some embodiments, the nucleic acid disclosed herein can include at least one chemically modified nucleotide. In some embodiments, the at least one chemically modified nucleotide includes a chemically modified nucleobase, a chemically modified ribose, a chemically modified phosphodiester bond, or a combination thereof.

[0448] In one embodiment, the at least one chemically modified nucleotide is a chemically modified nucleobase. In some embodiments, the mRNA can include a heterologous 5’ untranslated region (5’ UTR). In some embodiments, the mRNA can include a heterologous 3’ untranslated region (3’ UTR).

[0449] All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While compositions and methods of the present disclosure have been described in terms of preferred embodiments, it will be apparent to those with ordinary skill in the art that variations can be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are chemically related can be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.

[0450] By way of non-limiting illustration, examples of certain embodiments of the present disclosure are set forth below.

[0451] Example

[0452] To overcome the challenges of current cancer vaccines, we utilized lipid nanoparticle (LNP) delivery technology to deliver nucleic acids (DNA or RNA) to express single-chain trimers (SCTs), composed of peptides, β2-microglobulin (β2m), and MHC class I heavy chains, to bypass antigen processing and enable stable presentation of peptides on the surface of antigen-presenting cells, as well as to deliver universal MHC II-restricted peptides to activate host memory CD4 T cells, thereby enhancing tumor-specific CD8 T cell immunity.

[0453] Example 1: Kb-E6-SCT mRNA induces a robust antigen-specific CD8+ T cell immune response and exhibits potent anti-tumor activity in animal models

[0454] Design and validation of Kb-E6-SCT mRNA

[0455] To establish the ability of mRNA encoding Kb-E6-SCT to enhance the efficacy of mRNA vaccines against HPV E6 / E7-expressing tumors in animal models, we designed and expressed mRNA encoding SCT. SCT consists of peptide, β2m, and mouse MHC class I Kb. Kb-E6-SCT mRNA is expressed in the presence of Figure 1A The in vitro efficacy was studied and the results are shown in Figures 1B to 1E The linearized plasmid template is shown in Figure 1B In vitro transcription of SCT-E6 mRNA is shown in Figure 1C The results of the physicochemical characterization of the LNP formulations and the encapsulation efficacy of L2M6 are shown in Figures 1D to 1E middle. Figure 1D Particle size and polydispersity index are shown, and Figure 1E Zeta potential and encapsulation efficiency are shown. Figure 1F Shown is the expression of SCT-E6 in 293T cells after transfection.

[0456] Animal studies

[0457] C57BL / 6 mice were immunized twice intramuscularly with 1 μg of the indicated vaccine mRNA at two-week intervals. Seven days after the last immunization, the E6-specific T cell population was determined by ELISPOT and intracellular IFN-γ staining using E6-CD8 T cell peptide. The results are shown in Figures 2A to 2C middle.

[0458] The in vivo antitumor efficacy of mRNA-L2M6 prophylactic vaccination was determined. Figure 3A The following diagram is shown: 5 C57BL / c mice were immunized intramuscularly with 1 μg of mRNA / SCT-E6 on days 1 and 14, and subcutaneously with 1×10 5A TC1 tumor cell, which is derived from a well-established cancer cell line that has been transformed by the oncogenic human papillomavirus (HPV) E6 and E7 genes. Tumor growth and survival were monitored. Results are shown in Figure 3B and Figure 3C .

[0459] In vivo efficacy of therapeutic administration of mRNA-L2M6 vaccine was determined. Figure 4A The following figure is shown: 10 female C57BL / 6 mice were injected subcutaneously in the flank with 2x10^5 TC-1 tumor cells on days 1, 8, and 15 after tumor challenge, mice received three doses of L2M6 or PBS immunization. Tumor progression and long-term survival were monitored for each experimental group. Results are shown in Figure 4B and Figure 4C .

[0460] Conclusion:

[0461] In summary, our study shows that mice immunized with Kb-E6-SCT mRNA vaccine exhibited elevated E6-specific CD8+ T cell immune responses. More importantly, our findings revealed that the anti-tumor effect against E6-expressing tumor cells was significantly enhanced. This underscores the effectiveness of our strategy to bypass the antigen processing and presentation machinery, highlighting its potential to significantly boost the efficacy of cancer vaccines.

[0462] Example 2: Therapeutic mRNA vaccine elicits broad and specific CD8 T cell responses targeting HPV 16 E6 in HLA A2.1 transgenic mice

[0463] Design and validation of four mRNAs

[0464] 1. HPV-E6-E7-S2-mRNA (mRNA-1): This DNA construct comprises HPV-16 E6, E7 and the spike glycoprotein of SARS-CoV-2 (subunit S2) (SEQ ID NO: 498), which encodes the full-length HPV-16 E6 / E7 protein (SEQ ID NO: 497) with the known oncogenic transforming region inactivated and a truncated S2 protein, aiming to induce broad E6 and E7-specific T cell responses. In addition, it also enables stimulation of S2-specific CD4 T cell responses, which can enhance the anti-tumor function of CD8 T cells given the widespread exposure to SARS-CoV-2 infection or COVID-19 immunization globally.

[0465] 2. HLA A0201 mRNA (mRNA-2): This DNA construct encodes the HLA A0201 gene (SEQ ID NO: 500), facilitating the overexpression of its protein (SEQ ID NO: 499) to enhance T cell immunity.

[0466] 3. HLA A0201-E6-SCT mRNA (mRNA-3): This DNA construct encodes immunodominant T-cell epitopes derived from HPV16 E6, β2-microglobulin, and HLA A0201 (SEQ ID NOs: 17 and 18).

[0467] 4. HLA A0201-E7-SCT mRNA (mRNA-4): DNA construct encoding immunodominant T cell epitopes derived from HPV16 E7, β2-microglobulin and HLA A0201 (SEQ ID NOs: 19 and 20).

[0468] mRNA was transcribed from the indicated constructs and is shown in Figure 5 middle. Figures 6A to 6C In vitro validation of mRNAs 1-4 after transfection into 293T cells is shown.

[0469] Animal studies

[0470] HLA-A2.1 transgenic mice (C57BL / 6-Mcph1Tg(HLA-A2.1)1Enge / J, strain #003475) were purchased from Jackson Laboratory. Mice were immunized intramuscularly with 1 μg of the indicated vaccine twice, two weeks apart. Spleens were harvested and analyzed fourteen days after the final immunization. Figure 7A .

[0471] CD8+ T cell response analysis: Antigen-specific T cell populations were determined by stimulation with E6, E7 peptide, or E6-CD8 T cell peptide using ELISPOT and intracellular IFN-γ staining. Results are presented in Figures 7B to 7D middle.

[0472] In vitro CTL killing assay: The CD8 T cell response induced by HPV-16 mRNA was further evaluated by in vitro CTL killing assay. Figures 8A to 8B Fourteen days after two immunizations with mRNA 1 and mRNA-3, spleen cells were harvested from HLA-A2.1 transgenic mice. CD8+ T cells were isolated ( Figure 8A ) and co-cultured with tumor cells (TC1 / A2) expressing HPV-16E6 / E7 and transduced with HLA A2.1. Cell death was determined by FACS, as Figure 8B shown.

[0473] in conclusion:

[0474] In summary, the use of vaccines encoding HPV 16 E6 / E7 / S2 and SCT-E6 / E7 mRNAs has shown promising results in HLA A2.1 transgenic mice. The vaccines induced strong and specific CD8 T cell responses targeting the HPV E6 and E7 antigens. Furthermore, the activated CD8 T cells demonstrated the ability to effectively eliminate HLA A2 positive E6 / E7 expressing tumor cells in an in vitro setting. These findings suggest that the SCT technology employed in this vaccine has the potential to enhance the in vivo clearance of established HPV positive cancers in vivo.

Claims

1. A single-stranded trimer nucleic acid encoding a first T cell epitope, β2-microglobulin and an MHC class I heavy chain amino acid sequence.

2. The single-stranded trimeric nucleic acid according to claim 1, wherein the nucleic acid comprises plasmid DNA, minicircle DNA, microRNA, mRNA, self-amplifying RNA, circular RNA, DNA-initiated self-amplifying RNA, or a viral vector.

3. The single-stranded trimeric nucleic acid according to claim 1 or 2, wherein the T cell epitope comprises a viral, bacterial, parasitic or cancer T cell epitope. 4 . The single-stranded trimeric nucleic acid according to claim 1 , wherein the T cell epitope comprises a tumor-specific antigen (TSA), a tumor-associated antigen (TAA), or a virus-derived cancer antigen.

5. The single-stranded trimeric nucleic acid according to any one of claims 1 to 4; wherein the single-stranded trimeric nucleic acid comprises SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 498 or SEQ ID NO:

500.

6. The single-stranded trimeric nucleic acid according to any one of claims 1 to 5; wherein the first T cell epitope comprises an epitope listed in Table 2.

7. The single-stranded trimeric nucleic acid according to any one of claims 1 to 6, wherein the single-stranded trimeric nucleic acid encodes a single-stranded trimer having SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 497 or SEQ ID NO:

499.

8. The single-stranded trimeric nucleic acid according to any one of claims 1 to 7, wherein the single-stranded trimeric nucleic acid comprises a nucleic acid encoding an amino to carboxyl terminal sequential T cell epitope, the β2 microglobulin and an MHC heavy chain sequence.

9. The single-stranded trimeric nucleic acid according to any one of claims 1 to 8, wherein the MHC class I heavy chain comprises HLA-A1:01, HLA-A2:01, HLA-A2:03, HLA-A2:06, HLA-A2:07, HLA-A3:01, HLA-A9, HLA-A10, HLA-A11:01, HLA-A23:01, HLA-A24:02, HLA-A25:01, HLA-A26:01, HLA-A29:02, HLA-A30:01, HLA-A30:02, HLA-A31:01, HLA-A32:03, HLA-A33:04, HLA-A34:05, HLA-A35:06, HLA-A36:07, HLA-A37:08, HLA-A38:09, HLA-A39:01, HLA-A40:01, HLA-A41:01, HLA-A42:02, HLA-A43:03, HLA-A44:03, HLA-A45:01, HLA-A46:01, HLA-A47:02, HLA-A48:03, HLA-A49:04, HLA-A50:01, HLA-A51:01, HLA-A52:01, HLA-A53:02, 2:02, HLA-A33:03, HLA-A34:01, HLA-A34:02, HLA-A68:01, HLA-A68:02, HLA-A74:01, HLA-B5, HLA-B7:02, HLA-B 8:01, HLA-B-12, HLA-B13:01, HLA-B14:02, HLA-B15:01, HLA-B15:02, HLA-B15:03, HLA-B15:18, HLA-B18:01, HL A-B35:01, HLA-B38:02, HLA-B40:01, HLA-B40:02, HLA-B42:01, HLA-B44:02, HLA-B44:03, HLA-B45:01, HLA-B46 :01, HLA-B49:01, HLA-B51:01, HLA-B52:01, HLA-B53:01, HLA-B54:01, HLA-B55:02, HLA-B57:01, HLA-B58:01, H LA-B67:01, HLA-C1:02, HLA-C2:02, HLA-C3:02, HLA-C3:03, HLA-C3:04, HLA-C4:01, HLA-C5:01, HLA-C6:02, HLA -C7:01, HLA-C7:02, HLA-C8:01, HLA-C8:02, HLA-C12:03, HLA-C14:02, HLA-C16:01, HLA-C17:01 or HLA-C18:01 heavy chain. 10 . The single-stranded trimeric nucleic acid according to claim 1 , further comprising a second T cell epitope. The single-stranded trimeric nucleic acid according to claim 10 , wherein the second T cell epitope is an MHC class II restricted epitope.

12. The single-stranded trimeric nucleic acid according to claim 11, wherein the second T cell epitope comprises SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 or an epitope listed in Table 5.

13. The single-stranded trimeric nucleic acid according to any one of claims 1 to 12, wherein the single-stranded trimeric nucleic acid further comprises a flexible first linker between the first T cell epitope and the β2 microglobulin and a second linker between the β2 microglobulin and an MHC class I heavy chain sequence.

14. A composition comprising a single-chain trimer nucleic acid encoding a first T cell epitope, β2-microglobulin, and an MHC class I heavy chain sequence.

15. The composition of claim 14, wherein the nucleic acid comprises plasmid DNA, minicircle DNA, microRNA, mRNA, self-amplifying RNA, circular RNA, or DNA-initiated self-amplifying RNA.

16. The composition of claim 14 or 15, wherein the T cell epitope comprises a viral, bacterial, parasitic or cancer T cell epitope.

17. The composition of any one of claims 14 to 16, wherein the T cell epitope comprises a T cell epitope of a tumor specific antigen (TSA), a tumor associated antigen (TAA), or a virus-derived cancer antigen.

18. The composition of any one of claims 14 to 17; wherein the single-stranded trimeric nucleic acid comprises SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 498, or SEQ ID NO:

500.

19. The composition of any one of claims 14 to 18; wherein the first T cell epitope comprises an epitope listed in Table 2.

20. The composition of any one of claims 14 to 19, wherein the single-stranded trimer nucleic acid encodes a single-stranded trimer having SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 497, or SEQ ID NO:

499.

21. The composition of any one of claims 14 to 20, wherein the single-chain trimer nucleic acid comprises a nucleic acid encoding an amino to carboxyl terminal sequential T cell epitope, the β2 microglobulin, and the MHC heavy chain sequence.

22. The composition of any one of claims 14 to 21, wherein the MHC class I heavy chains include HLA-A1:01, HLA-A2:01, HLA-A2:03, HLA-A2:06, HLA-A2:07, HLA-A3:01, HLA-A9, HLA-A10, HLA-A11:01, HLA-A23:01, HLA-A24:02, HLA-A25:01, HLA-A26:01, HLA-A29:02, HLA-A30:01, HLA-A30:02, HLA-A31:01, HLA-A32:03, HLA-A33:04, HLA-A34:05, HLA-A35:06, HLA-A36:07, HLA-A37:08, HLA-A38:09, HLA-A39:01, HLA-A40:01, HLA-A41:01, HLA-A42:02, HLA-A43:03, HLA-A44:03, HLA-A45:01, HLA-A46:01, HLA-A47:02, HLA-A48:03, HLA-A49:04, HLA-A50:01, HLA-A51:01, HLA-A52:01, HLA-A53:02, HLA-A54:01 2:02, HLA-A33:03, HLA-A34:01, HLA-A34:02, HLA-A68:01, HLA-A68:02, HLA-A74:01, HLA-B5, HLA-B7:02, HLA-B 8:01, HLA-B-12, HLA-B13:01, HLA-B14:02, HLA-B15:01, HLA-B15:02, HLA-B15:03, HLA-B15:18, HLA-B18:01, HL A-B35:01, HLA-B38:02, HLA-B40:01, HLA-B40:02, HLA-B42:01, HLA-B44:02, HLA-B44:03, HLA-B45:01, HLA-B46 :01, HLA-B49:01, HLA-B51:01, HLA-B52:01, HLA-B53:01, HLA-B54:01, HLA-B55:02, HLA-B57:01, HLA-B58:01, H LA-B67:01, HLA-C1:02, HLA-C2:02, HLA-C3:02, HLA-C3:03, HLA-C3:04, HLA-C4:01, HLA-C5:01, HLA-C6:02, HLA -C7:01, HLA-C7:02, HLA-C8:01, HLA-C8:02, HLA-C12:03, HLA-C14:02, HLA-C16:01, HLA-C17:01 or HLA-C18:01 heavy chain.

23. The composition of any one of claims 14 to 20, further comprising a second T cell epitope.

24. The composition of claim 23, wherein the second T cell epitope is an MHC class II restricted epitope.

25. The composition of claim 23, wherein the second T cell epitope comprises SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, or an epitope listed in Table 5.

26. The composition of any one of claims 14 to 25, wherein the single-chain trimer nucleic acid further comprises a flexible first linker between the first T cell epitope and the β2 microglobulin and a second linker between the β2 microglobulin and the MHC class I heavy chain sequence.

27. The composition of any one of claims 14 to 26, wherein the composition is a nanoparticle, a lipid nanoparticle dispersion, a liposomal formulation, a lipid emulsion, a vaccine, a carrier, or any combination thereof.

28. The composition of any one of claims 14 to 27, wherein the composition is a lipid nanoparticle, wherein the lipid nanoparticle comprises 20% to 80% ionizable lipids, cationic lipids, or any combination thereof; 0% to 5% PEGylated lipids; 0% to 40% helper lipids; and 0% to 80% sterols.

29. A lipid nanoparticle comprising: 20% to 80% ionizable lipids, cationic lipids, or any combination thereof; 0% to 5% PEGylated lipids; 0% to 40% helper lipids; 0% to 80% sterols; and A single-chain trimer nucleic acid encoding a first T cell epitope, β2-microglobulin and an MHC class I heavy chain sequence is encapsulated in the lipid nanoparticle.

30. The lipid nanoparticle of claim 29, wherein the nucleic acid comprises plasmid DNA, minicircle DNA, microRNA, mRNA, self-amplifying RNA, circular RNA, or DNA-initiated self-amplifying RNA.

31. The lipid nanoparticle of claim 29 or 30, wherein the T cell epitope comprises a viral, bacterial, parasitic or cancer T cell epitope.

32. The lipid nanoparticle of any one of claims 29 to 31, wherein the T cell epitope comprises a T cell epitope of a tumor specific antigen (TSA), a tumor associated antigen (TAA), or a virus-derived cancer antigen.

33. The lipid nanoparticle of any one of claims 29 to 32; wherein the single-stranded trimeric nucleic acid comprises SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 498, or SEQ ID NO:

500.

34. The lipid nanoparticle of any one of claims 29 to 33; wherein the first T cell epitope comprises an epitope listed in Table 2.

35. The lipid nanoparticle of any one of claims 29 to 34, wherein the single-chain trimer nucleic acid encodes a single-chain trimer having SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 497, or SEQ ID NO:

499.

36. The lipid nanoparticle of any one of claims 29 to 35, wherein the single-stranded trimer nucleic acid comprises a nucleic acid encoding an amino to carboxyl terminal sequential T cell epitope, the β2 microglobulin, and the MHC heavy chain sequence.

37. The lipid nanoparticle of any one of claims 29 to 36, wherein the MHC class I heavy chain comprises HLA-A1:01, HLA-A2:01, HLA-A2:03, HLA-A2:06, HLA-A2:07, HLA-A3:01, HLA-A9, HLA-A10, HLA-A11:01, HLA-A23:01, HLA-A24:02, HLA-A25:01, HLA-A26:01, HLA-A29:02, HLA-A30:01, HLA-A30:02, HLA-A31:01, HLA-A32:03, HLA-A33:04, HLA-A34:05, HLA-A35:06, HLA-A36:07, HLA-A37:08, HLA-A38:09, HLA-A39:01, HLA-A40:01, HLA-A41:01, HLA-A42:02, HLA-A43:03, HLA-A44:03, HLA-A45:01, HLA-A46:01, HLA-A47:02, HLA-A48:03, HLA-A49:04, HLA-A50:01, HLA-A51:01, HLA-A52:01, HLA-A53:02, 2:02, HLA-A33:03, HLA-A34:01, HLA-A34:02, HLA-A68:01, HLA-A68:02, HLA-A74:01, HLA-B5, HLA-B7:02, HLA-B 8:01, HLA-B-12, HLA-B13:01, HLA-B14:02, HLA-B15:01, HLA-B15:02, HLA-B15:03, HLA-B15:18, HLA-B18:01, HL A-B35:01, HLA-B38:02, HLA-B40:01, HLA-B40:02, HLA-B42:01, HLA-B44:02, HLA-B44:03, HLA-B45:01, HLA-B46 :01, HLA-B49:01, HLA-B51:01, HLA-B52:01, HLA-B53:01, HLA-B54:01, HLA-B55:02, HLA-B57:01, HLA-B58:01, H LA-B67:01, HLA-C1:02, HLA-C2:02, HLA-C3:02, HLA-C3:03, HLA-C3:04, HLA-C4:01, HLA-C5:01, HLA-C6:02, HLA -C7:01, HLA-C7:02, HLA-C8:01, HLA-C8:02, HLA-C12:03, HLA-C14:02, HLA-C16:01, HLA-C17:01 or HLA-C18:01 heavy chain.

38. The lipid nanoparticle of any one of claims 29 to 37, further comprising a second T cell epitope.

39. The lipid nanoparticle of claim 38, wherein the second T cell epitope is an MHC class II restricted epitope.

40. The lipid nanoparticle of claim 38, wherein the second T cell epitope comprises SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, or an epitope listed in Table 5.

41. The lipid nanoparticle of any one of claims 29 to 40, wherein the single-stranded trimer nucleic acid further comprises a flexible first linker between the first T cell epitope and the β2 microglobulin and a second linker between the β2 microglobulin and the MHC class I heavy chain sequence.

42. A pharmaceutical composition comprising the single-stranded trimeric nucleic acid according to any one of claims 1 to 13, the composition according to any one of claims 14 to 28, or the lipid nanoparticle according to any one of claims 29 to 41.

43. A cell comprising the single-stranded trimeric nucleic acid according to any one of claims 1 to 13, the composition according to any one of claims 14 to 28, or the lipid nanoparticle according to any one of claims 29 to 41 or the pharmaceutical composition according to claim 42.

44. The cell of claim 37, wherein the cell comprises a dendritic cell, a B cell, a macrophage, or other cell derived from a subject that can be used for immunotherapy.

45. A method for activating and / or amplifying antigen-presenting cells, comprising co-culturing the antigen-presenting cells and cells comprising the single-stranded trimer nucleic acid according to any one of claims 1 to 13, the compositions according to any one of claims 14 to 28, or the lipid nanoparticles according to any one of claims 29 to 41, or the pharmaceutical composition according to claim 42.

46. ​​A method for treating a subject suffering from a viral infection, a bacterial infection, a parasitic infection and / or a cancer, the method comprising administering to the subject a therapeutically effective amount of a single-stranded trimeric nucleic acid according to any one of claims 1 to 13, a composition according to any one of claims 14 to 28, or a lipid nanoparticle according to any one of claims 29 to 41, a pharmaceutical composition according to claim 42, or a cell according to any one of claims 43 to 44.

47. A method for treating a subject suffering from a viral infection, a bacterial infection, a parasitic infection and / or a cancer, the method comprising obtaining antigen-presenting cells from the subject; contacting the antigen-presenting cells from the subject with a single-stranded trimeric nucleic acid according to any one of claims 1 to 13, a composition according to any one of claims 14 to 28, or a lipid nanoparticle according to any one of claims 29 to 41, or a pharmaceutical composition according to claim 42, thereby activating the antigen-presenting cells, and administering the activated cells to the subject.

48. The method of claim 45 or claim 47, wherein the antigen presenting cells comprise dendritic cells, B cells, macrophages, or other cells derived from a subject that can be used for immunotherapy.

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