Nucleic acid-based cancer vaccines and methods thereof

Abstract

This paper describes a composition and a method for using the same.The composition described herein can comprise a single-chain trimeric nucleic acid encoding a first T cell epitope, β2 microglobulin, and an MHC class I heavy chain sequence.The method described herein can be used to activate and / or expand antigen-presenting cells.The method described herein can also be used to treat or prevent viral infection, bacterial infection, parasitic infection, and / or cancer in subjects.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 431,571, filed December 9, 2022, the contents of which are incorporated herein in their entirety.

[0002] Sequence Listing Reference The Sequence Listing, created on December 8, 2023, and submitted on December 11, 2023 as a text file named "11538-006WO1_ST26.xml" with a file size of 679,893 bytes, is incorporated herein by reference in accordance with 37 C.FR § 1.52(e)(5). [Background technology]

[0003] Tumor-specific antigens (TSAs) or tumor-associated antigens (TAAs) are essential targets for cancer vaccines, which 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 results, and the main reasons for their ineffectiveness are at least the following: 1) These tumor antigen-derived peptides have low MHC binding affinity and / or abundance, and they must compete with numerous endogenous self-antigen- or other pathogen-derived peptides for binding to MHC I molecules. Because MHC I does not discriminate between self- and non-self-derived peptides, the processing and MHC I presentation efficiency of these tumor peptides is usually inefficient, resulting in clinically ineffective antitumor CD8 T cell responses. 2) Although most cancer vaccines are designed to activate antigen-specific CD8 T cell responses, increasing evidence supports the important role of CD4 T cells not only in cellular immunity but also in providing the help needed for tumor-specific cytotoxic T lymphocytes (CTLs), including activating antigen-presenting cells, promoting CD8 T cell homing to tumor tissue, and generating effective memory CD8 T cells. 3) The vaccine delivery platform is either not sufficient to express tumor antigens or is unable to deliver antigens to antigen-presenting cells (APCs), specialized cells that effectively activate T cells.

[0004] Thus, there remains a need for compositions and methods for treating and preventing bacterial infections, viral infections, parasitic infections, and / or cancer.

[0005] The compositions and methods disclosed herein address these and other needs. Summary of the Invention

[0006] Provided herein are single-stranded nucleic acid trimers, compositions comprising the single-stranded trimeric nucleic acids described herein, and methods of using the same.

[0007] Described herein are single-chain trimeric 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 trimeric nucleic acids encoding a first T cell epitope, β2 microglobulin, and an MHC class I heavy chain sequence.

[0008] In some embodiments, the single-chain trimer 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 can comprise 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.

[0009] In some embodiments, the single chain trimer nucleic acid can include nucleic acids encoding, in amino to carboxy terminal order, a T cell epitope, β2 microglobulin, and an MHC heavy chain sequence.

[0010] In some embodiments, the single-chain trimeric nucleic acid can further comprise a flexible first linker between the first T cell epitope and β2 microglobulin and a second linker between the β2 microglobulin and the MHC class I heavy chain sequence.

[0011] In some embodiments, the composition can further comprise a pharmaceutically acceptable carrier. In some aspects, pharmaceutical compositions are disclosed herein, which can be a single-chain trimeric 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 can be a nanoparticle, a lipid nanoparticle dispersion, a liposome formulation, a lipid emulsion, a vaccine, a vector, or any combination thereof.

[0012] In some embodiments, the pharmaceutical composition can include lipid nanoparticles comprising a single-chain trimeric nucleic acid (e.g., mRNA) encoding a first T cell epitope, β2 microglobulin, and an MHC class I heavy chain sequence.

[0013] 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.

[0014] Also described herein are methods for activating and / or expanding antigen-presenting cells, the method comprising co-culturing antigen-presenting cells with cells comprising the single-stranded trimeric nucleic acid described herein, the composition described herein, the lipid nanoparticle described herein, or the pharmaceutical composition described herein. In some embodiments, the antigen-presenting cells can be dendritic cells, B cells, macrophages, or other cells derived from a subject that can be used for immunotherapy.

[0015] Also described herein are methods of treating a subject having a viral infection, a bacterial infection, a parasitic infection, and / or cancer, the method 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.

[0016] Also described herein are methods for treating a subject with a viral infection, a bacterial infection, a parasitic infection, and / or 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 described herein, a composition described herein, a lipid nanoparticle described herein, or a pharmaceutical composition described herein to thereby activate the antigen-presenting cells, and administering the activated antigen-presenting cells to the subject. In some embodiments, the antigen-presenting cells can include dendritic cells, B cells, macrophages, or other cells derived from the subject that can be used for immunotherapy.

[0017] The details of one or more embodiments of the disclosure are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the disclosure will be apparent from the description and drawings, and from the claims. [Brief explanation of the drawings]

[0018] [Figure 1A] 1 shows the design and in vitro efficacy of Kb-E6-SCT mRNA. 2 shows the design of SCT-E6 mRNA. [Figure 1B] 1 shows the design and in vitro potency of Kb-E6-SCT mRNA. A linearized plasmid template is shown. [Figure 1C] 1 shows the design and in vitro efficacy of Kb-E6-SCT mRNA. 2 shows in vitro transcription of SCT-E6 mRNA. [Figure 1D]The design and in vitro efficacy of Kb-E6-SCT mRNA are shown. The physicochemical properties of lipid nanoparticle (LNP) formulations and the encapsulation efficacy of L2M6 are shown (L2M6 refers to an LNP formulation of Kb-E6-SCT mRNA using the ionizable lipid ARV-T1). The particle size and polydispersity index are shown. [Figure 1E] The design and in vitro efficacy of Kb-E6-SCT mRNA are shown. The physicochemical properties of lipid nanoparticle (LNP) formulations and the encapsulation efficacy of L2M6 are shown (L2M6 refers to an LNP formulation of Kb-E6-SCT mRNA using the ionizable lipid ARV-T1). Zeta potential and encapsulation efficiency are also shown. [Figure 1F] 1 shows the design and in vitro efficacy of Kb-E6-SCT mRNA. 2 shows the expression of SCT-E6 in 293T cells after transfection. [Figure 2A] In vivo validation: C57BL / 6 mice were immunized intramuscularly with 1 μg of mRNA of the indicated LNP-formulated vaccine twice, 2 weeks apart. Seven days after the final immunization, E6-CD8 T cell peptide was used to determine E6-specific T cell populations by ELISPOT (Figures 2A-2B) (2A) images and (2B) graphs, and intracellular IFN-gamma staining (Figures 2C-2D). [Figure 2B] In vivo validation: C57BL / 6 mice were immunized intramuscularly with 1 μg of mRNA of the indicated LNP-formulated vaccine twice, 2 weeks apart. Seven days after the final immunization, E6-CD8 T cell peptide was used to determine E6-specific T cell populations by ELISPOT (Figures 2A-2B) (2A) images and (2B) graphs, and intracellular IFN-gamma staining (Figures 2C-2D). [Figure 2C] In vivo validation: C57BL / 6 mice were immunized intramuscularly with 1 μg of mRNA of the indicated LNP-formulated vaccine twice, 2 weeks apart. Seven days after the final immunization, E6-CD8 T cell peptide was used to determine E6-specific T cell populations by ELISPOT (Figures 2A-2B) (2A) images and (2B) graphs, and intracellular IFN-gamma staining (Figures 2C-2D). [Figure 3A] Figure 3A shows the in vivo antitumor efficacy of prophylactic vaccination with mRNA-L2M6. Figure 3A is a diagram of five C57BL / c mice immunized intramuscularly with 1 μg of mRNA / SCT-E6 on days 1 and 14, and then injected subcutaneously with 1×10 TC1 tumor cells on day 21. Tumor growth (Figure 3B) and survival (Figure 3C) were monitored. [Figure 3B] Figure 3A shows the in vivo antitumor efficacy of prophylactic vaccination with mRNA-L2M6. Figure 3A is a diagram of five C57BL / c mice immunized intramuscularly with 1 μg of mRNA / SCT-E6 on days 1 and 14, and then injected subcutaneously with 1×10 TC1 tumor cells on day 21. Tumor growth (Figure 3B) and survival (Figure 3C) were monitored. [Figure 3C] Figure 3A shows the in vivo antitumor efficacy of prophylactic vaccination with mRNA-L2M6. Figure 3A is a diagram of five C57BL / c mice immunized intramuscularly with 1 μg of mRNA / SCT-E6 on days 1 and 14, and then injected subcutaneously with 1×10 TC1 tumor cells on day 21. Tumor growth (Figure 3B) and survival (Figure 3C) were monitored. [Figure 4A] Figure 4A shows the in vivo efficacy of therapeutic administration of mRNA-L2M6 vaccine. Figure 4A shows the experimental scheme of 10 female C57BL / 6 mice subcutaneously injected with 2 x 10^5 TC-1 tumor cells into the flank on days 1, 8, and 15 after tumor challenge. The mice received three doses of L2M6 or PBS immunization. Figure 4B shows a graph of tumor progression. Figure 4C shows a graph of long-term survival for each experimental group. [Figure 4B] Figure 4A shows the in vivo efficacy of therapeutic administration of mRNA-L2M6 vaccine. Figure 4A shows the experimental scheme of 10 female C57BL / 6 mice subcutaneously injected with 2 x 10^5 TC-1 tumor cells into the flank on days 1, 8, and 15 after tumor challenge. The mice received three doses of L2M6 or PBS immunization. Figure 4B shows a graph of tumor progression. Figure 4C shows a graph of long-term survival for each experimental group. [Figure 4C]Figure 4A shows the in vivo efficacy of therapeutic administration of mRNA-L2M6 vaccine. Figure 4A shows the experimental scheme of 10 female C57BL / 6 mice subcutaneously injected with 2 x 10^5 TC-1 tumor cells into the flank on days 1, 8, and 15 after tumor challenge. The mice received three doses of L2M6 or PBS immunization. Figure 4B shows a graph of tumor progression. Figure 4C shows a graph of long-term survival for each experimental group. [Figure 5] In vitro synthesis of HPV mRNA vaccines. HPV mRNAs 1 to 3 were synthesized by in vitro transcription with T7 RNA polymerase and run on a 0.8% MOPS agarose gel. [Figure 6A] In vitro validation of the HPV mRNA vaccine is shown. HPV mRNAs 1–4 were transfected into 293T cells, and expression was detected at 48 hours. (6A–6B) The expression levels of HPV mRNAs 1 (6A) and 2–4 (6B) were detected by flow cytometry using anti-Sars-S2 (6A) and anti-HLA-A2 (6B) antibodies, respectively. (6C) Expression of HPV mRNA-1 was detected by Western blot using E7 antibody. [Figure 6B] In vitro validation of the HPV mRNA vaccine is shown. HPV mRNAs 1–4 were transfected into 293T cells, and expression was detected at 48 hours. (6A–6B) The expression levels of HPV mRNAs 1 (6A) and 2–4 (6B) were detected by flow cytometry using anti-Sars-S2 (6A) and anti-HLA-A2 (6B) antibodies, respectively. (6C) Expression of HPV mRNA-1 was detected by Western blot using E7 antibody. [Figure 6C]In vitro validation of the HPV mRNA vaccine is shown. HPV mRNAs 1–4 were transfected into 293T cells, and expression was detected at 48 hours. (6A–6B) The expression levels of HPV mRNAs 1 (6A) and 2–4 (6B) were detected by flow cytometry using anti-Sars-S2 (6A) and anti-HLA-A2 (6B) antibodies, respectively. (6C) Expression of HPV mRNA-1 was detected by Western blot using E7 antibody. [Figure 7A] These results demonstrate that HPV mRNA vaccines induce robust T cell and antibody responses in hHLA-A2 transgenic mice. (7C-7D) Splenocytes were isolated from mouse spleens and subjected to ELISPOT assays using stimulation with the E6 mixed peptide pool, E7 mixed peptide pool, and E6 single peptide pool. Representative images of ELISpot plate readouts are shown in 7C, and IFNγ-producing T lymphocytes were quantified as shown in 7D. (7E-7I) Splenocytes were isolated from mouse spleens and subjected to flow cytometry intracellular staining using stimulation with the E6+E7 mixed peptide pool (7F-7G) or E6 single peptide pool (7H-7I). [Figure 7B] These results demonstrate that HPV mRNA vaccines induce robust T cell and antibody responses in hHLA-A2 transgenic mice. (7C-7D) Splenocytes were isolated from mouse spleens and subjected to ELISPOT assays using stimulation with the E6 mixed peptide pool, E7 mixed peptide pool, and E6 single peptide pool. Representative images of ELISpot plate readouts are shown in 7C, and IFNγ-producing T lymphocytes were quantified as shown in 7D. (7E-7I) Splenocytes were isolated from mouse spleens and subjected to flow cytometry intracellular staining using stimulation with the E6+E7 mixed peptide pool (7F-7G) or E6 single peptide pool (7H-7I). [Figure 7C]These results demonstrate that HPV mRNA vaccines induce robust T cell and antibody responses in hHLA-A2 transgenic mice. (7C-7D) Splenocytes were isolated from mouse spleens and subjected to ELISPOT assays using stimulation with the E6 mixed peptide pool, E7 mixed peptide pool, and E6 single peptide pool. Representative images of ELISpot plate readouts are shown in 7C, and IFNγ-producing T lymphocytes were quantified as shown in 7D. (7E-7I) Splenocytes were isolated from mouse spleens and subjected to flow cytometry intracellular staining using stimulation with the E6+E7 mixed peptide pool (7F-7G) or E6 single peptide pool (7H-7I). [Figure 7D] These results demonstrate that HPV mRNA vaccines induce robust T cell and antibody responses in hHLA-A2 transgenic mice. (7C-7D) Splenocytes were isolated from mouse spleens and subjected to ELISPOT assays using stimulation with the E6 mixed peptide pool, E7 mixed peptide pool, and E6 single peptide pool. Representative images of ELISpot plate readouts are shown in 7C, and IFNγ-producing T lymphocytes were quantified as shown in 7D. (7E-7I) Splenocytes were isolated from mouse spleens and subjected to flow cytometry intracellular staining using stimulation with the E6+E7 mixed peptide pool (7F-7G) or E6 single peptide pool (7H-7I). [Figure 7E] These results demonstrate that HPV mRNA vaccines induce robust T cell and antibody responses in hHLA-A2 transgenic mice. (7C-7D) Splenocytes were isolated from mouse spleens and subjected to ELISPOT assays using stimulation with the E6 mixed peptide pool, E7 mixed peptide pool, and E6 single peptide pool. Representative images of ELISpot plate readouts are shown in 7C, and IFNγ-producing T lymphocytes were quantified as shown in 7D. (7E-7I) Splenocytes were isolated from mouse spleens and subjected to flow cytometry intracellular staining using stimulation with the E6+E7 mixed peptide pool (7F-7G) or E6 single peptide pool (7H-7I). [Figure 7F]These results demonstrate that HPV mRNA vaccines induce robust T cell and antibody responses in hHLA-A2 transgenic mice. (7C-7D) Splenocytes were isolated from mouse spleens and subjected to ELISPOT assays using stimulation with the E6 mixed peptide pool, E7 mixed peptide pool, and E6 single peptide pool. Representative images of ELISpot plate readouts are shown in 7C, and IFNγ-producing T lymphocytes were quantified as shown in 7D. (7E-7I) Splenocytes were isolated from mouse spleens and subjected to flow cytometry intracellular staining using stimulation with the E6+E7 mixed peptide pool (7F-7G) or E6 single peptide pool (7H-7I). [Figure 7G] These results demonstrate that HPV mRNA vaccines induce robust T cell and antibody responses in hHLA-A2 transgenic mice. (7C-7D) Splenocytes were isolated from mouse spleens and subjected to ELISPOT assays using stimulation with the E6 mixed peptide pool, E7 mixed peptide pool, and E6 single peptide pool. Representative images of ELISpot plate readouts are shown in 7C, and IFNγ-producing T lymphocytes were quantified as shown in 7D. (7E-7I) Splenocytes were isolated from mouse spleens and subjected to flow cytometry intracellular staining using stimulation with the E6+E7 mixed peptide pool (7F-7G) or E6 single peptide pool (7H-7I). [Figure 7H] These results demonstrate that HPV mRNA vaccines induce robust T cell and antibody responses in hHLA-A2 transgenic mice. (7C-7D) Splenocytes were isolated from mouse spleens and subjected to ELISPOT assays using stimulation with the E6 mixed peptide pool, E7 mixed peptide pool, and E6 single peptide pool. Representative images of ELISpot plate readouts are shown in 7C, and IFNγ-producing T lymphocytes were quantified as shown in 7D. (7E-7I) Splenocytes were isolated from mouse spleens and subjected to flow cytometry intracellular staining using stimulation with the E6+E7 mixed peptide pool (7F-7G) or E6 single peptide pool (7H-7I). [Figure 7I]These results demonstrate that HPV mRNA vaccines induce robust T cell and antibody responses in hHLA-A2 transgenic mice. (7C-7D) Splenocytes were isolated from mouse spleens and subjected to ELISPOT assays using stimulation with the E6 mixed peptide pool, E7 mixed peptide pool, and E6 single peptide pool. Representative images of ELISpot plate readouts are shown in 7C, and IFNγ-producing T lymphocytes were quantified as shown in 7D. (7E-7I) Splenocytes were isolated from mouse spleens and subjected to flow cytometry intracellular staining using stimulation with the E6+E7 mixed peptide pool (7F-7G) or E6 single peptide pool (7H-7I). [Figure 8A] These results demonstrate that HPV mRNA induced a strong cell-mediated immune response. hHLA-A2 transgenic mice were immunized intramuscularly with 5 μg of LNP-formulated mRNA vaccine (HPV mRNA 1+3) on Days 0 and 14. Mouse spleens were collected on Day 28. Splenocytes were isolated from the mouse spleens. (8A-8B) Mouse CD8 T cells were isolated using a kit and detected by flow cytometry using an anti-mouse CD8 T cell antibody. (8C) T cell-mediated immune responses were evaluated by a cytotoxic T lymphocyte assay (CTL assay). Tumor target cells (T), TA2-Luc stable cells (a TC-1 cell line overexpressing hHLA-A2 and luciferase), were seeded onto a 96-well plate on Day 0 at a cell suspension of 2 × 104 cells / well. Assume the cancer cells doubled overnight. Isolated CD8 T cells from A as effector cells (E) were added to tumor target cells at 1 × 10 / well (E:T = 2.5:1) on D1. Cells were subjected to flow cytometry-based cytotoxicity evaluation using Annexin V and Helix NP on D3. [Figure 8B]These results demonstrate that HPV mRNA induced a strong cell-mediated immune response. hHLA-A2 transgenic mice were immunized intramuscularly with 5 μg of LNP-formulated mRNA vaccine (HPV mRNA 1+3) on Days 0 and 14. Mouse spleens were collected on Day 28. Splenocytes were isolated from the mouse spleens. (8A-8B) Mouse CD8 T cells were isolated using a kit and detected by flow cytometry using an anti-mouse CD8 T cell antibody. (8C) T cell-mediated immune responses were evaluated by a cytotoxic T lymphocyte assay (CTL assay). Tumor target cells (T), TA2-Luc stable cells (a TC-1 cell line overexpressing hHLA-A2 and luciferase), were seeded onto a 96-well plate on Day 0 at a cell suspension of 2 × 104 cells / well. Assume the cancer cells doubled overnight. Isolated CD8 T cells from A as effector cells (E) were added to tumor target cells at 1 × 10 / well (E:T = 2.5:1) on D1. Cells were subjected to flow cytometry-based cytotoxicity evaluation using Annexin V and Helix NP on D3. [Figure 8C] These results demonstrate that HPV mRNA induced a strong cell-mediated immune response. hHLA-A2 transgenic mice were immunized intramuscularly with 5 μg of LNP-formulated mRNA vaccine (HPV mRNA 1+3) on Days 0 and 14. Mouse spleens were collected on Day 28. Splenocytes were isolated from the mouse spleens. (8A-8B) Mouse CD8 T cells were isolated using a kit and detected by flow cytometry using an anti-mouse CD8 T cell antibody. (8C) T cell-mediated immune responses were evaluated by a cytotoxic T lymphocyte assay (CTL assay). Tumor target cells (T), TA2-Luc stable cells (a TC-1 cell line overexpressing hHLA-A2 and luciferase), were seeded onto a 96-well plate on Day 0 at a cell suspension of 2 × 104 cells / well. Assume the cancer cells doubled overnight. Isolated CD8 T cells from A as effector cells (E) were added to tumor target cells at 1 × 10 / well (E:T = 2.5:1) on D1. Cells were subjected to flow cytometry-based cytotoxicity evaluation using Annexin V and Helix NP on D3. DETAILED DESCRIPTION OF THE INVENTION

[0019] Like reference symbols in the various drawings indicate like elements.

[0020] Although several embodiments of the present disclosure have been described, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

[0021] definition To facilitate understanding of the disclosure set forth herein, several terms are defined below. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

[0022] General definition As used in this specification and the claims that follow, the terms "comprise" (and its forms, derivatives, or variations, such as "comprising" and "comprises") and "include" (and its forms, derivatives, or variations, such as "including" and "includes") are inclusive (i.e., open-ended) and do not exclude additional elements or steps. For example, the terms "comprise" and / or "comprising," as used herein, specify the presence of specified properties, integers, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other properties, integers, steps, operations, elements, components, and / or groups thereof. Unless otherwise noted, all numbers expressing quantities of ingredients, reaction conditions, geometries, dimensions, and the like used in the specification and claims are to be understood as at least and are not intended to limit the application of the doctrine of equivalents to the scope of the claims, but should be construed in light of the number of significant digits and ordinary rounding approaches.

[0023] Thus, these terms are intended to cover not only the recited element(s) or step(s), but may also include other elements or steps not expressly recited. Furthermore, as used herein, the use of the terms "a," "an," and "the" when used in conjunction with elements may mean "one," but are also consistent with the meanings of "one or more," "at least one," and "one or more than one." Thus, an element preceded by "a" or "an" does not, without further constraints, preclude the presence of additional identical elements.

[0024] Ranges can be expressed herein as from "about" one particular value and / or to "about" another particular value. "About" means within 5% of a value, e.g., within 4, 3, 2, or 1% of a value. When such ranges are expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, it will be understood that the particular value forms another embodiment by use of the antecedent "about." It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that, as several values ​​are disclosed herein, each value is also disclosed herein as "about" that particular value in addition to the value itself. For example, if the value "10" is disclosed, then "about 10" is also disclosed. A range can be interpreted to include both the start and end of the range. For example, a range of 10% to 20% (i.e., a range of 10%-20%) can include 10%, can also include 20%, and can include percentages between 10% and 20%, unless expressly stated otherwise herein.

[0025] As used herein, the terms "may," "optionally," and "optionally may" are used interchangeably and are meant to include cases where the condition occurs as well as cases where the condition does not occur. Thus, for example, a statement that a formulation "may include an excipient" is meant to include cases where the formulation includes an excipient as well as cases where the formulation does not include an excipient.

[0026] Where combinations, subsets, groups, etc. of elements (e.g., combinations of components in a composition or combinations of steps in a method) are disclosed, it is understood that specific reference to each of the various individual and collective combinations and permutations of those elements may not be explicitly disclosed, but each is specifically contemplated and described herein.

[0027] "Administration" to a subject includes any route of introducing or delivering an agent to a subject. Administration can be by any suitable route, including oral, topical, transcutaneous, transdermal, intra-joint, intra-arteriolar, intradermal, intraventricular, intralesional, intranasal, rectal, vaginal, by inhalation, via an implanted reservoir, parenteral (e.g., subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intraperitoneal, intrahepatic, intralesional, and intracranial injection or infusion techniques), and the like. As used herein, "concurrent administration," "administration in combination," "co-administration," or "administered simultaneously" means that compounds are administered at the same time or essentially sequentially. In the latter case, the two compounds are administered sufficiently close in time that the results observed are indistinguishable from those that would be achieved if the compounds were administered at the same time. "Systemic administration" refers to the introduction or delivery of an agent to a subject via a route that introduces or delivers the agent to a wide area of ​​the subject's body (e.g., more than 50% of the body), for example, through an entrance into the circulatory or lymphatic system. In contrast, "local administration" refers to the introduction or delivery of an agent to a subject via a route that introduces or delivers the agent to the area of ​​the administration point or an area immediately adjacent thereto, and does not introduce the agent systemically in therapeutically significant amounts. For example, a locally administered agent is readily detectable in the local vicinity of the administration point, but is undetectable or detectable in only trace amounts in distant parts of the subject's body. Administration includes self-administration and administration by another.

[0028] 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 both therapeutic effects, i.e., the treatment of a disorder or other undesirable physiological condition, and prophylactic effects, i.e., the prevention of a disorder or other undesirable physiological condition. These 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 term "beneficial agent" or "active agent" is used, then, or when a particular agent is specifically identified, it should be understood that the term includes the agent itself as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, complexes, active metabolites, isomers, fragments, analogs, and the like.

[0029] "Reduction" can refer to any change that results in a lower amount of symptoms, diseases, compositions, conditions, or activities. A substance is also understood to reduce the genetic output of a gene when the genetic output of a gene product containing the substance is less than the output of the gene product without the substance. For example, a reduction can also be a change in the symptoms of a disorder, such that the symptoms are less than previously observed. A reduction can be any individual, median, or average decrease in a statistically significant amount of a condition, symptom, activity, or composition. Thus, a reduction can be a 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 the reduction is statistically significant.

[0030] "Inhibit," "inhibiting," and "inhibition" refer to a decrease in an activity, response, condition, disease, or other biological parameter. This can include, but is not limited to, the complete elimination of the activity, response, condition, or disease. It can also include, for example, a 10% reduction in the activity, response, condition, or disease compared to native or control levels. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount in between, compared to native or control levels.

[0031] "Inactivate," "inactivating," and "inactivation" mean to reduce or eliminate an activity, response, condition, disease, or other biological parameter resulting from the chemical (covalent bond formation) between a ligand and its biological target.

[0032] "Reduce" or other forms of the word, such as "reducing" or "reduction," refer to a decrease in an event or characteristic (e.g., tumor growth). This is typically relative to some standard or expected value, in other words, it is relative, but it is understood that reference to a standard or relative value is not necessarily required. For example, "reducing tumor growth" refers to a reduction in the rate of tumor growth compared to a standard or control.

[0033] As used herein, the term "treating" or "treatment" of a subject includes administering a drug to a subject for the purpose of preventing, curing, alleviating, mitigating, relieving, altering, correcting, improving, ameliorating, stabilizing, or affecting a disease or disorder, or the symptoms of a disease or disorder. The terms "treating" and "treatment" can 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 correcting damage.

[0034] "Prevent" or other forms of the word, such as "preventing" or "prevention," mean stopping a particular event or characteristic, stabilizing or delaying the development or progression of a particular event or characteristic, or minimizing the likelihood that a particular event or characteristic will occur. Prevent typically does not require a comparison with a control, for example, because it is more absolute than reduce. As used herein, something may be reduced but not prevented, but something that is reduced may be prevented. Similarly, something may be prevented but not reduced, but something that is prevented may be reduced. When reduce or prevent is used, it should be understood that the use of other words is also expressly disclosed unless specifically indicated otherwise. For example, the term "prevent" or "suppress" can refer to a treatment that prevents or delays the onset of a disease or condition, or reduces the severity of a disease or condition. Thus, if a treatment can treat a disease in a subject with symptoms of the disease, it can also prevent or suppress the disease in a subject who does not yet suffer from some or all of the symptoms. As used herein, the term "preventing" a disorder or unwanted physiological event in a subject specifically refers to preventing the occurrence of the symptoms and / or their underlying causes, and the subject may or may not exhibit increased susceptibility to the disorder or event.

[0035] The term "effective amount" of a therapeutic agent refers to a non-toxic but sufficient amount of a beneficial agent to produce a desired effect. The amount of a beneficial agent that is "effective" 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, an appropriate "effective" amount for any subject can be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, unless otherwise specified, the "effective amount" of a beneficial agent can also refer to an amount that covers both a therapeutically effective amount and a prophylactically effective amount.

[0036] The "effective amount" of a drug required to achieve a therapeutic effect may vary depending on factors such as the age, sex, and weight of the subject. Dosage regimens can be adjusted to provide the optimal therapeutic response. For example, the dose may be divided into several doses administered daily, or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

[0037] 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 undesirable physiological condition. The therapeutically effective amount and prophylactically effective amount of a given therapeutic agent will typically vary depending on factors such as the type and severity of the disorder or disease being treated, as well as the age, sex, and weight of the subject. The term "therapeutically effective amount" can also refer to the amount of therapeutic agent, or the rate of delivery of the therapeutic agent (e.g., amount over time), effective to promote a desired therapeutic effect. The exact desired therapeutic effect will vary according to the condition being treated, the subject's tolerance, the agent and / or agent formulation being administered (e.g., the potency of the therapeutic agent (drug), the concentration of the drug in the formulation, etc.), and various other factors understood by those skilled in the art.

[0038] As used herein, a "pharmaceutically acceptable" ingredient may refer to a biologically or otherwise undesirable ingredient, i.e., an ingredient that may be incorporated into a pharmaceutical formulation of the present invention 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 ingredients of the formulation in which it is contained. When the term "pharmaceutically acceptable" is used to refer to an excipient, this term generally means that the ingredient has met the necessary standards of toxicology and manufacturing testing or is included in the inactive ingredient guide prepared by the U.S. Food and Drug Administration.

[0039] A "pharmaceutically acceptable carrier" (sometimes referred to as a "carrier") refers to a carrier or excipient that is generally safe and non-toxic and useful in preparing a pharmaceutical or therapeutic composition, and includes carriers that are 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. As used herein, the term "carrier" encompasses, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations, and materials further described herein.

[0040] As used herein, "pharmaceutically acceptable salts" refer to derivatives of the disclosed compounds, in which the parent compound is modified by making its inorganic and organic, non-toxic, acid or base addition salts. Salts of the present compounds can be synthesized from parent compounds containing a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid form of these compounds with a stoichiometric amount of an appropriate base (e.g., Na, Ca, Mg, or K hydroxide, carbonate, bicarbonate, etc.), or by reacting the free base form of these compounds with a stoichiometric amount of an appropriate acid. Such reactions are typically carried out in water or an organic solvent, or a mixture of the two. Generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are typical, where feasible. Salts of the present compounds also include solvates of the compounds and solvates of the compound salts.

[0041] Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines, alkali or organic salts of acidic residues such as carboxylic acids, etc. Pharmaceutically acceptable salts include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids, and the quaternary ammonium salts. For example, conventional non-toxic acid salts include salts derived from inorganic acids such as, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, and the like, as well as salts prepared from organic acids such as, for example, acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, mesylic acid, esylic acid, besylic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethionic acid, HOOC-(CH)-COOH (where n is 0-4), or using different acids that produce the same counterion. Lists of additional suitable salts can be found, for example, in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., p. 1418 (1985).

[0042] Also, as used herein, "pharmacologically active" (or simply "active") can refer to derivatives or analogs (e.g., salts, esters, amides, complexes, metabolites, isomers, fragments, etc.) that have the same type of pharmacological activity as the parent compound and to approximately the same extent, as in "pharmacologically active" derivatives or analogs.

[0043] A "control" is a substitute subject or sample used in an experiment for comparison purposes. Controls can be "positive" or "negative."

[0044] As used herein, "subject" means an individual. Thus, "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. "Subject" can also include mammals, such as primates or humans. Thus, a subject can be a human or a veterinary patient. The term "patient" refers to a subject under the care of 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 carried out at a dosage and for a period of time effective to treat the subject. In some embodiments, the subject is a human.

[0045] Genetic Definition As used herein, the term "nucleic acid" refers to a polymer composed of nucleotides, eg, deoxyribonucleotides or ribonucleotides.

[0046] As used herein, the terms "ribonucleic acid" and "RNA" refer to a polymer composed of ribonucleotides.

[0047] As used herein, the terms "deoxyribonucleic acid" and "DNA" refer to a polymer composed of deoxyribonucleotides.

[0048] The term "oligonucleotide" refers to a single- or double-stranded nucleotide multimer of about 2 to a maximum of 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 triester method by Matteucci, et al., J. Am. Chem. Soc., 103:3185 (1981), both of which are incorporated herein by reference, or by other chemical methods using either a commercial automated oligonucleotide synthesizer or VLSIPS™ technology. When an oligonucleotide is referred to as "double-stranded," it will be understood by those skilled in the art that a pair of oligonucleotides is typically present in an array of hydrogen-bonded helices, such as those associated with DNA. In addition to 100% complementary forms of double-stranded oligonucleotides, the term "double-stranded" as used herein is also meant to refer to those forms that include structural features such as bulges and loops, which are more fully described in biochemistry texts such as Stryer, Biochemistry, Third Ed., (1988), which is incorporated herein by reference for all purposes.

[0049] The term "polynucleotide" refers to a single- or double-stranded polymer composed of nucleotide monomers. Polynucleotide sequences can be modified, for example, by using base modifications or end-capping, to enhance efficacy and / or reduce immune response. In other embodiments, unmodified polynucleotide sequences are used. For example, a polynucleotide can be an RNA sequence or a DNA sequence. In some embodiments, an mRNA can contain optimized codons. Codon optimization can reduce the formation of secondary structures and improve translation efficiency. In certain embodiments, codon optimization involves GC enrichment of the coding region. In certain embodiments, codon optimization involves codon quality enrichment of the coding region. In certain aspects, an mRNA can contain one or more regions or portions that act or function as untranslated regions (UTRs) of a gene. UTRs are transcribed but not translated. In an mRNA, the 5' UTR begins at the transcription start site and follows, but does not include, the start codon. The 3' UTR begins immediately after the stop codon and continues until the transcription termination signal. The use of human-derived UTRs can facilitate the expression of polypeptides in cells. In some embodiments, the polynucleotide comprises at least one chemically modified nucleotide. In some embodiments, the at least one chemically modified nucleotide comprises a chemically modified nucleic acid base, a chemically modified ribose, a chemically modified phosphodiester linkage, or a combination thereof. In some embodiments, when used, the polynucleotide sequence comprises a modified nucleoside such as 5-methylcysteine ​​or psudouridine.

[0050] As used herein, "modified" refers to an altered state or structure of a molecule of the invention. Molecules can be modified in many ways, including chemically, structurally, and functionally. In one embodiment, a polynucleotide of the invention is "chemically modified," for example, by the introduction of non-natural nucleosides and / or nucleotides relative to the natural ribonucleotides A, U, G, and C. Nucleoside and / or nucleotide modifications used in the invention can be naturally occurring (i.e., include nucleotides and / or nucleosides other than the natural ribonucleotides A, U, G, and C) or artificial. Non-canonical nucleotides, such as cap structures, differ in chemical structure from A, G, C, and U ribonucleotides, but are not considered "modified." As used herein, a "structural" modification is one in which two or more linked nucleosides are inserted, deleted, duplicated, inverted, or randomized in a polynucleotide without significant chemical modification of the nucleotides themselves. Because chemical bonds are necessarily broken and reconstructed to cause structural modification, structural modification has chemical nature, and therefore is chemical modification.However, structural modification will cause different sequences of nucleotides.When the polynucleotide of the present invention is chemically and / or structurally modified, the polynucleotide can be referred to as " modified nucleotide ".

[0051] In some embodiments, the nucleic acids disclosed herein can comprise at least one chemically modified nucleotide. In some embodiments, the at least one chemically modified nucleotide comprises a chemically modified nucleobase, a chemically modified ribose, a chemically modified phosphodiester linkage, or a combination thereof.

[0052] In one embodiment, the at least one chemically modified nucleotide is a chemically modified nucleobase.

[0053] In one embodiment, the chemically modified nucleobase is 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-carboxymethylesteruridine (5camU), pseudouridine (Ψ), N 1 -Methylpseudouridine (me 1 Ψ), N 6 -Methyladenosine (me 6 A), or thienoguanosine ( th G).

[0054] 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 -Methylpseudouridine (me 1 Ψ).

[0055] The structures of these modified nucleobases are shown below. [ka]

[0056] In one embodiment, at least one chemically modified nucleotide is a chemically modified ribose.

[0057] In one embodiment, the chemically modified ribose is selected from 2'-O-methyl (2'-O-Me), 2'-fluoro (2'-F), 2'-deoxy-2'-fluoro-beta-D-arabinonucleic 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).

[0058] The structures of these modified riboses are shown below. [ka]

[0059] In one embodiment, at least one chemically modified nucleotide is a chemically modified phosphodiester linkage.

[0060] In one embodiment, the chemically modified phosphodiester linkage is selected from phosphorothioate (PS), boranophosphate, phosphodithioate (PS2), 3',5'-amide, N3'-phosphoramidate (NP), phosphodiester (PO), or 2',5'-phosphodiester (2',5'-PO). In one embodiment, the chemically modified phosphodiester linkage is phosphorothioate.

[0061] The structures of these modified phosphodiester linkages are shown below. [ka]

[0062] 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).

[0063] 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 approximately the 20 standard naturally occurring amino acids, although natural and synthetic amino acids that are not members of the standard 20 amino acids can also be used. The standard 20 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 (Ile, 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 the alphabetical representation of a polypeptide molecule.

[0064] Conservative substitution of amino acids in proteins and polypeptides is known in the art.For example, replacing one amino acid residue with another biologically and / or chemically similar amino acid residue is known to those skilled in the art as conservative substitution.For example, conservative substitution is replacing one hydrophobic residue with another hydrophobic residue, or replacing one polar residue with another polar residue.Substitutions include, for example, Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr and other combinations.Such conservatively substituted variations of each clearly disclosed sequence are included in the polypeptides provided herein.

[0065] Substantial changes in protein function or immunological identity are made by selecting substitutions that are less conservative, i.e., by selecting residues that are more significantly different in their effect on maintaining (a) the structure of the polypeptide backbone in the region of the substitution, e.g., as a sheet or helix conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. In general, the substitutions expected to result in the greatest changes in protein properties will be those in which (a) a hydrophilic residue, e.g., seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; (b) a cysteine ​​or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) a residue having no side chain, e.g., glycine, in which case (e) the substitution increases the number of sites of sulfation and / or glycosylation.

[0066] "Variant" refers to a molecule that has a substantially similar structure. Thus, 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 respective reference sequence. A variant protein has a modified sequence in which one or more amino acids in the reference sequence are deleted or substituted, or one or more amino acids are inserted into the sequence of the reference amino acid sequence. As a result of the modification, 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 that is at least 95% identical has no more than five modifications, i.e., any combination of deletions, insertions, or substitutions, per 100 amino acids of the reference sequence.

[0067] The term "complementary" refers to the topological compatibility or matching together of the interacting surfaces of a probe molecule and its target. Thus, a target and its probe can be described as complementary, and further, the contact surface properties are complementary to each other.

[0068] The term "hybridization" refers to the process of establishing non-covalent, sequence-specific interactions between two or more complementary strands of nucleic acid, resulting in a single hybrid, which in the case of two strands is called a duplex.

[0069] The term "identical" or "percent identity," in the context of two or more nucleic acid or polypeptide sequences, when measured using the BLAST or BLAST 2.0 sequence comparison algorithm with default parameters described below, or by manual alignment and visual inspection (see, e.g., the NCBI website), refers to a specified percentage of amino acid residues or nucleotides that are the same or are the same (i.e., about 60% identity over a specified region when compared and aligned for best correspondence over a window or designated region of comparison). "Substantially identical" refers to two or more sequences or subsequences that contain at least one sequence or subsequence with at least one of the following identity (preferably 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity). Such sequences are then said to be "substantially identical." This definition also refers to or can apply to the complement of a test sequence. This definition also includes sequences with deletions and / or additions, as well as sequences with substitutions. As explained below, preferred algorithms can account for gaps, etc. Preferably, 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 10-50 amino acids or 20-50 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 to those in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be accomplished in a variety of ways that are within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software.Appropriate parameters for measuring alignment, including algorithms needed to achieve maximal alignment over the full-length of the sequences being compared, can be determined by known methods.

[0070] For sequence comparison, typically, one sequence serves as reference sequence, and test sequence is compared to it.When using sequence comparison algorithm, test and reference sequence are input into computer, and if necessary, subsequence coordinate is designated, and sequence algorithm program parameters are designated.Preferably, default program parameters can be used, or alternative parameters can be designated.Then, sequence comparison algorithm calculates the sequence identity percentage for test sequence compared with reference sequence based on program parameters.

[0071] Examples of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that, when aligned with words of the same length in a database sequence, either match or meet some positive threshold score T. T is referred to as the neighbor word score threshold (Altschul et al. (1990) J. Mol. Biol. 215:403-410). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. Word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of word hits in each direction is terminated when the cumulative alignment score drops by a quantity X from its maximum achieved value, when the cumulative score falls below zero due to the accumulation of one or more negative-scoring residue alignments, or when 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 as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands.For amino acid sequences, the BLASTP program uses as defaults a word length of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) with an alignment (B) of 50, an expectation (E) of 10, M=5, N=4, and a comparison of both strands.

[0072] The BLAST algorithm also performs statistical analysis of the similarity between two sequences (see, for example, Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One of the similarity measures provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences will occur by chance. For example, a nucleic acid is considered to be similar to a reference sequence if the minimum sum probability in the comparison between the test nucleic acid and the reference nucleic acid is less than about 0.2, more preferably less than about 0.01.

[0073] The term "nucleobase" refers to the portion of a nucleotide that has Watson / Crick base pairing functionality. The most common naturally occurring nucleobases, adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T), have hydrogen-bonding functionality that joins one nucleic acid strand to another in a sequence-specific manner.

[0074] Reference will now be made in detail to certain embodiments of the disclosed materials, compounds, compositions, articles and methods, examples of which are illustrated in the accompanying examples and figures.

[0075] single-stranded trimeric nucleic acid 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.

[0076] In some embodiments, the single-stranded trimer nucleic acid can comprise a nucleic acid sequence selected from any of 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, as well as homologs having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity to the nucleic acid sequence of 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.

[0077] In some embodiments, the single-chain trimer nucleic acid can encode an amino acid selected from any one 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, as well as homologs having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity to the amino acid sequence 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.

[0078] In some embodiments, the first T cell epitope can include an epitope listed in Tables 1-3, or a homologue 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 Table 2.

[0079] In some embodiments, the first T cell epitope can include an epitope listed in Table 2, or a homologue 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 Table 2.

[0080] In some embodiments, the single chain trimer nucleic acid can include nucleic acids encoding, in amino to carboxy terminal order, a T cell epitope, β2 microglobulin, and an MHC heavy chain sequence.

[0081] Table 1 lists the DNA and protein sequences for exemplary cancer vaccine constructs. Table 1 also includes universal CD4 T cell epitopes 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.

[0082] 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 that can activate pre-existing memory CD4 T cells, which can effectively promote the anti-tumor activity of CD8 T cells. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] Table 1-8 Table 1-9 Table 1-10 Table 2-1 Table 2-2 Table 2-3 Table 2-4 Table 2-5 Table 3-1 Table 3-2 Table 3-3 Table 3-4 Table 3-5 Table 3-6

[0083] The expression of MHC genes expressed in HLA-A1:01 HLA-A2:01HLA -A2:03、HLA-A2:06、HLA-A2:07、HLA-A3:01、HLA-A9、HLA-A 10、HLA-A11:01、HLA-A23:01、HLA-A24:02、HLA-A25:01、HLA-A26:02、HLA-A30:01、HLA-23A-A30 1:01、HLA-A32:02、HLA-A33:03、HLA-A34:01、HLA-A34:02、HLA-A68:01、HLA-A68:02、HLA-A74:01-B、、A5-B、 2、HLA-B8:01、HLA-B-12、HLA-B13:01、HLA-B14:02、HLA-B15:01、HLA-B15:02、HLA-B15:03、HLA-B15:1-18、HLA-B15:1-18 、HLA-B35:01、HLA-B38:02、HLA-B40:01、HLA-B40:02、HLA-B42:01、HLA-B44:02、HLA-B44:03、HLA-45:01:01 01、HLA-B49:01、HLA-B51:01、HLA-B52:01、HLA-B53:01、HLA-B54:01、HLA-B55:02、HLA-B57:01、HL6A-B58、HLA-16B-B58 7:01、HLA-C1:02、HLA-C2:02、HLA-C3:02、HLA-C3:03、HLA-C3:04、HLA-C4:01、HLA-C5:01、HLA-C6:1A-7:02C、 A-C7:02、HLA-C8:01、HLA-C8:02、HLA-C12:03、HLA-C14:0 2、HLA-C16:01、HLA-C17:01 and HLA-C18:01 were reported in the circulation.

Figure 4-1

Figure 4-2

[0084] In some embodiments, the single-chain trimer 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 an epitope 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, or an epitope listed in Table 5, or a homologue 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. [Table 5-1] [Table 5-2] [Table 5-3]

[0085] In some embodiments, the single-chain trimeric nucleic acid can further comprise a flexible first linker between the first T cell epitope and β2 microglobulin and a second linker between the β2 microglobulin and the MHC class I heavy chain sequence.

[0086] In some embodiments, the nucleic acid can comprise a plasmid DNA, a minicircle DNA, a microRNA, an mRNA, a self-amplifying RNA, a circle RNA, a DNA-triggered 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.

[0087] composition Described herein are compositions comprising a single chain trimeric nucleic acid encoding a first T cell epitope, β2 microglobulin, and an MHC class I heavy chain sequence.

[0088] In some embodiments, the single-stranded trimer nucleic acid can comprise a nucleic acid sequence selected from any of 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, as well as homologs having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity to the nucleic acid sequence of 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.

[0089] In some embodiments, the single-chain trimer nucleic acid can encode an amino acid selected from any one 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, as well as homologs having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity to the amino acid sequence 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.

[0090] In some embodiments, the first T cell epitope can include an epitope listed in Tables 1-3, or a homologue 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 1-3.

[0091] In some embodiments, the first T cell epitope can include an epitope listed in Table 2, or a homologue 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 Table 2.

[0092] In some embodiments, the single chain trimer nucleic acid can include nucleic acids encoding, in amino to carboxy terminal order, a T cell epitope, β2 microglobulin, and an MHC heavy chain sequence.

[0093] In some embodiments, the single-chain trimer 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 an epitope 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, or an epitope listed in Table 5, or a homologue 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.

[0094] In some embodiments, the single-chain trimeric nucleic acid can further comprise a flexible first linker between the first T cell epitope and β2 microglobulin and a second linker between the β2 microglobulin and the MHC class I heavy chain sequence.

[0095] Preferred MHC class I heavy chains are 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-A3 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-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.

[0096] Suitable MHC class II heavy chains can include, but are not limited to, HLA-DP, HLA-DQ, or HLA-DR.

[0097] In some embodiments, the composition can further comprise a pharmaceutically acceptable carrier. In some aspects, pharmaceutical compositions are disclosed herein, comprising a single-chain trimeric 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 can be a nanoparticle, a lipid nanoparticle dispersion, a liposome formulation, a lipid emulsion, a vaccine, a vector, or any combination thereof.

[0098] In some embodiments, the pharmaceutical composition can include lipid nanoparticles comprising a single-chain trimeric nucleic acid (e.g., mRNA) encoding a first T cell epitope, β2 microglobulin, and an MHC class I heavy chain sequence.

[0099] Also described herein are cells comprising the compositions described herein. In some embodiments, the cells can comprise 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 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 virus-derived cancer antigen.

[0100] Delivery method Suitable nucleic acid delivery vehicles are well known in the art and can include, but are not limited to, lipid-based (e.g., liposomal formulations, lipoplexes, or lipid nanoparticles (LNPs)), viral-based, or physical methods such as injection, microinjection, electroporation, ultrasound, gene guns, hydrodynamic application, or any combination thereof.

[0101] nanoparticles Described herein are nanoparticles comprising a single-chain trimeric nucleic acid (eg, mRNA) encoding a first T cell epitope, β2 microglobulin, and an MHC class I heavy chain sequence.

[0102] In some embodiments, the nanoparticles can be lipid nanoparticles. In some embodiments, the nanoparticles can be lipid-polycation complexes, referred to as cationic lipid nanoparticles. By way of non-limiting example, the polycation can include a cationic peptide or polypeptide, such as, but not limited to, polylysine, polyornithine, and / or polyarginine. In some embodiments, the lipid nanoparticles can include a non-cationic lipid, such as, but not limited to, cholesterol or dioleoylphosphatidylethanolamine (DOPE).

[0103] In some embodiments, described herein are lipid nanoparticles comprising a single-chain trimeric nucleic acid (e.g., mRNA) encoding a first T cell epitope, β2 microglobulin, and an MHC class I heavy chain sequence.

[0104] Lipid nanoparticle formulation can be influenced by biophysical parameters, including but not limited to, the selection of cationic lipid components, the degree of cationic lipid saturation, the nature of PEGylation, the ratio of all components, and size.In one example by Semple et al. (Nature Biotech.2010 28:172-176), lipid nanoparticles can further comprise 57% cationic lipid, 7% dipalmitoyl phosphatidylcholine, 34% cholesterol, and 1.5% PEG-c-DMA.In another example, by changing the composition of cationic lipid, siRNA can be more effectively delivered to various antigen-presenting cells (Basha et al. Mol Ther.2011 19:2186-2200).

[0105] In some embodiments, the lipid nanoparticle formulation may comprise 35-45% cationic lipid, 40-50% cationic lipid, 50-60% cationic lipid, and / or 55-65% cationic lipid. In some embodiments, the lipid to nucleic acid (e.g., mRNA) ratio in the lipid nanoparticle may be 5:1 to 20:1, 10:1 to 25:1, 15:1 to 30:1, and / or at least 30:1.

[0106] Lipid nanoparticle formulations typically include lipids, particularly ionizable cationic lipids, and further include neutral lipids, sterols, and molecules that can reduce particle aggregation, such as PEG or PEG-modified lipids.

[0107] In some embodiments, the lipid nanoparticle formulation comprises at least one ionizable lipid, cationic lipid, or any combination thereof; a neutral lipid selected from DSPC, DPPC, POPC, DOPE, and SM; (iii) a sterol, e.g., cholesterol; and (iv) a PEGylated lipid, e.g., PEG-DMG or PEG-cDMA, in a molar ratio of 20-60% cationic lipid: 5-25% neutral lipid: 25-55% sterol; 0.5-15% PEGylated lipid.

[0108] In some embodiments, the lipid nanoparticle formulation comprises 25% to 75% cationic lipid on a molar basis, e.g., 35 to 65%, 45 to 65%, 60%, 57.5%, 50%, or 40% on a molar basis, 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).

[0109] 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.

[0110] 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.

[0111] 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.

[0112] 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.

[0113] 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.

[0114] 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.

[0115] 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.

[0116] 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.

[0117] In some embodiments, the lipid nanoparticle formulation comprises 57.5% cationic lipid selected from PEGylated lipids, on a molar basis 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% neutral lipid, 31.5% sterol, and 3.5% PEG or PEG-modified lipid.

[0118] In some embodiments, the lipid nanoparticle formulation comprises a lipid mixture at 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 at a molar ratio of 20-60% cationic lipid: 5-25% neutral lipid: 25-55% cholesterol: 0.5-15% PEG-modified lipid.

[0119] In some embodiments, the molar lipid 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., For example, 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).

[0120] Non-limiting examples of lipid nanoparticle compositions and methods for making them are described, for example, 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.

[0121] In some embodiments, lipid nanoparticle formulations may comprise cationic lipids, PEGylated lipids, and structural lipids, optionally including non-cationic lipids. As a non-limiting example, lipid nanoparticles may comprise 40-60% cationic lipids, 5-15% non-cationic lipids, 1-2% PEGylated lipids, and 30-50% structural lipids. As another non-limiting example, lipid nanoparticles may comprise 50% cationic lipids, 10% non-cationic lipids, 1.5% PEGylated lipids, and 38.5% structural lipids. As yet another non-limiting example, lipid nanoparticles may comprise 55% cationic lipids, 10% non-cationic lipids, 2.5% PEGylated lipids, and 32.5% structural lipids.

[0122] In some embodiments, the lipid nanoparticle formulations described herein may be four-component lipid nanoparticles. The lipid nanoparticles may comprise cationic lipids, non-cationic lipids, PEGylated lipids, and structured lipids. As a non-limiting example, the lipid nanoparticles may comprise 40-60% cationic lipids, 5-15% non-cationic lipids, 1-2% PEGylated lipids, and 30-50% structured lipids. As another non-limiting example, the lipid nanoparticles may comprise 50% cationic lipids, 10% non-cationic lipids, 1.5% PEGylated lipids, and 38.5% structured lipids. As yet another non-limiting example, the lipid nanoparticles may comprise 55% cationic lipids, 10% non-cationic lipids, 2.5% PEGylated lipids, and 32.5% structured lipids.

[0123] In some embodiments, the lipid nanoparticle formulations described herein may contain cationic lipids, non-cationic lipids, PEG lipids, and structural lipids. As a non-limiting example, the lipid nanoparticles may contain 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 may contain 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 may contain 50% cationic lipid DLin-MC3-DMA, 10% non-cationic lipid DSPC, 1.5% PEG lipid PEG-DMG, and 38.5% structural lipid cholesterol. As yet another non-limiting example, the lipid nanoparticles comprise 55% cationic lipid L319, 10% non-cationic lipid DSPC, 2.5% PEGylated lipid PEG-DMG, and 32.5% structural lipid cholesterol.

[0124] In some embodiments, the nanoparticles can be 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 can be those described in U.S. Patent No. 10,933,127, such as nanoparticles comprising a compound according to Formula (I), (Ia), (II), (IIa), (IIb), (IIc), (IId), or (IIe), as set forth in columns 101 through 187.

[0125] In some embodiments, the nanoparticles (e.g., lipid nanoparticles) have an average diameter of 10 to 500 nm, 20 to 400 nm, 30 to 300 nm, or 40 to 200 nm, or 50 to 150 nm, 50 to 200 nm, 80 to 100 nm, or 80 to 200 nm.

[0126] The lipid nanoparticles (LNPs) described herein can be made in a sterile environment.

[0127] In some embodiments, the LNP formulation may be formulated into a nanoparticle, such as a nucleic acid-lipid particle. As a non-limiting example, the lipid particle may include one or more active or therapeutic agents, one or more cationic lipids comprising 50 mol% to 85 mol% of the total lipids present in the particle, one or more non-cationic lipids comprising 13 mol% to 49.5 mol% of the total lipids present in the particle, and one or more conjugated lipids that inhibit particle aggregation, comprising 0.5 mol% to 2 mol% of the total lipids present in the particle.

[0128] The nanoparticle formulation may include a phosphate conjugate. The phosphate conjugate may increase the in vivo circulation time of the nanoparticle and / or increase the targeted delivery of the nanoparticle. As a non-limiting example, the phosphate conjugate may include a compound of any one of the formulas set forth in International Application No. 2013 / 033438, the contents of which are incorporated herein by reference in their entirety.

[0129] The nanoparticle formulation may include 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. 2013 / 0059360, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the 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. 2013 / 0072709, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the polymer conjugates may have pendant side groups containing ring moieties, such as, but not limited to, the polymer conjugates described in U.S. Patent Publication No. 2013 / 0196948, the contents of which are incorporated herein by reference in their entirety.

[0130] The nanoparticle formulations may include conjugates to enhance delivery of the nanoparticles of the present disclosure in a subject.

[0131] In some embodiments, a composition comprises a polynucleotide of the present disclosure and a conjugate that may have a degradable linkage.Non-limiting examples of conjugates include an aromatic moiety containing an ionizable hydrogen atom, a spacer moiety, and a water-soluble polymer.Non-limiting examples of pharmaceutical compositions comprising conjugates with degradable linkages and methods for delivering such pharmaceutical compositions are described in U.S. Patent Publication No. 2013 / 0184443, the contents of which are incorporated herein by reference in their entirety.

[0132] In some embodiments, the lipid nanoparticles can comprise 20% to 80% ionized lipids, cationic lipids, or any combination thereof, 0% to greater than 5% polyethylene glycol lipids, 0% to greater than 20% helper lipids, 20% to 80% sterol, and a single-chain trimeric nucleic acid encoding a first T cell epitope, beta-2 microglobulin, and an MHC class I heavy chain sequence encapsulated in the lipid nanoparticles.

[0133] In some embodiments, the lipid nanoparticles can comprise 20% to 80% ionized lipids, cationic lipids, or any combination thereof, 0% to greater than 5% polyethylene glycol lipids, 0% to greater than 20% helper lipids, 20% to 80% sterol, and a single-chain trimeric nucleic acid (e.g., mRNA) encoding a first T cell epitope, beta-2 microglobulin, and an MHC class I heavy chain sequence encapsulated in the lipid nanoparticles.

[0134] In one embodiment, the nanoparticles can comprise 40% to 60% ionized lipids, cationic lipids, or any combination thereof, 1% to 2% polyethylene glycol lipids, 8% to 12% helper lipids, 35% to 40% sterol, and a single-chain trimeric nucleic acid (e.g., mRNA) encoding a first T cell epitope, beta-2 microglobulin, and an MHC class I heavy chain sequence encapsulated in the lipid nanoparticles.

[0135] In one embodiment, the nanoparticles can comprise 40% to 60% ionized lipids, cationic lipids, or any combination thereof, 1% to 2% polyethylene glycol lipids, 8% to 12% helper lipids, 35% to 40% sterol, and a single-chain trimeric nucleic acid (e.g., mRNA) encoding a first T cell epitope, beta-2 microglobulin, and an MHC class I heavy chain sequence encapsulated in the lipid nanoparticles.

[0136] In some embodiments, the single-stranded trimer nucleic acid can comprise a nucleic acid sequence selected from any of 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, as well as homologs having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity to the nucleic acid sequence of 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.

[0137] In some embodiments, the single-chain trimer nucleic acid can encode an amino acid selected from any one 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, as well as homologs having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.8%, or 99.9%) identity to the amino acid sequence 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.

[0138] In some embodiments, the first T cell epitope can include an epitope listed in Tables 1-3, or a homologue 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 1-3.

[0139] In some embodiments, the first T cell epitope can include an epitope listed in Table 2, or a homologue 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 Table 2.

[0140] In some embodiments, the single chain trimer nucleic acid can include nucleic acids encoding, in amino to carboxy terminal order, a T cell epitope, β2 microglobulin, and an MHC heavy chain sequence.

[0141] In some embodiments, the single-chain trimer 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 an epitope 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, or an epitope listed in Table 5, or a homologue 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.

[0142] In some embodiments, the single-chain trimeric nucleic acid can further comprise a flexible first linker between the first T cell epitope and β2 microglobulin and a second linker between the β2 microglobulin and the MHC class I heavy chain sequence.

[0143] Preferred MHC class I heavy chains are 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-A3 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-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.

[0144] Suitable MHC class II heavy chains can include, but are not limited to, HLA-DP, HLA-DQ, or HLA-DR.

[0145] 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.

[0146] neutral lipid In some embodiments, the lipid nanoparticle formulation comprises 0.5% to 15% neutral lipid on a molar basis, e.g., 3-12%, 5-10%, or 15%, 10%, or 7.5% on a molar basis. 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% sterol on a molar basis (e.g., 15-45%, 20-40%, 40%, 38.5%, 35%, or 31% on a molar basis). A non-limiting example of a sterol is cholesterol. In some embodiments, the lipid nanoparticle formulation comprises 0.5% to 20% PEG or PEG-modified lipid on a molar basis (e.g., 0.5-10%, 0.5-5%, 1.5%, 0.5%, 1.5%, 3.5%, or 5% on a molar basis). In embodiments, the PEG or PEG-modified lipid comprises PEG molecules with an average molecular weight of 2,000 Da. In some embodiments, the PEG or PEG-modified lipid comprises PEG molecules with an average molecular weight of less than 2,000, e.g., approximately 1,500 Da, approximately 1,000 Da, or approximately 500 Da. Non-limiting examples of PEG-modified lipids include PEG-distearoylglycerol (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 herein by reference in their entirety).

[0147] Zwitterionic lipids In some embodiments, the composition may be encapsulated in, linked to, and / or associated with zwitterionic lipids. Non-limiting examples of zwitterionic lipids and methods of using zwitterionic lipids are described in U.S. Patent Publication No. 2013 / 0216607, the contents of which are incorporated herein by reference in their entirety. In some embodiments, zwitterionic lipids can be used in the liposomes and lipid nanoparticles described herein.

[0148] cationic lipids Suitable cationic lipids include 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 US2013 / 0150625); 2-amino-3-[(9Z)-octadec-9-en-1-yloxy]-2-{[(9Z)-octadec-9-en-1-yloxy]methyl}propan-1-ol (compound 2 in US2013 / 0150625); 2-amino-3-[(9Z,12Z)-octadec-9-en-1-yloxy]-2-{[(9Z)-octadec-9-en-1-yloxy]methyl}propan-1-ol 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 US2013 / 0150625); or any pharmaceutically acceptable salt or stereoisomer thereof. Non-limiting examples of cationic lipids include (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-13,16-dien-5-amine, (12Z,15Z)-N,N-dimethylhenicosa-12,15-dien-4-amine, and (14Z,17Z)-N,N-dimethyltricosa-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-dimethyltricosa-14,17-dien-4-amine, (19Z,22Z)-N,N-dimethyloctacosa-19,22-dien-9-amine, (18Z,21Z)-N,N-dimethylheptacosa-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-Dimethylhentriaconta-22,25-dien-10-amine, (21Z,24Z)-N,N-Dimethyltriaconta-21,24-dien-9-amine, (18Z)-N,N-Dimethylheptacosa-18-en-10-amine, (17Z)-N,N-Dimethylhexacosa-17-en-9-amine, (19Z,22Z)-N,N-Dimethyloctacos- N,N-dimethylheptacosa-19,22-dien-7-amine, N,N-dimethylheptacosa-10-amine, (20Z,23Z)-N-ethyl-N-methylnonacosa-20,23-dien-10-amine, 1-[(11Z,14Z)-1-nonylcosa-11,14-dien-1-yl]pyrrolidine, (20Z)-N,N-dimethylheptacosa-20-en-10-amine, (15Z)-N,N-dimethylheptacosa-15-en-10-amine, (14Z)-N,N-dimethylnonacosa-14-en-10-amine, (17Z)-N,N-dimethylnonacosa-17-en-1 -10-amine, (24Z)-N,N-dimethyltritriacont-24-en-10-amine, (20Z)-N,N-dimethylnonacos-20-en-10-amine, (22Z)-N,N-dimethylhentriacont-22-en-10-amine, (16Z)-N,N-dimethylpentacos-16-en-8-amine, (12Z,15Z)-N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine, (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine, N,N-dimethyl-1-[(1 S,2R)-2-octylcyclopropyl]eptadecan-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]henicosan-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]hexadecan-8-amine, N,N-Dimethyl-[(1R,2S)-2-undecylcyclopropyl]tetradecan-5-amine, N,N-Dimethyl-3-{7-[(1S,2R)-2-octylcyclopropyl]heptyl}dodecan-1-amine, 1-[(1R,2S)-2-heptylcyclopropyl]-N,N-dimethyloctadecane-9-amine, 1-[(1S,2R)-2-decylcyclopropyl]-N,N-dimethylpentadecan-6-amine, N,N-Dimethyl-1- R1S,2R)-2-Octylcyclopropyl-1-pentadecan-8-amine, R1N,N-Dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine, S1N,N-Dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine, 1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}pyrrolidine, (2S)-N,N-Dimethyl 1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-[(5Z)-oct-5-en-1-yloxy]propan-2-amine, 1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}azetidine, (2S)-1-(hexyloxy)-N,N-dimethyl-3-R9Z,12Z)-octadeca-9,12-dien-1-yloxylpropan-2-amine, (2S)-1-(heptyloxy)-N,N-dimethyl-3-R9Z,12Z)-octadeca-9,12-dien-1-yloxylpropan-2-amine, (2S)-1-(heptyloxy)-N,N-dimethyl-3-R9Z,12Z)-octadeca-9,12-dien-1-yloxylpropan-2-amine, N,N-dimethyl-1-(nonyloxy)-3-R(9Z,12Z)-octadeca-9,12-dien-1-yloxylpropan-2-amine, N,N-dimethyl-1-[(9Z)-octadec-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)-Icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(pentyloxy)propan-2-amine, (2S)-1-(hexyloxy)-3-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethylpropan-2-amine, 1-[(11Z,14Z)-icosa-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)-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-di Methyl-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-methoyloctyl)oxy]-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, (2R)-1-[(3,7-dimethyloctyl)oxy]-N,N-dimethyl-3-R9Z,12Z)-octadeca-9 ,12-dien-1-yloxylpropan-2-amine, N,N-dimethyl-1-(octyloxy)-3-({8-R1S,25)-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 may be a cationic lipid, such as, but not limited to, DLin-DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, and aminoalcohol lipids. The aminoalcohol cationic lipid may be a lipid described in U.S. Patent Publication No. 2013 / 0150625, the entire contents of which are incorporated herein by reference, and / or a lipid made by the method described therein. In some embodiments, the nanoparticles described herein may include an amine cationic lipid, such as those described in International Patent Application No. 2013 / 059496, 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.

[0149] In some embodiments, the cationic lipids may be low molecular weight cationic lipids such as those described in U.S. Patent Application No. 2013 / 0090372, the contents of which are incorporated herein by reference in their entirety.

[0150] Ionized lipids Exemplary ionizable lipids are described in U.S. Patent Publication Nos. 2016 / 0311759, 2015 / 0376115, 2016 / 0151284, 2017 / 0210697, 2015 / 0140070, 2013 / 0178541, 2013 / 0303587, 2015 / 0141678, 2015 / 0239926, 2016 / 0376224, 2017 / 0119904, 2012 / 0149894, 2015 / 0057373, 2013 / 0090372, 2013 / 0 No. 274523, No. 2013 / 0274504, No. 2013 / 0274504, No. 2009 / 0023673, No. 2012 / 0128760, 2010 / 03241240, 2014 / 0200257, 2015 / 0203446, 2 018 / 0005363, 2014 / 0308304, 2013 / 0338210, 2012 / 0101148, No. 2012 / 0027796, No. 2012 / 0058144, No. 2013 / 0323269, No. 2011 / 0117125, Same No. 2011 / 0256175, No. 2012 / 0202871, No. 2011 / 0076335, No. 2006 / 0083780 No. 2013 / 0123338, No. 2015 / 0064242, No. 2006 / 0051405, No. 2013 / 00659 No. 39, No. 2006 / 0008910, No. 2003 / 0022649, No. 2010 / 0130588, No. 2013 / 011 No. 6307, No. 2010 / 0062967, No. 2013 / 0202684, No. 2014 / 0141070, No. 2014 / 02 No. 55472, No. 2014 / 0039032, No. 2018 / 0028664, No. 2016 / 0317458, No. 2013 / No. 0195920, No. 2022 / 0062175, No. 2021 / 0121411, No. 2022 / 0009878, No. 2022 / 0040325, 2012 / 61657480, 2016 / 0074514, 2013 / 0330401, 2 019 / 0185410, 2012 / 61617468, 2019 / 0032087, 2015 / 62184188,Same No. 2019 / 0127318, No. 2021 / 0002813, No. 2020 / 0345641, No. 2014 / 61944336, No. 2012 / 6165 No. 7480, No. 2021 / 0059953, No. 2022 / 0162521, No. 2022 / 0235377, No. 2018 / 0085474, No. 2018 Nos. 2020 / 0129445, 2021 / 0145982, 2021 / 0378980, 2020 / 0254086, 2021 / 0346306, and 2018 / 0000953, the contents of all of which are incorporated herein by reference in their entireties.

[0151] In some embodiments, the nanoparticles comprise 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).

[0152] The ionizable lipids can be present in a molar ratio ranging from any of the minimum values ​​described above to any of the maximum values ​​described above. For example, in some embodiments, the 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%).

[0153] Helper lipids In some embodiments, the nanoparticles comprise a helper lipid. In some embodiments, the helper lipid can be a non-cationic lipid. In some embodiments, the non-cationic lipid can be 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-phosphoethanolamine), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine ...distearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPC), 1,2-distearoyl-sn-glycero-3- The non-cationic lipid may include, but is not limited to, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphotidylcholine (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.

[0154] 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%.

[0155] Polyethylene glycol lipids In some embodiments, the nanoparticles contain polyethylene glycol lipids (PEG lipids). The PEG lipids are incorporated to form a hydrophilic outer layer and stabilize the particles. Non-limiting examples of polyethylene glycol lipids include PEG-modified lipids such as PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol. Representative polyethylene glycol lipids include DMG-PEG, DLPE-PEG, DMPE-PEG, DPPC-PEG, and DSPE-PEG. In one embodiment, the polyethylene glycol lipid is 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol (DMG-PEG). In one embodiment, the polyethylene glycol lipid is 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene 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 moiety, for example, DMG-PEG2000 or DMG-PEG5000.

[0156] In some embodiments, the nanoparticles comprise polyethylene glycol lipids at a molar ratio of 0% to 5%. In some embodiments, the nanoparticles comprise polyethylene glycol lipids at a molar ratio of about 0%, 0.25%, 0.5%, 0.75%, 1%, 1.5%, 2%, 3%, 4%, or 5%. In one embodiment, the nanoparticles comprise polyethylene glycol lipids at a molar ratio of 0.75%.

[0157] In some embodiments, the ratio of PEG in a lipid nanoparticle formulation may be increased or decreased, and / or the carbon chain length of the PEGylated lipid may be changed from C14 to C18 to modify the pharmacokinetics and / or biodistribution of the lipid nanoparticle formulation. As a non-limiting example, the lipid nanoparticle formulation may 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% of PEG-c-DOMG (R-3-[(ω-methoxy-poly(ethylene glycol)2000)carbamoyl)]-1,2-dimyristyloxypropyl-3-amine) (also referred to herein as PEG-DOMG) relative to the cationic lipid, DSPC, and cholesterol. In some embodiments, PEG-c-DOMG may be replaced with 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).

[0158] In some embodiments, the LNP formulation may contain PEG-c-DOMG at a lipid molar ratio of 3%. In some embodiments, the LNP formulation may contain PEG-c-DOMG at a lipid molar ratio of 1.5%.

[0159] In some embodiments, the pharmaceutical composition may comprise at least one of the PEGylated lipids described in WO 2012 / 099755, the contents of which are incorporated herein by reference in their entirety.

[0160] In some embodiments, an LNP formulation may contain PEG-DMG2000 (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000). In some embodiments, an LNP formulation may contain PEG-DMG2000, a cationic lipid known in the art, and at least one other component. In some embodiments, an LNP formulation may contain PEG-DMG2000, a cationic lipid known in the art, DSPC, and cholesterol. As a non-limiting example, an LNP formulation may contain PEG-DMG2000, DLin-DMA, DSPC, and cholesterol. As another non-limiting example, an LNP formulation can contain PEG-DMG2000, 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 incorporated herein by reference in their entirety).

[0161] sterols In some embodiments, nanoparticles comprise sterol.Sterol is well known to those skilled in the art and generally refers to those compounds that have perhydrocyclopentanophenanthrene ring system and have one or more OH substituents.Examples of sterol include but are not limited to cholesterol, campesterol, ergosterol, sitosterol, etc.

[0162] In some embodiments, the sterol is selected from cholesterol-based lipids. In some embodiments, the one or more cholesterol-based lipids are selected from cholesterol, PEGylated cholesterol, DC-Choi (N,N-dimethyl-N-ethylcarboxamidocholesterol), 1,4-bis(3-N-oleylamino-propyl)piperazine, or a combination thereof.

[0163] Sterols can be used to modulate particle permeability and fluidity based on their function in cell membranes, hi one embodiment, the sterol is cholesterol.

[0164] In some embodiments, the nanoparticles comprise 20% to 80% molar sterol. In some embodiments, the nanoparticles comprise 25%, 30%, 35%, 40%, 45%, or 50% molar sterol. In one embodiment, the nanoparticles comprise 40% molar sterol.

[0165] The nanoparticle formulation may be a carbohydrate nanoparticle comprising a carbohydrate carrier and a nucleic acid sequence (e.g., mRNA) as described herein. Non-limiting examples of carbohydrate carriers include, but are not limited to, anhydride-modified phytoglycogen or glycogen-type materials, photoglycogen octenyl succinate, phytoglycogen beta-dextrin, and anhydride-modified phytoglycogen beta-dextrin. (See, for example, International Publication No. 2012 / 109121, the contents of which are incorporated herein by reference in their entirety.)

[0166] The nanoparticle formulations of the present disclosure can be coated with surfactants or polymers to improve particle delivery. In some embodiments, nanoparticles can be coated with a hydrophilic coating, such as, but not limited to, a PEG coating and / or a coating with a neutral surface charge. Hydrophilic coatings can be useful for delivering nanoparticles with larger payloads, such as, but not limited to, RNA (e.g., mRNA), into the central nervous system. As a non-limiting example, nanoparticles with hydrophilic coatings and methods for making such nanoparticles are described in U.S. Patent Publication No. 2013 / 0183244, the contents of which are incorporated herein by reference in their entirety.

[0167] In some embodiments, the lipid nanoparticles of the present disclosure can be hydrophilic polymer particles. Non-limiting examples of hydrophilic polymer particles and methods for making hydrophilic polymer particles are described in U.S. Patent Publication No. 2013 / 0210991, the contents of which are incorporated herein by reference in their entirety.

[0168] In some embodiments, the lipid nanoparticles of the present disclosure can be hydrophobic polymer particles.

[0169] In some embodiments, an immune response can be elicited by delivering lipid nanoparticles that can contain nanoseeds, polymers, and immunogens. (U.S. Publication No. 2012 / 0189700 and International Publication No. 2012 / 099805; each of which is incorporated herein by reference in its entirety.) The polymer may encapsulate or partially encapsulate the nanoseeds. The immunogen may be a recombinant protein, modified RNA, and / or polynucleotide as described herein. In some embodiments, the lipid nanoparticles can be formulated for use in vaccines, such as, but not limited to, those against pathogens.

[0170] Lipid nanoparticles may be engineered to modify the surface properties of the particles so that they can penetrate mucosal barriers. Mucus is located on mucosal tissues, including, but not limited to, the oral cavity (e.g., the membranes of the cheeks and esophagus, and tonsillar tissue), eyes, gastrointestinal (e.g., the stomach, small intestine, large intestine, colon, and rectum), nasal, respiratory (e.g., membranes of the nose, pharynx, trachea, and bronchi), and reproductive (e.g., membranes of the vagina, cervix, and urethra). Nanoparticles larger than 10-200 nm, which are preferred for their higher drug encapsulation efficiency and ability to provide sustained delivery of a wide range of drugs, are considered too large to rapidly diffuse through mucosal barriers. Because mucus is continuously secreted, excreted, discarded, or digested and recycled, most trapped particles can be removed from mucosal tissue within seconds or hours. Large polymer nanoparticles (200-500 nm diameter) densely coated with low-molecular-weight polyethylene glycol (PEG) diffused 4-6 times less through mucus than the same particles 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). Nanoparticle transport can be determined by the rate of penetration and / or using fluorescence microscopy techniques, including, but not limited to, fluorescence recovery after photobleaching (FRAP) and high-resolution multiparticle tracking (MPT). As a non-limiting example, compositions capable of penetrating mucosal barriers can be prepared as described in U.S. Pat. No. 8,241,670 or International Patent Publication No. 2013 / 110028, the contents of each of which are incorporated herein by reference in their entirety.

[0171] Lipid nanoparticles engineered to penetrate mucus may comprise a polymeric material (i.e., a polymer core) and / or a polymer-vitamin conjugate and / or a triblock copolymer. The polymeric material may include, but is not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, poly(styrenes), polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates. The polymeric material may be biodegradable and / or biocompatible. Non-limiting examples of biocompatible polymers are described in International Patent Publication No. 2013 / 116804, the contents of which are incorporated herein by reference in their entirety. The polymeric material may additionally be irradiated. As a non-limiting example, the polymeric material may be gamma-irradiated (see, for example, International Application No. 2012 / 82165, the entire contents of which are incorporated herein by reference). 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-co-glycolic acid) (PLGA), poly(L-lactic-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), and poly(D,L-lactide-co-PEO ... Poly(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(ester amides), polyamides, poly(ester ethers), 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), polyvinylpyrrolidone, polysiloxanes, polystyrene (PS), polyurethanes, derivatized celluloses such as alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitrocellulose, hydroxypropyl cellulose, carboxymethyl cellulose, polymers of acrylic acid such as poly(methyl (meth)acrylate) (PMMA), poly(ethyl (meth)acrylate), poly(butyl (meth)acrylate), poly(isobutyl (meth)acrylate), etc. Poly(meth)acrylate), poly(hexyl(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(ortho)esters, poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone), PEG-PLGA-PEG and trimethylene carbonate, polyvinylpyrrolidone. The lipid nanoparticles may be coated with or associated with copolymers such as, but not limited to, block copolymers (such as the branched polyether-polyamide block copolymers described in International Publication No. WO 2013 / 012476, which is incorporated herein by reference in its entirety), and (poly(ethylene glycol))-(poly(propylene oxide))-(poly(ethylene glycol)) triblock copolymers (see, e.g., U.S. Publication Nos. 2012 / 0121718 and 2010 / 0003337 and U.S. Patent No. 8,263,665, the contents of each of which are incorporated herein by reference in their entirety). The copolymers may be:The polymer may be generally regarded as safe (GRAS), and the lipid nanoparticles may be formed in such a way that no new chemical substances are created.For example, the lipid nanoparticles may include poloxamer-coated PLGA nanoparticles, which can still rapidly penetrate human mucus without creating new chemical substances (Yang et al. Angew. Chem. Int. Ed. 2011 50:2597-2600; the contents of which are incorporated herein by reference in their entirety).A non-limiting scalable method for producing nanoparticles that can penetrate human mucus is described by Xu et al. (see, for example, J Control Release 2013,170(2):279-86, the contents of which are incorporated herein by reference in their entirety).

[0172] The vitamin of the polymer-vitamin conjugate can be vitamin E. The vitamin portion of the conjugate can be substituted with other suitable moieties, 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).

[0173] Lipid nanoparticles engineered to penetrate mucus may 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 dimethyldioctadecylammonium bromide), sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamer), mucolytic agents (e.g., N-acetylcysteine, Artemisia princeps, bromelain, papain, Clerodendrum, acetylcysteine, bromhexine, carbocysteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin β4 dornase alpha, neltenexin, erdosteine), and various DNases, including rhDNase. The surface-altering agent may be embedded or entrapped on the surface of the particle, or may be disposed on the surface of the lipid nanoparticle (e.g., by coating, adsorption, covalent attachment, or other process) (see, e.g., U.S. Publication Nos. 2010 / 0215580, 2008 / 0166414, and US2013 / 0164343, the contents of each of which are incorporated herein by reference in their entirety).

[0174] In some embodiments, the mucus-penetrating lipid nanoparticles may contain at least one polynucleotide as described herein. The polynucleotide may be encapsulated in the lipid nanoparticles and / or disposed on the surface of the particles. The polynucleotide may be covalently coupled to the lipid nanoparticles. The mucus-penetrating lipid nanoparticle formulation may contain multiple nanoparticles.

[0175] In some embodiments, the composition can be formulated as solid lipid nanoparticles. Solid lipid nanoparticles (SLNs) can be spherical with an average diameter of 10 to 1000 nm. SLNs have a solid lipid core matrix that can solubilize lipophilic molecules and can be stabilized with surfactants and / or emulsifiers. In some embodiments, the lipid nanoparticles can be self-assembled lipid-polymer nanoparticles (Zhang et al., ACS Nano, 2008, 2(8), pp. 1696-1702; the contents of which are incorporated herein by reference in their entirety). As a non-limiting example, the SLNs can be those described in International Patent Publication No. 2013 / 105101, the contents of which are incorporated herein by reference in their entirety. As another non-limiting example, the SLNs can be made by the methods or processes described in International Patent Publication No. 2013 / 105101, the contents of which are incorporated herein by reference in their entirety.

[0176] In some embodiments, the compositions of the present disclosure may be encapsulated in nanoparticles, which may be prepared using methods described herein, including but not limited to, WO 2010 / 005740, WO 2010 / 030763, WO 2010 / 005721, WO 2010 / 005723, WO 2012 / 054923, U.S. Publication Nos. 2011 / 0262491, 2010 / 0104645, 2010 / 0087337, 2010 / 0068285, and 2011 / 0274 759, 2010 / 0068286, 2012 / 0288541, 2013 / 0123351, and 2013 / 0230567, 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, polymeric nanoparticles can be identified by the methods described in U.S. Publication No. 2012 / 0140790, the contents of which are incorporated herein by reference in their entirety.

[0177] In some embodiments, nanoparticles can be formulated for sustained release. As used herein, "sustained release" refers to a pharmaceutical composition or compound that is adapted to a release rate over a specific period of time. The period can include, but is not limited to, hours, days, weeks, months, and years. As a non-limiting example, sustained release nanoparticles can include the polymers and nucleic acid sequences of the present disclosure (see International Publication No. 2010 / 075072, and U.S. Publication Nos. 2010 / 0216804, 2011 / 0217377, and 2012 / 0201859, the contents of each of which are incorporated herein by reference in their entirety). In another non-limiting example, sustained release formulations can include agents that enable sustained bioavailability, such as, but not limited to, crystals, macromolecular gels, and / or microparticle suspensions (see U.S. Patent Publication No. 2013 / 0150295, the contents of each of which are incorporated herein by reference in their entirety).

[0178] In some embodiments, the nanoparticles of the present disclosure may comprise a polymer matrix. By way of non-limiting example, the nanoparticles may comprise two or more polymers, such as, but not limited to, polyethylene, polycarbonate, polyanhydride, polyhydroxy acid, polypropyl fumerate, 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-co-L-lysine), poly(4-hydroxy-L-proline ester), or a combination thereof.

[0179] In some embodiments, the nanoparticles comprise a diblock copolymer. In some embodiments, the diblock copolymer may comprise PEG in combination with a polymer such as, but not limited to, polyethylene, polycarbonate, polyanhydride, polyhydroxy acid, polypropyl fumerate, 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-co-L-lysine), poly(4-hydroxy-L-proline ester), or a combination thereof. In yet another embodiment, the diblock copolymer may be a high-X diblock copolymer such as those described in International Patent Publication No. 2013 / 120052, the contents of which are incorporated herein by reference in their entirety.

[0180] As a non-limiting example, the nanoparticle comprises a PLGA-PEG block copolymer (see U.S. Publication No. 2012 / 0004293 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 nanoparticle is a stealth nanoparticle comprising a diblock copolymer of PEG and PLA or PEG and PLGA (see U.S. Patent No. 8,246,968 and WO 2012 / 166923, the contents of each of which are incorporated herein by reference in their entirety). In yet another non-limiting example, the nanoparticle is a stealth nanoparticle or a target-specific stealth nanoparticle as described in U.S. Publication No. 2013 / 0172406, the contents of which are incorporated herein by reference in their entirety.

[0181] In some embodiments, the nanoparticles may comprise multi-block copolymers (see, e.g., U.S. Pat. Nos. 8,263,665 and 8,287,910, and U.S. Patent Publication No. 2013 / 0195987, the contents of each of which are incorporated by reference herein in their entirety).

[0182] In yet another non-limiting example, the lipid nanoparticles comprise the block copolymer PEG-PLGA-PEG (e.g., thermosensitive hydrogel (PEG-PLGA-PEG) has been used as a TGF-beta 1 gene delivery vehicle in Lee et al. Thermosensitive Hydrogel as a TGF-β1 Gene Delivery Vehicle Enhances Diabetic Wound Healing. Pharmaceutical Research, 2003 20(12):1995-2000; Li et al. Controlled Gene Delivery System Based on Thermosensitive Biodegradable Hydrogel. Pharmaceutical Research 2003 20(6):884-888; 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 incorporated herein by reference in their entirety.) The nucleic acid (e.g., mRNA) vaccines of the present disclosure can be formulated in lipid nanoparticles comprising PEG-PLGA-PEG block copolymers.

[0183] In some embodiments, the nanoparticles may comprise multi-block copolymers (see, e.g., U.S. Pat. Nos. 8,263,665 and 8,287,910, and U.S. Patent Publication No. 2013 / 0195987, the contents of each of which are incorporated by reference herein in their entirety).

[0184] In some embodiments, the block copolymers described herein can be included in a polyion complex comprising a non-polymeric micelle and the block copolymer (see, e.g., U.S. Publication No. 2012 / 0076836, the contents of which are incorporated herein by reference in their entirety).

[0185] In some embodiments, the nanoparticles may comprise at least one acrylic polymer, including, but not limited to, acrylic acid, methacrylic acid, acrylic and methacrylic acid copolymers, methyl methacrylate copolymers, ethoxyethyl methacrylate, cyanoethyl methacrylate, aminoalkyl methacrylate copolymers, poly(acrylic acid), poly(methacrylic acid), polycyanoacrylate, and combinations thereof.

[0186] In some embodiments, the nanoparticles 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 that described in International Application No. 2013 / 032829 or U.S. Patent Publication No. 2013 / 0121954, 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 described herein.

[0187] 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) acid-poly(ethylene) glycol copolymer (see, e.g., International Patent Publication No. 2013 / 044219, 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, e.g., International Patent Publication No. 2013 / 044219, the contents of which are incorporated herein by reference in their entirety).

[0188] In some embodiments, the nanoparticles can include at least one cationic polymer described herein and / or known in the art.

[0189] In some embodiments, the nanoparticles may comprise at least one amine-containing polymer, such as, but not limited to, polylysine, polyethyleneimine, poly(amidoamine) dendrimers, poly(beta-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.

[0190] 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 ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), and combinations thereof. In some embodiments, the degradable polyester may comprise PEG conjugation to form a PEGylated polymer.

[0191] In some embodiments, the compositions may be formulated in colloidal nanocarriers such as those described in U.S. Patent Publication No. 2013 / 0197100, the contents of which are incorporated herein by reference in their entirety.

[0192] In some embodiments, the nanoparticles can be optimized for oral administration. The nanoparticles can include at least one cationic biopolymer, such as, but not limited to, chitosan or its derivatives. As a non-limiting example, the nanoparticles can be formulated by the method described in U.S. Publication No. 2012 / 0282343, the contents of which are incorporated herein by reference in their entirety.

[0193] In some embodiments, the LNPs comprise the lipid KL52 (an amino lipid disclosed in U.S. Application Publication No. 2012 / 0295832, the contents of which are incorporated herein by reference in their entirety. The activity and / or safety of LNP administration (e.g., as measured by examining one or more of ALT / AST, white blood cell count, and cytokine induction) may be improved by the incorporation of such a lipid. KL52-containing LNPs may be administered intravenously and / or in one or more doses. In some embodiments, administration of KL52-containing LNPs results in equal or improved mRNA and / or protein expression compared to LNPs containing MC3.

[0194] In some embodiments, the lipid nanoparticles may be the limit-size lipid nanoparticles described in International Patent Publication No. 2013 / 059922, the contents of which are incorporated herein by reference in their entirety. Limit-size lipid nanoparticles may comprise a lipid bilayer surrounding an aqueous core or a hydrophobic core, wherein the lipid bilayer may comprise phospholipids such as, but not limited to, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, cerebroside, C8-C20 fatty acid diacylphosphatidylcholine, and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC). In some embodiments, limit-size lipid nanoparticles may comprise polyethylene glycol lipids such as, but not limited to, DLPE-PEG, DMPE-PEG, DPPC-PEG, and DSPE-PEG.

[0195] In some embodiments, the composition can be formulated into nanoparticles comprising an inner core containing non-cellular material and an outer surface containing a cell membrane. The cell membrane can be derived from a cell or a virus. As a non-limiting example, the nanoparticles can be made by the method described in International Patent Publication No. 2013 / 052167, the contents of which are incorporated herein by reference in their entirety. As another non-limiting example, the nanoparticles described in International Patent Publication No. 2013 / 052167 can be used to deliver the compositions described herein, the contents of which are incorporated herein by reference in their entirety.

[0196] In some embodiments, the compositions may be formulated in porous nanoparticle-supported lipid bilayers (protocells), which are described in International Patent Publication No. WO 2013 / 056132, the contents of which are incorporated herein by reference in their entirety.

[0197] In some embodiments, the compositions described herein can be formulated into polymeric nanoparticles such as those described in U.S. Patent Nos. 8,420,123 and 8,518,963, and European Patent No. 2073848B1, or polymeric nanoparticles prepared 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 polymeric nanoparticles can have a high glass transition temperature, such as those described in U.S. Patent No. 8,518,963, or nanoparticles prepared by the methods described therein, the contents of which are incorporated herein by reference in their entirety. As another non-limiting example, polymeric nanoparticles for oral and parenteral formulations can be prepared by the methods described in European Patent No. 2073848B1, the contents of which are incorporated herein by reference in their entirety.

[0198] In some embodiments, the compositions described herein can be formulated into nanoparticles for use in imaging. The nanoparticles can be liposomal nanoparticles, such as those described in U.S. Patent Publication No. 2013 / 0129636, the entire contents of which are incorporated herein by reference. As a non-limiting example, the liposomes can contain gadolinium(III) 2-{4,7-bis-carboxymethyl-10-[(N,N-distearylamidomethyl-N'-amido-methyl]-1,4,7,10-tetra-azacyclododec-1-yl}-acetate and a neutral, fully saturated phospholipid component (see, for example, U.S. Patent Publication No. 2013 / 0129636, the contents of which are incorporated herein by reference in their entirety).

[0199] In some embodiments, nanoparticles that may be used in the present disclosure are formed by the methods described in U.S. Patent Application No. 2013 / 0130348, the contents of which are incorporated herein by reference in their entirety.

[0200] In some embodiments, the compositions of the present disclosure may be formulated in swellable nanoparticles, including, but 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.

[0201] The compositions of the present disclosure may be formulated in polyanhydride nanoparticles, such as, but not limited to, those described in U.S. Pat. No. 8,449,916, the contents of which are incorporated herein by reference in their entirety.

[0202] The nanoparticles and microparticles of the present disclosure can be geometrically engineered to modulate macrophage and / or immune responses. In some embodiments, the geometrically engineered particles can have various shapes, sizes, and / or surface charges to incorporate the polynucleotides of the present disclosure for targeted delivery. Other physical properties that the geometrically engineered particles may have include, but are not limited to, fenestration, angled arms, asymmetry, and surface roughness, charges that can modify interactions with cells and tissues. As a non-limiting example, the nanoparticles of the present disclosure can be produced by the method described in International Publication No. 2013 / 082111, the contents of which are incorporated herein by reference in their entirety.

[0203] In some embodiments, the nanoparticles of the present disclosure may be water-soluble nanoparticles, such as, but not limited to, those described in International Publication No. 2013 / 090601, the contents of which are incorporated herein by reference in their entirety. The nanoparticles may be inorganic nanoparticles that are dense and have zwitterionic ligands to exhibit good water solubility. The nanoparticles may also have a small hydrodynamic diameter (HD), stability over time, pH, and salinity, and low levels of nonspecific protein binding.

[0204] In some embodiments, the nanoparticles of the present disclosure can be developed by the methods described in U.S. Patent Publication No. 2013 / 0172406, the contents of which are incorporated herein by reference in their entirety.

[0205] 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. 2013 / 0172406, 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. 2013 / 0172406, the contents of which are incorporated herein by reference in their entirety.

[0206] In some embodiments, stealth or target-specific stealth nanoparticles can include a polymer matrix. The polymer matrix can include two or more polymers, such as, but not limited to, polyethylene, polycarbonate, polyanhydride, polyhydroxy acid, polypropyl fumerate, 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.

[0207] In some embodiments, the nanoparticles can be nanoparticle-nucleic acid hybrid structures having a dense nucleic acid layer. As a non-limiting example, nanoparticle-nucleic acid hybrid structures can be made by the methods described in U.S. Patent Publication No. 2013 / 0171646, the contents of which are incorporated herein by reference in their entirety. The nanoparticles can include nucleic acids, such as, but not limited to, polynucleotides described herein and / or known in the art.

[0208] At least one of the nanoparticles of the present disclosure may be embedded in a core nanostructure or may be coated with a low-density porous 3D structure or coating that can carry or associate with at least one payload within or on the surface of the nanostructure. Non-limiting examples of nanostructures comprising at least one nanoparticle are described in International Patent Publication No. 2013 / 123523, the contents of which are incorporated herein by reference in their entirety.

[0209] In some embodiments, the composition can be delivered using smaller LNPs. Such particles include, but are not limited to, particles smaller than 0.1 μm, 1.0 μm, 5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 85 μm, 90 μm, 95 μm, 100 μm, 125 μm, 150 μm, 175 μm, 200 μm, 225 μm, 250 μm, 275 μm, 300 μm, 325 ... The diameter may include diameters from less than 0.1 μm up to 100 nm, such as less than 0 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.

[0210] In some embodiments, the composition has a thickness of 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 to 50 nm, Smaller LNPs may be delivered using LNPs that may include diameters of 20-50 nm, 30-50 nm, 40-50 nm, 20-60 nm, 30-60 nm, 40-60 nm, 20-70 nm, 30-70 nm, 40-70 nm, 50-70 nm, 60-70 nm, 20-80 nm, 30-80 nm, 40-80 nm, 50-80 nm, 60-80 nm, 20-90 nm, 30-90 nm, 40-90 nm, 50-90 nm, 60-90 nm, and / or 70-90 nm.

[0211] In some embodiments, such LNPs are synthesized using methods that involve a microfluidic mixer. Examples of microfluidic mixers may include, but are not limited to, slit interdigital micromixers, including those manufactured by Microinnova (Allerheiligen bei Wildon, Austria), and / or staggered herringbone micromixers (SHM) (Zhigaltsev, IV et al., 2012). Bottom-up design and synthesis of limit-size lipid nanoparticle systems with aqueous and triglyceride cores using millisecond microfluidic mixing has been published (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 (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 method of producing LNPs involving SHM further includes mixing at least two input streams, where the mixing occurs via microstructure-induced chaotic advection (MICA). According to this method, fluid streams flow through channels present in a herringbone pattern, causing rotational flow and mixing of the fluids around each other. The method can also include a surface for fluid mixing, where the surface changes orientation during fluid circulation.Methods for producing 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.

[0212] In some embodiments, the compositions of the present disclosure may be formulated into lipid nanoparticles made using a micromixer, such as, but not limited to, a Slit Interdigital Microstructured Mixer (SIMM-V2) or a Standard Slit Interdigital Micro Mixer (SSIMM) or a Caterpillar (CPMM) or an Impinging-jet (IJMM) from Institut für Mikrotechnik Mainz GmbH, Mainz Germany.

[0213] In some embodiments, the compositions of the present disclosure can be formulated into lipid nanoparticles made using microfluidic technology (see, for example, 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). By way of non-limiting example, controlled microfluidic formulations include passive methods for mixing steady pressure-driven flow in microchannels at low Reynolds numbers (see, for example, Abraham et al. Chaotic Mixer for Microchannels. Science, 2002 295:647-651, the contents of which are incorporated herein by reference in their entirety).

[0214] In some embodiments, the compositions of the present disclosure can be formulated into lipid nanoparticles made using a micromixer chip, such as those from Harvard Apparatus (Holliston, Mass.) or Dolomite Microfluidics (Royston, UK). Micromixer chips can be used for rapid mixing of two or more fluid streams using a split and recombine mechanism.

[0215] In some embodiments, the compositions of the present disclosure may have a particle size of, 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, 30 to 90 nm, 30 to 100 nm The lipid nanoparticles may be formulated into lipid nanoparticles having diameters of 10-100 nm, such as 40-50 nm, 40-60 nm, 40-70 nm, 40-80 nm, about 40 to about 90 nm, about 40-100 nm, 50-60 nm, 50-70 nm, 50-80 nm, 50-90 nm, 50-100 nm, 60-70 nm, 60-80 nm, 60-90 nm, 60-100 nm, 70-80 nm, 70-90 nm, 70-100 nm, 80-90 nm, 80-100 nm, and / or 90-100 nm.

[0216] In some embodiments, the lipid nanoparticles may have a diameter of 10 to 500 nm.

[0217] In some embodiments, lipid nanoparticles can have a diameter of 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.

[0218] Also described herein is a composition comprising an effective amount of nanoparticles described herein and a pharmaceutically acceptable carrier.In some embodiments, the pharmaceutical composition can comprise a pharmaceutically acceptable carrier and nanoparticles that comprise a first T cell epitope, β2 microglobulin, and a single-chain trimeric nucleic acid (e.g., mRNA) that is encapsulated in the nanoparticles and that encodes a first MHC heavy chain sequence.In some embodiments, the pharmaceutical composition can comprise a pharmaceutically acceptable carrier and lipid nanoparticles that comprise a first T cell epitope, β2 microglobulin, and a single-chain trimeric nucleic acid (e.g., mRNA) that is encapsulated in the lipid nanoparticles and that encodes a first MHC heavy chain sequence.

[0219] Also described herein is a composition comprising an effective amount of nanoparticles described herein and a pharmaceutically acceptable carrier.In some embodiments, the pharmaceutical composition can comprise a pharmaceutically acceptable carrier and nanoparticles that comprise a single-stranded nucleic acid dimer (e.g., mRNA) that encodes a T cell epitope and a primary MHC heavy chain sequence, encapsulated in the nanoparticle.In some embodiments, the pharmaceutical composition can comprise a pharmaceutically acceptable carrier and lipid nanoparticles that comprise a single-stranded nucleic acid dimer (e.g., mRNA) that encodes a T cell epitope and a primary MHC heavy chain sequence, encapsulated in the lipid nanoparticle.

[0220] Liposomes, lipoplexes, or lipid nanoparticles can be used to improve the efficiency of polynucleotide-directed protein production. Because these formulations may be able to increase cell transfection with RNA (e.g., mRNA) polynucleotides and / or increase the translation of the encoded protein. One such example involves the use of lipid encapsulation to enable effective systemic delivery of polyplexed 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.

[0221] Liposomes are artificially prepared vesicles that may be composed primarily of lipid bilayers and can be used as delivery vehicles for the administration of nutrients and pharmaceutical formulations. Liposomes can be of different sizes, including, but not limited to, multilamellar vesicles (MLVs), which can be hundreds of nanometers in diameter and contain a series of concentric bilayers separated by narrow aqueous compartments; small unilamellar vesicles (SUVs), which can be less than 50 nm in diameter; and large unilamellar vesicles (LUVs), which can be 50-500 nm in diameter. Liposome designs can include, but are not limited to, opsonins or ligands to improve liposome attachment to non-healthy tissues or to activate events such as endocytosis. Liposomes can also contain low or high pH to improve the delivery of pharmaceutical formulations.

[0222] Liposome formation may depend on physicochemical properties such as, but not limited to, the encapsulated pharmaceutical agent and liposome components, the nature of the medium in which the lipid vesicles are dispersed, the effective concentration of the encapsulated substance and its potential toxicity, any additional processes involved during application and / or delivery of the vesicles, optimization of the size, polydispersity and shelf life, and batch-to-batch reproducibility of the vesicles for the intended use, and the feasibility of large-scale production of the liposome product in a safe and efficient manner.

[0223] In some embodiments, the pharmaceutical compositions described herein may include liposomes such as, but not limited to, those formed from 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA) liposomes, DiLa2 liposomes from Marina Biotech (Bothell, Wash.), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), and MC3 (US 2010 / 0324120, incorporated herein by reference in its entirety), as well as liposomes capable of delivering small molecule drugs, such as, but not limited to, DOXIL® from Janssen Biotech, Inc. (Horsham, Pa.).

[0224] In some embodiments, the pharmaceutical compositions described herein may comprise liposomes, such as, but not limited to, those formed from the synthesis of stabilized plasmid-lipid particles (SPLPs) or stabilized nucleic acid lipid particles (SNALPs), which have been previously described and shown to be suitable for oligonucleotide delivery in vitro and in vivo (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 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. 2013 / 0122104, all of which are incorporated herein in their entireties. The original manufacturing method by Wheeler et al. was the detergent dialysis method, which was later improved by Jeffs et al. and referred to as the spontaneous vesicle formation method. Liposome formulations consist of three to four lipid components in addition to the polynucleotide. As an example, the liposomes can contain, but are not limited to, 55% cholesterol, 20% disteroylphosphatidylcholine (DSPC), 10% PEG-S-DSG, and 15% 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA) as described by Jeffs et al.As another example, a particular liposome formulation may contain, but is not limited to, 48% cholesterol, 20% DSPC, 2% PEG-c-DMA, and 30% cationic lipid, where the cationic lipid may be 1,2-distearoyl-N,N-dimethylaminopropane (DSDMA), DODMA, DLin-DMA, or 1,2-dilinolenyloxy-3-dimethylaminopropane (DLenDMA) as described by Heyes et al.

[0225] In some embodiments, the liposome formulation may contain about 25.0% to about 40.0% cholesterol, about 30.0% to about 45.0% cholesterol, about 35.0% to about 50.0% cholesterol, and / or about 48.5% to about 60% cholesterol. In some embodiments, the formulation may contain a percentage of cholesterol 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 formulation may contain about 5.0% to about 10.0% DSPC and / or about 7.0% to about 15.0% DSPC.

[0226] In some embodiments, the compositions may be formulated in liposomes such as, but not limited to, DiLa2 liposomes (Marina Biotech, Bothell, Wash.), SMARTICLES® (Marina Biotech, Bothell, Wash.), neutral DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine)-based liposomes (e.g., siRNA delivery for ovarian cancer (Landen et al. Cancer Biology & Therapy 2006 5(12)1708-1713); incorporated herein by reference in its entirety), and hyaluronan-coated liposomes (Quiet Therapeutics, Israel).

[0227] In some embodiments, the compositions may be formulated in lipid vesicles which may have crosslinks between functionalized lipid bilayers.

[0228] In some embodiments, the composition may be formulated into a lipid-polythione complex. Formation of the lipid-polythione complex may be achieved by methods known in the art and / or methods such as those described in U.S. Publication No. 2012 / 0178702, the entire contents of which are incorporated herein by reference. By way of non-limiting example, the polycation may comprise a cationic peptide or polypeptide, such as, but not limited to, polylysine, polyornithine, and / or polyarginine. In some embodiments, the composition may be formulated into a lipid-polycation complex, which may further comprise a non-cationic lipid, such as, but not limited to, cholesterol or dioleoylphosphatidylethanolamine (DOPE).

[0229] In some embodiments, the compositions are based on siRNA-lipoplex technologies, including, but not limited to, the ATUPLEX™ system, the DACC system, the DBTC system, and other siRNA-lipoplex technologies from Silence Therapeutics (London, United Kingdom), STEMFECT™ from STEMGENT® (Cambridge, Mass.), and polyethylenimine (PEI) or protamine-based targeted and non-targeted delivery of nucleic acids (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.,2011J.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 incorporated herein by reference in their entirety).

[0230] In some embodiments, such formulations can also be constructed or modified so that they are passively or actively directed in vivo to different cell types, 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. An example of passive targeting of formulations to liver cells includes DLin-DMA, DLin-KC2-DMA, and DLin-MC3-DMA-based lipid nanoparticle formulations, which have been shown to bind to apolipoprotein E and promote the binding and uptake of these formulations into liver cells in vivo (Akinc et al. Mol Ther. 2010 18:1357-1364, the contents of which are incorporated herein by reference in their entirety).Formulations can also be selectively targeted through the expression of different ligands on their surface, as exemplified by, but not limited to, folate, 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 incorporated herein by reference in their entirety).

[0231] 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 conforms to a specific release pattern to produce a therapeutic outcome. In some embodiments, the compositions may be encapsulated in 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 surround, enclose, or encase. In the context of formulations of compounds of the present disclosure, encapsulation can be substantial, complete, or partial. The term "substantially encapsulated" means that at least 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, greater than 99.9, or greater than 99.999% of the pharmaceutical composition or compound of the present disclosure can be enclosed, surrounded, or encased within the 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 may be enclosed, surrounded, or enveloped within the delivery agent. Advantageously, encapsulation may be determined by measuring the escape or activity of the pharmaceutical composition or compound of the present disclosure using fluorescence and / or electron microscopy. 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 greater than 99.99% of the pharmaceutical composition or compound of the present disclosure is encapsulated within the delivery agent.

[0232] In some embodiments, the controlled release formulation may include, but is not limited to, a triblock copolymer. 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).

[0233] In some embodiments, the composition may be encapsulated in lipid nanoparticles or rapidly eliminated lipid nanoparticles, which may then be encapsulated in a polymer, hydrogel, and / or surgical sealant described herein and / or known in the art. By way of non-limiting example, the polymer, hydrogel, or surgical sealant may be PLGA, ethylene vinyl acetate (EVAc), poloxamer, surgical sealants such as GELSITE® (Nanotherapeutics, Inc. Alachua, Fla.), HYLENEX® (Halozyme Therapeutics, San Diego, Calif.), fibrinogen polymers (Ethicon Inc. Cornelia, Ga.), TISSELL® (Baxter International, Inc. Deerfield, Ill.), PEG-based sealants, and COSEAL® (Baxter International, Inc. Deerfield, Ill.).

[0234] In some embodiments, the compositions described herein formulated for controlled release and / or targeted delivery may also include at least one degradable polyester that may contain polycationic side chains. Degradable polyesters include, but are not limited to, poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), and combinations thereof. In some embodiments, the degradable polyester may include PEG conjugation to form a PEGylated polymer.

[0235] In some embodiments, the compositions described herein that are formulated for controlled release and / or targeted delivery may also include at least one PEG and / or PEG-related polymer derivative as described in U.S. Pat. No. 8,404,222, the contents of which are incorporated herein by reference in their entirety.

[0236] In some embodiments, the compositions described herein that are formulated for controlled release delivery may be controlled release polymer systems as described in US2013 / 0130348, the contents of which are incorporated herein by reference in their entirety.

[0237] In some embodiments, synthetic nanocarriers can be formulated for targeted release. In some embodiments, synthetic nanocarriers are formulated to release polynucleotides at a specific pH and / or after a desired time interval. As a non-limiting example, synthetic nanoparticles can be formulated to release RNA (e.g., mRNA) vaccines after 24 hours and / or at a pH of 4.5 (see International Publication Nos. 2010 / 138193 and 2010 / 138194 and U.S. Publication Nos. 2011 / 0020388 and 2011 / 0027217, each of which is incorporated herein by reference in its entirety).

[0238] In some embodiments, synthetic nanocarriers can be formulated for controlled and / or sustained release of the nucleic acids described herein. By way of non-limiting example, synthetic nanocarriers for sustained release can be formulated by methods known in the art, described herein, and / or as described in WO 2010 / 138192 and U.S. 2010 / 0303850, each of which is incorporated herein by reference in its entirety.

[0239] In some embodiments, synthetic nanocarriers can be formulated for use as vaccines. By way of non-limiting example, synthetic nanocarriers can include, but are not limited to, those nanocarriers described in International Publication Nos. 2011 / 150264, 2011 / 150249, 2012 / 024621, 2012 / 02629, and 2012 / 024632, as well as U.S. Publication Nos. 2011 / 0293701, 2011 / 0293723, 2012 / 0064110, 2012 / 0058153, and 2012 / 0058154, the contents of each of which are incorporated herein by reference in their entirety. Vaccine dosage forms may be selected by methods described herein, known in the art, and / or described in WO 2011 / 150258 and U.S. 2012 / 0027806, the contents of each of which are incorporated herein by reference in their entirety.

[0240] In some embodiments, the compositions may be formulated in colloidal nanocarriers such as those described in U.S. Patent Publication No. 2013 / 0197100, the contents of which are incorporated herein by reference in their entirety.

[0241] In some embodiments, compositions of the present disclosure can be formulated for delivery using drug-encapsulated microspheres as described in International Patent Publication No. 2013 / 063468 or U.S. Patent No. 8,440,614, the contents of each of which are incorporated herein by reference in their entirety. The microspheres can contain compounds of formula (I), (II), (III), (IV), (V), or (VI) as described in International Patent Publication No. 2013 / 063468, the contents of which are incorporated herein by reference in their entirety. In some embodiments, amino acids, peptides, polypeptides, lipids (APPLs) are useful in delivering RNA (e.g., mRNA) polynucleotides of the present disclosure to cells (see International Patent Publication No. 2013 / 063468, the contents of which are incorporated herein by reference in their entirety).

[0242] In some embodiments, the composition can be delivered, localized, and / or concentrated at a specific location using the delivery methods described in International Patent Publication No. 2013 / 063530, the contents of which are incorporated herein by reference in their entirety. As a non-limiting example, a subject can be administered empty polymer particles before, simultaneously with, or after delivery of a composition described herein to the subject. Upon contact with the subject, the empty polymer particles change volume and remain, become embedded, immobilized, or encapsulated at a specific location in the subject.

[0243] In some embodiments, the composition may be formulated in an active agent release system (see, e.g., U.S. Patent Publication No. 2013 / 0102545, the contents of which are incorporated herein by reference in their entirety). The active agent release system may include 1) at least one nanoparticle bound to an oligonucleotide inhibitor chain hybridized with a catalytically active nucleic acid, and 2) a compound bound to at least one substrate molecule bound to a therapeutically active agent (e.g., a polynucleotide described herein), wherein the therapeutically active agent is released by cleavage of the substrate molecule by the catalytically active nucleic acid.

[0244] In some embodiments, the compositions may be associated with cationic or polycationic compounds, such as protamine, nucleolin, spermine, or spermidine, or other cationic peptides or proteins, such as poly-L-lysine (PLL), polyarginine, basic polypeptides, 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(s), Pep-1, L-oligomer, calcitonin peptide(s), antennapedia-derived peptides (especially from Drosophila antennapedia), pAntp, plsl, FGF, lactoferrin, transportan, buforin-2, Bac715- 24, SynB, SynB(1), pVEC, hCT-derived peptides, SAP, cell-penetrating peptides (CPPs) including histones, cationic polysaccharides such as chitosan, polybrene, cationic polymers such as polyethyleneimine (PEI), cationic lipids such as DOTMA: [1-(2,3-thioleyloxy)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: dimyristoxypropyldimethylhydroxyethylammonium bromide, DOTAP: dioleoyloxy-3-(trimethylammonio)propane, DC-6-14: O,O-ditetradecanoyl-N-alpha-trimethylammonioacetyl)diethanolamine chloride, CLIP1: rac-[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammonium chloride, CLIP6: rac-[2(2,3-dihexadecyloxypropyloxymethyloxy)ethyl]-trimethylammonium, CLIP9: rac-[2(2,3-dihexadecyloxypropyloxysuccinyloxy)ethyl]-trimethylammonium, Oligofectamine, or Cationic or polycationic polymers, such as modified polyamino acids, such as beta-amino acid polymers or inverse polyamides, modified polyethylenes, such as PVP (poly(N-ethyl-4-vinylpyridinium bromide)), modified acrylates, such as pDMAEMA (poly(dimethylaminoethyl methyl acrylate)), modified amidoamines, such as pAMAM (poly(amidoamine)), modified polybeta-amino esters (PBAE), such as diamine-terminated 1,4 butanediol diacrylate-co-5-amino-1-pentanol polymers, dendrimers, such as polypropylene Examples of suitable polymers include polyallylamine dendrimers or pAMAM-based dendrimers, polyimine(s), such as PEI (poly(ethyleneimine)), poly(propyleneimine), polyallylamine, sugar-based polymers, such as cyclodextrin-based polymers, dextran-based polymers, chitosan, silane-based polymers, such as PMOXA-PDMS copolymers, and block polymers consisting of a combination of one or more cationic blocks (e.g., selected from the cationic polymers mentioned above) and one or more hydrophilic or hydrophobic blocks (e.g., polyethylene glycol).

[0245] In other embodiments, the composition is not associated with a cationic or polycationic compound.

[0246] vaccine The present specification describes a vaccine comprising the composition described herein.In some embodiments, the present specification describes a vaccine comprising a composition comprising a first T cell epitope, β2 microglobulin, and a single-chain trimer nucleic acid (e.g., mRNA) encoding a first MHC heavy chain sequence.

[0247] Also described herein, in some embodiments, is a vaccine comprising a composition comprising a single-stranded nucleic acid dimer (eg, mRNA) encoding a T cell epitope and a primary MHC heavy chain sequence.

[0248] In some embodiments, the vaccine can be formulated in the nanoparticles described herein. In some embodiments, the vaccine can be formulated in the lipid nanoparticles described herein. In some embodiments, the vaccine can be formulated in the lipid-polycation complexes described herein, referred to as cationic lipid nanoparticles. The vaccines of the present disclosure can be formulated using one or more liposomes, lipoplexes, or lipid nanoparticles described herein. In some embodiments, the pharmaceutical composition of the vaccine can include the liposomes described herein. In some embodiments, the vaccine can be formulated in lipid vesicles that may have crosslinks between functionalized lipid bilayers. In some embodiments, the vaccine can be formulated in lipid-polythione complexes.

[0249] In some embodiments, described herein are vaccines comprising lipid nanoparticles comprising a single-chain trimeric nucleic acid (e.g., mRNA) encoding a first T cell epitope, β2 microglobulin, and a first MHC heavy chain sequence. Also described herein are vaccines in some embodiments, comprising lipid nanoparticles comprising a single-chain nucleic acid dimer (e.g., mRNA) encoding a T cell epitope and a primary MHC heavy chain sequence.

[0250] Method of administration The compositions used in the methods described herein can be administered by any suitable method and technique known to those skilled in the art now or in the future.For example, the active ingredients described herein can be formulated in physiologically or pharmaceutically acceptable form and can be administered by any suitable route known in the art, including, for example, oral and parenteral administration routes.As used herein, the term "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intraperitoneal and intrasternal administration, such as by injection.The administration of the active ingredients of these compositions can be administered in a single dose, or can be administered continuously and at different intervals, as can be easily determined by those skilled in the art.

[0251] "Excipients" include any and all solvents, diluents or other liquid vehicles, dispersing or suspending aids, surface active agents, isotonicity agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, etc., as suitable for the particular dosage form desired. General considerations in formulation and / or manufacturing can be found, for example, in Remington's Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980), and Remington: The Science and Practice of Pharmacy, 21st Edition (Lippincott Williams & Wilkins, 2005).

[0252] Exemplary excipients include, but are not limited to, any non-toxic, inert solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation auxiliary.Some examples of materials that can function as excipients include 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; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; 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; Tween Excipients include, but are not limited to, detergents such as 80; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening agents, flavorings and fragrances, preservatives, and antioxidants, and may be present in the composition according to the judgment of the formulator. As will be understood by those skilled in the art, excipients can be selected based on what the composition is useful for. For example, with pharmaceutical compositions, the choice of excipient will depend on the route of administration, the agent being delivered, the time course of delivery of the agent, etc., and can be administered to humans and / or animals orally, rectally, parenterally, intracisternally, intravaginally, intranasally, intraperitoneally, topically (by powder, cream, ointment, or drops), bucally, or as an oral or nasal spray. In some embodiments, the active compounds disclosed herein are administered topically.

[0253] Exemplary diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen 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.

[0254] Exemplary granulating and / or dispersing agents include potato starch, corn starch, tapioca starch, sodium starch glycolate, clay, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose, and wood products, natural sponge, cation exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethylcellulose, cross-linked sodium carboxymethylcellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water-insoluble starch, calcium carboxymethylcellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, and the like, and combinations thereof.

[0255] Exemplary surfactants and / or emulsifiers include natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, 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, ethylhexyl hydroxybenzoate ... 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, polyethylene glycol fatty acid esters (e.g., Cremophor), polyoxyethylene ethers (e.g., polyoxyethylene lauryl ether [Brij30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic F68, Poloxamer 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, 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., acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, isapol husk mucilage, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum), and larch arabogalactan), alginates, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylates, waxes, water, alcohol, and the like, and / or combinations thereof.

[0256] Exemplary preservatives include antioxidants, chelating agents, antibacterial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and other preservatives.

[0257] 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.

[0258] Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA) and its salts and hydrates (e.g., edetate sodium, edetate disodium, edetate trisodium, edetate calcium disodium, edetate dipotassium, 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 antibacterial preservatives include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.

[0259] Exemplary antifungal preservatives include butylparaben, methylparaben, ethylparaben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid.

[0260] Exemplary alcohol preservatives include ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoates, and phenylethyl alcohol.

[0261] 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, cetrimide, 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, methylparaben, 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.

[0262] Exemplary buffering agents include citrate buffer solution, acetate buffer solution, phosphate buffer solution, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, calcium diphosphate, phosphoric acid, calcium triphosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, potassium diphosphate, potassium monophosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, sodium diphosphate, sodium monophosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and the like, and combinations thereof.

[0263] Exemplary lubricants include magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl betaine, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and the like, and combinations thereof.

[0264] Exemplary natural oils include almond, apricot kernel, avocado, babassu, bergamot, blackcurrant seed, borage, cade, chamomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cottonseed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazelnut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon. Exemplary synthetic oils include limonene, macadamia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savory, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, camellia, vetiver, walnut, and wheat germ oil. Exemplary synthetic oils include butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and combinations thereof.

[0265] Additionally, the composition may further comprise a polymer. Exemplary polymers contemplated herein include cellulosic polymers and copolymers, such as cellulose ethers, such as methylcellulose (MC), hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), hydroxypropylmethylcellulose (HPMC), methylhydroxyethyl cellulose (MHEC), methylhydroxypropyl cellulose (MHPC), carboxymethyl cellulose (CMC), and various salts thereof, including, for example, the sodium salt, hydroxyethyl carboxymethyl cellulose (HECMC) and various salts thereof, carboxymethyl hydroxyethyl cellulose (CMHE), carboxymethyl hydroxyethyl cellulose (CMHE), and various salts thereof. C) and its various salts, other polysaccharides and polysaccharide derivatives such as starch, dextran, dextran derivatives, chitosan, and various gums including alginic acid and its various salts, carrageenan, xanthan gum, guar gum, gum arabic, karaya gum, ghatti gum, konjac, and tragacanth gum, 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(epsilon).-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, polyethyleneamine, and polypyridine, polyethylene glycol Polyglycol (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, but are not limited to these.

[0266] Additionally, the composition may further comprise an emulsifier. Exemplary emulsifiers include polyethylene glycol (PEG), polypropylene glycol, polyvinyl alcohol, poly-N-vinylpyrrolidone, and copolymers thereof, poloxamer nonionic surfactants, neutral water-soluble polysaccharides (e.g., dextran, Ficoll, cellulose), non-cationic poly(meth)acrylates, non-cationic polyacrylates such as poly(meth)acrylic acid, and their ester amides and hydroxyalkyl amides, natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, 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., 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 [Myrj45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and Solutol), sucrose fatty acid esters, polyethylene glycol fatty acid 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 F68, Poloxamer 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, and / or combinations thereof. In certain embodiments, the emulsifier is cholesterol.

[0267] Liquid compositions include emulsions, microemulsions, solutions, suspensions, syrups and elixirs.In addition to active compounds, liquid compositions can contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed, peanut, corn, germ, olive, castor and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan, and their mixtures.In addition to inert diluents, oral compositions can also contain adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavorings and fragrances.

[0268] Injectable compositions, for example, injectable aqueous or oily suspensions, can be formulated according to known techniques using suitable dispersants or wetting agents and suspending agents. Sterile injectable preparations can also be injectable solutions, suspensions, or emulsions in non-toxic parenterally acceptable diluents or solvents, for example, as solutions in 1,3-butanediol. Acceptable vehicles and solvents for pharmaceutical or cosmetic compositions include water, Ringer's solution, USP, and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally used as solvents or suspending media. Any bland fixed oil can be used, including synthetic monoglycerides or diglycerides. In addition, fatty acids such as oleic acid can be used in the preparation of injectables. In certain embodiments, particles are suspended in a carrier fluid containing 1% (w / v) sodium carboxymethylcellulose and 0.1% (v / v) Tween 80. Injectable compositions can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0269] 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 will melt in the rectum or vaginal cavity and release the particles.

[0270] Solid composition includes capsule, tablet, pill, powder and granule.In this solid composition, particle is mixed with at least one excipient and / or a) filler or extender such as starch, lactose, sucrose, glucose, mannitol and silicic acid, b) binder such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidinone, sucrose and acacia, c) moisturizing agent such as glycerol, d) disintegrating agent such as agar-agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain silicate and sodium carbonate, e) solution retardant such as paraffin, f) absorption accelerator such as quaternary ammonium compound, g) wetting agent such as cetyl alcohol and glycerol monostearate, h) absorbent such as kaolin and bentonite clay, and i) lubricant such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and their mixture. In the case of capsules, tablets, and pills, the dosage forms may also contain buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar, as well as high molecular weight polyethylene glycols and the like.

[0271] Tablets, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulation art. They may optionally contain opacifying agents and can be of a composition that releases the active ingredient(s) only, or preferentially, in a certain part of the gastrointestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type can also be used as fillers in soft and hard-filled gelatin capsules using excipients such as lactose or milk sugar, and high molecular weight polyethylene glycols, etc.

[0272] Compositions for topical or transdermal administration include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, or patches. The active compound is mixed with an excipient and any necessary preservatives or buffers, if any, which may be required.

[0273] The ointments, pastes, creams, and gels may contain, in addition to the active compound, excipients such as animal and vegetable fats, oils, waxes, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc, and zinc oxide, or mixtures thereof.

[0274] Powders and sprays can contain, in addition to the active compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates, and polyamide powder, or mixtures of these substances. Sprays can additionally contain conventional propellants, such as chlorofluorohydrocarbons.

[0275] Transdermal patches have the additional advantage of providing controlled delivery of compounds to the body.Such dosage forms can be prepared by dissolving or distributing nanoparticles in a suitable medium.Absorption enhancers can also be used to increase the flux of compounds across the skin.The rate can be controlled by providing a rate-controlling membrane or dispersing particles in a polymer matrix or gel.

[0276] The compound can be incorporated into microparticles, nanoparticles, or a combination thereof, which provide controlled release of the compound and / or additional active agents. For example, the compound can be incorporated into polymer microparticles which provide controlled release of drug(s). The release of the drug(s) is controlled by the diffusion of the drug(s) from the microparticles and / or the degradation of the polymer particles by hydrolysis and / or enzymatic degradation. Suitable polymers include ethylcellulose and other natural or synthetic cellulose derivatives.

[0277] Polymers that dissolve slowly in an aqueous environment and form gels, such as hydroxypropylmethylcellulose 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-hydroxybutyric acid (PHB) and its copolymers, poly-4-hydroxybutyric acid (P4HB) and its copolymers, polycaprolactone and its copolymers, and combinations thereof.

[0278] Alternatively, the compound can be incorporated into microparticles prepared from materials that are insoluble in aqueous solutions or that dissolve slowly in aqueous solutions but can be degraded in the GI tract by means including enzymatic degradation, the surfactant action of bile acids, and / or mechanical erosion. As used herein, the term "slowly soluble in water" refers to a material that does not dissolve in water within 30 minutes. Preferred examples include fats, fatty substances, waxes, wax-like substances, and mixtures thereof. Suitable fats and fatty substances include fatty acids and derivatives, including, but not limited to, fatty alcohols (such as lauryl, myristylstearyl, cetyl, or cetostearyl alcohol), fatty acid esters, fatty acid glycerides (monoglycerides, diglycerides, and triglycerides), and hydrogenated fats. Specific examples include, but are not limited to, hydrogenated vegetable oils, hydrogenated cottonseed oils, hydrogenated castor oils, hydrogenated oils available under the trade name Sterotex®, stearic acid, cocoa butter, and stearyl alcohol. Suitable waxes and wax-like materials include natural or synthetic waxes, hydrocarbons, and conventional waxes. Specific examples of waxes include beeswax, glycowax, castor wax, carnauba wax, paraffin, and candelilla wax. As used herein, a wax-like material is defined as any material that is typically solid at room temperature and has a melting point of about 30 to 300°C.

[0279] In some cases, it may be desirable to modify the water penetration rate into microparticles.For this purpose, rate-controlling (wicking) agents 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., hydroxypropylmethyl-cellulose, hydroxypropylcellulose, methylcellulose, and carboxymethyl-cellulose), alginic acid, lactose, and talc.In addition, pharmaceutically acceptable surfactants (e.g., lecithin) can be added to facilitate the disintegration of such microparticles.

[0280] Water-insoluble proteins, such as zein, can also be used as materials for forming drug-containing microparticles. Additionally, water-soluble proteins, polysaccharides, and combinations thereof can be formulated into microparticles with drugs and then crosslinked to form an insoluble network. For example, cyclodextrin can be complexed with individual drug molecules and then crosslinked.

[0281] The encapsulation or incorporation of drugs into carrier materials to produce drug-containing microparticles can be achieved through known pharmaceutical formulation techniques.When formulated in fat, wax, or wax-like materials, the carrier material is typically heated above its melting temperature, and the drug is added to form a mixture containing drug particles suspended in the carrier material, drug dissolved in the carrier material, or a mixture thereof.The microparticles can then be formulated through several methods, including but not limited to, solidification, extrusion, spray cooling, or aqueous dispersion processes.In a preferred process, wax is heated above its melting temperature, the drug is added, and the molten wax-drug mixture is solidified under constant stirring as the mixture cools.Alternatively, the molten wax-drug mixture can be extruded and spheronized to form pellets or beads.These processes are known in the art.

[0282] For some carrier materials, it may be desirable to use solvent evaporation techniques to create drug-containing microparticles. In this case, the drug and carrier material are co-dissolved in a mutual solvent, after which the microparticles can be created by several techniques, including, but not limited to, forming an emulsion in water or other suitable medium, spray drying, or by evaporating the solvent from a bulk solution and milling the resulting material.

[0283] In some embodiments, the drug(s) in particulate form are uniformly dispersed in a water-insoluble or slowly water-soluble material. To minimize the size of drug particles in the composition, the drug powder itself may be milled to produce fine particles before formulation. Jet milling, a process known in the pharmaceutical field, can be used for this purpose. In some embodiments, the drug in particulate form is uniformly dispersed in a wax or wax-like substance by heating the wax or wax-like substance above its melting point and adding the drug particles while stirring the mixture. In this case, a pharmaceutically acceptable surfactant may be added to the mixture to facilitate dispersion of the drug particles.

[0284] Particles can also be coated with one or more modified-release coatings. Solid esters of fatty acids hydrolyzed by lipase can be spray-coated onto 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 spray coating or wet granulation techniques. In addition to naturally water-insoluble materials, some substrates of digestive enzymes can be treated with a cross-linking procedure, resulting in the formation of an insoluble network. Many methods for cross-linking proteins, initiated by both chemical and physical means, have been reported. One of the most common methods for achieving cross-linking is the use of chemical cross-linking agents. Examples of chemical cross-linking agents include aldehydes (gluteraldehyde and formaldehyde), epoxy compounds, carbodiimides, and genipin. In addition to these cross-linking agents, oxidized and natural sugars have been used to cross-link gelatin. Cross-linking can also be achieved using enzymatic means. For example, transglutaminase has been approved as a GRAS substance for cross-linking seafood products. Finally, crosslinking can be initiated by physical means such as heat treatment, UV irradiation, and gamma irradiation.

[0285] To prepare a coating layer of crosslinked protein surrounding microparticles or drug-containing particles, water-soluble protein can be spray-coated on the microparticles, and then crosslinked by one of the methods described above. Alternatively, drug-containing microparticles can be microencapsulated in protein by coacervation-phase separation (for example, by adding salt), and then crosslinked. Some suitable proteins for this purpose include gelatin, albumin, casein, and gluten.

[0286] 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 undergo cross-linking in the presence of multivalent cations. Complexes between oppositely charged polysaccharides can also form. For example, pectin and chitosan can complex through electrostatic interactions.

[0287] In certain embodiments, it may be desirable to provide the patient in need with the continuous delivery of one or more compounds.For intravenous or intraarterial route, this can be achieved by using drip system, such as by intravenous administration.For local application, it can be repeatedly applied, or patch can be used to provide the continuous administration of compound for a long period of time.

[0288] The compounds described herein can be incorporated into injectable / implantable solid or semi-solid implants, such as polymeric implants. In one embodiment, the compounds are incorporated into a polymer that is liquid or paste at room temperature but exhibits an increase in viscosity upon contact with an aqueous medium, such as physiological fluid, to form a semi-solid or solid material. Exemplary polymers include, but are not limited to, hydroxyalkanoic acid polyesters derived from the copolymerization of at least one unsaturated hydroxy fatty acid copolymerized with a hydroxyalkanoic acid. The polymer can be melted, mixed with the active agent, and cast or injection molded into a device. Such melt processing requires a polymer with a melting point below the temperature at which the substance to be delivered and the polymer decompose or become reactive. Devices can also be prepared by solvent casting, in which the polymer is dissolved in a solvent, the drug is dissolved or dispersed in the polymer solution, and then the solvent is evaporated. The solvent process requires the polymer to be soluble in an organic solvent. Another method is compression molding of a mixed powder of polymer and drug, or polymer particles loaded with the active agent.

[0289] 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 biodegradable polymers, such as polyanhydrides, polyhydroalkanoic acids (PHAs), PLA, PGA, PLGA, polycaprolactone, polyesters, polyamides, polyorthoesters, polyphosphazenes, proteins and polysaccharides, such as collagen, hyaluronic acid, albumin, and gelatin, and combinations thereof, and compressed into a solid device, such as a disk, wafer, or extruded into a device, such as a rod.

[0290] The release of a compound from an implant can be altered by the choice of polymer, the molecular weight of the polymer, and / or modifying the polymer, e.g., by creating pores and / or incorporating hydrolyzable linkages, to increase degradation. Methods for modifying the properties of biodegradable polymers to change the release profile of a compound from an implant are well known in the art.

[0291] In some embodiments, the compound or pharmaceutical composition can be administered locally. In some embodiments, the compound is incorporated into a delivery system such as a gel, nanoparticles, microparticles, or an implant (e.g., a rod, disk, wafer, or orthopedic implant) for sustained release. In some embodiments, the compound can be administered using an implantable system for local delivery comprising the compound incorporated within a gel, nanoparticles, microparticles, or implant. In some embodiments, the pharmaceutical composition comprises a delivery system such as a gel, nanoparticles, microparticles, or an implant (e.g., a rod, disk, wafer, or orthopedic implant) for sustained release of paroxetine or a pharmaceutically acceptable salt or derivative thereof.

[0292] The active ingredient can be administered in the amount, time, and route deemed necessary to achieve the desired results. 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 administration method, its mode of activity, etc. Whether the active ingredient is the active compound itself or the active compound combined with a drug, it is preferably formulated into a unit dosage form for ease of administration and uniformity of dosage. However, it will be understood that the total daily amount of the active ingredient will be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any specific subject will depend on various factors, including the disorder being treated and the severity of the disorder; the activity of the active ingredient used; the specific composition used; the patient's age, weight, general health, sex, and diet; the administration time, administration route, and excretion rate of the specific active ingredient used; the duration of treatment; drugs used in combination with or simultaneously with the specific active ingredient used; and similar factors well known in the medical arts.

[0293] The active ingredient can be administered by any route. In some embodiments, the active ingredient is administered by various routes, including oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, subcutaneous, intracerebroventricular, transdermal, intradermal, rectal, intravaginal, intraperitoneal, topical (by powder, ointment, cream, and / or drops), mucosal, nasal, buccal, enteral, sublingual, intratracheal instillation, bronchial instillation, and / or inhalation, and / or oral spray, nasal spray, and / or aerosol. Generally, the most suitable administration route will depend on various factors, including the properties of the active ingredient (for example, its stability in the gastrointestinal environment), the condition of the subject (for example, whether the subject can tolerate oral administration), etc.

[0294] 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 the side effect or disorder, the identity of the specific compound(s), the mode of administration, etc. For example, the amount administered to a child or adolescent can be determined by a practitioner or person skilled in the art and may be lower than or the same as the amount administered to an adult.

[0295] Useful dosages of the compositions disclosed herein can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known in the art.

[0296] The dosage range for administering the composition is large enough to bring about the desired effect that symptoms or disorders are affected.Dosage should not be so large as to cause harmful side effects, such as undesirable cross-reaction, anaphylactic reaction, etc. Generally, dosage varies according to the age, condition, sex, and degree of disease of the patient, and can be determined by those skilled in the art.Dosage can be adjusted by individual physicians in the event of any contraindications.Dosage can vary and can be administered in one or more doses per day for one or several days.

[0297] Compositions described herein are typically formulated into unit dosage form for ease of administration and dosage uniformity.However, it will be understood that the total daily amount of composition used can be determined by attending physician within the scope of sound medical judgment.The specific therapeutically effective dose level, prophylactically effective dose level or suitable imaging dose level for any specific patient will depend on various factors, including the disorder being treated and the severity of the disorder; the activity of the specific compound used; the specific composition used; the age, weight, general health condition, sex and diet of the patient; the administration time, administration route and excretion rate of the specific compound used; treatment period; the drug used in combination with or simultaneously with the specific compound used; and similar factors well known in the medical field.

[0298] In some embodiments, the compositions are administered at a dose of 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. ...1 mg / kg, etc. of a subject's body weight per day, one or more times per day, per week, per month, etc. to achieve a desired therapeutic, diagnostic, prophylactic, or imaging effect. The desired dosage may be administered at dosage levels sufficient to deliver 0.1 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 (see, e.g., the unit dose ranges set forth in WO 2013 / 078199, the contents of which are incorporated herein by reference in their entirety). The desired dosage may be delivered three times a day, twice a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, every four weeks, every two months, every three months, every six months, etc. In some embodiments, the desired dosage may be delivered using multiple administrations (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more administrations). When multiple administrations are used, split-dose regimens such as those described herein may be used. In exemplary embodiments, the compositions may be administered at a dosage level sufficient to deliver 0.0005 mg / kg to 0.01 mg / kg, e.g., about 0.0005 mg / kg to about 0.0075 mg / kg, e.g., about 0.0005 mg / kg, about 0.001 mg / kg, about 0.002 mg / kg, about 0.003 mg / kg, about 0.004 mg / kg, or about 0.005 mg / kg.

[0299] In some embodiments, the composition may be administered once or twice (or more) at a dosage 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.

[0300] In some embodiments, the composition is administered twice (e.g., on days 0 and 7, 0 and 14, 0 and 21, 0 and 28, 0 and 60, 0 and 90, 0 and 120, 0 and 150, 0 and 180, 0 and 3 months, 0 and 6 months, 0 and 9 months, 0 and 12 months, 0 and 18 months, 0 and 2 years, 0 and 5 years, or 0 and 10 years) at 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, The compound may be administered at a dosage level sufficient to deliver a total dose of, or equal to, 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, 0.775 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. Higher and lower dosages and administration frequencies are encompassed by the present disclosure. For example, the compositions described herein can be administered three or four times.

[0301] In some embodiments, the composition may be administered twice (e.g., on days 0 and 7, 0 and 14, 0 and 21, 0 and 28, 0 and 60, 0 and 90, 0 and 120, 0 and 150, 0 and 180, 0 and 3 months, 0 and 6 months, 0 and 9 months, 0 and 12 months, 0 and 18 months, 0 and 2 years, 0 and 5 years, or 0 and 10 years) at a total dose of 0.010 mg, 0.025 mg, 0.100 mg, or 0.400 mg, or at a dosage level sufficient to deliver that total dose.

[0302] In some embodiments, a composition (e.g., a vaccine) for use in a method of vaccinating a subject is administered to a subject as a single dosage of 10 μg / kg to 400 μg / kg of nucleic acid vaccine (in an amount effective to vaccinate the subject). In some embodiments, a composition (e.g., a vaccine) for use in a method of vaccinating a subject is administered to a subject as a single dosage of 10 μg to 400 μg of nucleic acid vaccine (in an amount effective to vaccinate the subject). In some embodiments, a composition (e.g., a vaccine) for use in a method of vaccinating a subject is administered to a subject as a single dosage of 25 to 1000 μg (e.g., a single dosage of mRNA encoding an infectious agent and mRNA encoding a T cell epitope, or an infectious agent and mRNA encoding at least one universal T cell epitope). In some embodiments, a composition (e.g., a vaccine) is administered to a subject in a single dosage 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, a composition (e.g., a vaccine) can be administered in a single dosage 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, a composition (e.g., a vaccine) for use in a method of vaccinating a subject is administered to the subject as two dosages, the combination being equivalent to 25 to 1000 μg of the composition (e.g., vaccine).

[0303] The compositions (e.g., vaccine pharmaceutical compositions) described herein 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).

[0304] How to use Described herein are methods for activating and / or expanding antigen-presenting cells, the methods comprising co-culturing antigen-presenting cells with cells comprising a single-stranded trimeric nucleic acid described herein, a composition described herein, a nanoparticle described herein, a vaccine described herein, or a pharmaceutical composition described herein. In some embodiments, the methods for activating and / or expanding antigen-presenting cells comprise co-culturing antigen-presenting cells and cells comprising a lipid nanoparticle described herein.

[0305] Also described herein are methods of treating a subject having a viral infection, a bacterial infection, a parasitic infection, and / or cancer, the method comprising administering to the subject a therapeutically effective amount of a single-stranded trimeric 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. 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 method comprising administering to the subject a therapeutically effective amount of a lipid nanoparticle described herein.

[0306] In some embodiments, described herein are methods of treating a subject with cancer, the method comprising administering to the subject a therapeutically effective amount of a single-stranded trimeric 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. In some embodiments, described herein are methods of treating a subject with cancer, the method comprising administering to the subject a therapeutically effective amount of a lipid nanoparticle described herein.

[0307] Also described herein are methods of treating a subject having a viral infection, a bacterial infection, a parasitic infection, and / or 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 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.

[0308] In some embodiments, methods are described for treating a subject having a viral infection, a bacterial infection, a parasitic infection, and / or cancer, the method comprising obtaining antigen-presenting cells from the subject, contacting the antigen-presenting cells from the subject with lipid nanoparticles described herein to thereby activate the antigen-presenting cells, and administering the activated antigen-presenting cells to the subject.

[0309] In some embodiments, described herein are methods of treating a subject having 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 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.

[0310] In some embodiments, a method is described for treating a subject having cancer, the method comprising obtaining antigen-presenting cells from the subject, contacting the antigen-presenting cells from the subject with lipid nanoparticles described herein to thereby activate the antigen-presenting cells, and administering the activated antigen-presenting cells to the subject.

[0311] Also described herein are methods for preventing a viral infection, bacterial infection, parasitic infection, and / or cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a single-stranded trimeric 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. In some embodiments, described herein are methods for preventing a viral infection, bacterial infection, parasitic infection, and / or cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a lipid nanoparticle described herein.

[0312] In some embodiments, described herein are methods of preventing a subject from having cancer, the method comprising administering to the subject a therapeutically effective amount of a single-stranded 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. In some embodiments, described herein are methods of preventing a subject from having cancer, the method comprising administering to the subject a therapeutically effective amount of a lipid nanoparticle described herein.

[0313] Also described herein is a method for preventing a viral infection, a bacterial infection, a parasitic infection, and / or cancer in a subject, 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 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.

[0314] In some embodiments, a method is described for preventing a subject from having a viral infection, a bacterial infection, a parasitic infection, and / or cancer, the method comprising obtaining antigen-presenting cells from the subject, contacting the antigen-presenting cells from the subject with lipid nanoparticles described herein to thereby activate the antigen-presenting cells, and administering the activated antigen-presenting cells to the subject.

[0315] In some embodiments, a method of preventing a subject from having cancer is described, 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 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.

[0316] In some embodiments, a method is described for preventing a subject from having cancer, the method comprising obtaining antigen-presenting cells from the subject, contacting the antigen-presenting cells from the subject with lipid nanoparticles described herein to thereby activate the antigen-presenting cells, and administering the activated antigen-presenting cells to the subject.

[0317] In some embodiments, the antigen-presenting cells may be dendritic cells, B cells, macrophages, or other cells derived from the subject that can be used for immunotherapy.

[0318] In some embodiments, the cancer is a circulating cancer cell (circulating tumor cell). In some embodiments, the cancer is a metastatic cancer cell. In some embodiments, the compositions and methods described herein are used to treat both localized and metastatic tumors. In some embodiments, the compositions and methods described herein are useful for treating or preventing metastasis or recurrence of cancer. In some embodiments, the compositions and methods described herein are useful for preventing recurrence of resected solid tumors. In some embodiments, the compositions and methods described herein are useful for preventing metastasis of resected solid tumors.

[0319] In one aspect, the methods described herein are directed to the treatment of cancer, such as, among others, melanoma, lung cancer (including lung adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, large cell carcinoma, bronchioloalveolar 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 carcinoma); colorectal cancer (colon cancer, rectal carcinoma, colorectal adenocarcinoma); anal cancer; pancreatic cancer (including pancreatic adenocarcinoma, pancreatic islet cell carcinoma, neuroendocrine tumors); prostate cancer; prostate adenocarcinoma; ovarian cancer (serous tumors, endometrioid tumors, and ovarian epithelial carcinoma including mucinous cystadenocarcinoma or superficial epithelial-stromal tumors, sex cord-stromal tumors); liver and bile duct cancer (bile duct carcinoma (including hepatocellular carcinoma, cholangiocarcinoma, and hemangioma); esophageal cancer (including esophageal adenocarcinoma and squamous cell carcinoma); oral and oropharyngeal squamous cell carcinoma; salivary gland adenoid cystic carcinoma; bladder cancer; bladder carcinoma; uterine cancer (including endometrial adenocarcinoma, ocular, uterine serous adenocarcinoma, uterine clear cell carcinoma, uterine sarcoma, leiomyosarcoma, and mixed Müllerian tumor); glioma, glioblastoma, medulloblastoma, and other tumors of the brain; kidney cancer (including renal cell carcinoma, clear cell carcinoma, and Wilms' tumor); head and neck cancer (including squamous cell carcinoma); stomach cancer (gastric carcinoma, gastric adenocarcinoma, and gastrointestinal stromal tumor); testicular cancer; germ cell tumors; neuroendocrine tumors; cervical cancer; carcinoids of the gastrointestinal tract, breast, and other organs; signet ring cell Cancer; including sarcoma, fibrosarcoma, hemangioma, hemangiomatosis, hemangiopericytoma, pseudoangiomatous stromal hyperplasia, myofibroblastoma, fibromatosis, inflammatory myofibroblastic tumor, lipoma, angiolipoma, granular cell tumor, neurofibroma, schwannoma, angiosarcoma, liposarcoma, rhabdomyosarcoma, osteosarcoma, leiomyoma, leiomyosarcoma, skin, melanoma, neck, retinoblastoma, head and neck cancer, pancreatic, brain, thyroid, sperm Cancer of the follicular, kidney, bladder, soft tissue, adrenal gland, urethra, penis; myxosarcoma, chondrosarcoma, osteosarcoma, chordoma, malignant fibrous histiocytoma, lymphangiosarcoma, mesothelioma, mesenchymal tumors including squamous cell carcinoma; epidermoid carcinoma, malignant skin adnexal tumor, adenocarcinoma, hepatocellular carcinoma, renal cell carcinoma, renal cell tumor, cholangiocarcinoma, transitional cell carcinoma, choriocarcinoma, seminoma, embryonal cell carcinoma, anaplastic glioma;Used to treat glioblastoma multiforme, neuroblastoma, medulloblastoma, malignant meningioma, malignant schwannoma, neurofibrosarcoma, parathyroid carcinoma, medullary carcinoma of the thyroid, bronchial carcinoid, pheochromocytoma, pancreatic islet cell carcinoma, malignant carcinoid, malignant paraganglioma, melanoma, Merkel cell neoplasm, cystosarcoma phyllodes, salivary carcinoma, thymic carcinoma, and cancer of the vagina;

[0320] In one embodiment, the bacterial organism capable of causing a bacterial infection is selected from the group consisting of Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Streptococcus pyrogenes, 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, Leptospirosis Interrogans, Borrelia burgdorferi, and Camphylobacter jejuni may be included, but are not limited to these.Viruses that can cause viral infections include metapneumoviruses, e.g., human metapneumovirus (hMPV), parainfluenza viruses, e.g., 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-N), and others. HCoV-HK1, HCoV-NH, HCoV-HKU1), poxvirus (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, and the like. Other organisms that can cause infection include fungal, protozoan, and parasitic organisms such as Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia ricketsii, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydial psittaci, Chlamydial trachomatis, Plasmodium falciparum, Trypanosoma brucei, Entamoeba histolytica, Toxoplasma gondii, Trichomonas vaginalis, Schistosoma mansoni, and the like.

[0321] In some embodiments, the method can further comprise administering an additional active agent. In some embodiments, an additional active agent.

[0322] As used herein, "active agent" refers to a therapeutic, diagnostic, or prophylactic agent. As discussed herein, therapeutic agents can be released from the disclosed compounds, compositions, and systems in a biologically active form.

[0323] As used herein, the term "therapeutic agent" is further understood to refer to one or more therapeutic agents, active ingredients, or substances that can be used to treat a medical condition. A therapeutic agent includes any synthetic or naturally occurring biologically active compound or composition that, when administered to an organism (human or non-human animal), induces a desired pharmacological, immunogenic, and / or physiological effect through local and / or systemic action. Thus, the term encompasses those compounds or chemicals traditionally considered to be drugs, vaccines, and biopharmaceuticals, including molecules such as proteins, peptides, hormones, nucleic acids, gene constructs, and the like. Examples of therapeutic agents are described in well-known references, such as the Merck Index (14th ed.), Physicians' Desk Reference (64th ed.), and The Pharmacological Basis of Therapeutics (12th ed.), and include, but are not limited to, medicines; vitamins and minerals, such as essential amino acids, calcium, iron, potassium, zinc, vitamin B12, and the like; substances used to treat, prevent, diagnose, cure, or mitigate a disease or disorder; substances that affect the structure or function of the body; or prodrugs that become biologically active or more active after being placed in a physiological environment. For example, the term "therapeutic agent" includes compounds or compositions for use in all of the major therapeutic areas, including adjuvants; antimicrobial agents (including antibiotics, antivirals, antiparasitics, and antifungals), anti-inflammatory agents (including steroids and nonsteroidal anti-inflammatory agents), anticoagulants, ophthalmic agents, gastrointestinal agents, antiplatelet agents, and antiseptics, steroidal agents, antitumor agents, anticancer agents, antigens, antibodies (e.g., cetuximab, anti-CD24 antibodies, panitumumab, and bevacizumab), birth control agents, progestational agents, anticholinergic agents, nutritional agents, analgesics, and analgesic combinations, such as acetaminophen, acetylsalicylic acid, and the like; anesthetic agents, such as lidocaine, xylocaine, and the like, anorexics, such as dexadrine, phendimetrazine tartrate, and the like;Antiepileptics, local and general anesthetics, hypnotics, sedatives, antipsychotics, neuroleptics, antidepressants, such as isocarboxazid, amoxapine, etc.; anxiolytics, antagonists, neuroleptics, anticholinergics and cholinergics, antimuscarinic and muscarinic agents, antiparkinsonian agents, antialzheimer's agents, antiadrenergic agents, antiarrhythmic agents, antihypertensive agents, hormones, such as insulin, progestins, estrogens, corticoids, glucocorticoids, androgens, etc.; as well as nutrients, antiarthritis drugs. Antiinflammatory drugs, such as methylprednisolone, ibuprofen, etc.; antiasthmatic drugs, such as terbutaline sulfate, theophylline, ephedrine, etc.; anticonvulsants, such as phenyloin sodium, diazepam, etc.; antiallergic drugs, antihistamines, such as diphenhydramine HCl, chlorpheniramine maleate, etc.; antiemetics, antineoplastic drugs, antipruritics, antipyretics; antispasmodics, such as belladonna alkaloids, dicyclomine hydrochloride, etc.; cardiovascular drugs, such as prazosin HCl, nitroglycerin, propranolol, etc. nolol HCl, hydralazine HCl, pancrelipase, succinate dehydrogenase, etc.; vasoactive agents, cardiovascular preparations (including calcium channel blockers, beta blockers, beta agonists, and antiarrhythmics), antihypertensives, diuretics, e.g., furosemide, spironolactone, etc.; vasodilators; central nervous system stimulants; cough and cold preparations; decongestants; diagnostic agents; bone growth stimulants and bone resorption inhibitors; muscle relaxants; psychostimulants; sedatives; tranquilizers, e.g., thorazine, diazepam, chlorpromazine antiulcer drugs, such as rantidine HCl, cimetidine HCl, etc.; antiasthmatics, antidiarrheals, antiobesity agents, antithrombotic agents, antitussives, antiuricemic agents, antianginal agents, appetite suppressants, expectorants, hyperglycemic agents, hypoglycemic agents, thyroid and antithyroid agents, tissue growth agents, uterine relaxants, immunomodulatory agents including cytokines, interleukins, interferons, colony-stimulating factors, tumor necrosis factors, etc.; immunosuppressants, such as rapamycin, tacrolimus, etc.; immunological agents;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 peptides, growth-releasing factors, angiotensin, FSH, EGF, bone morphogenetic proteins (BMPs), erythropoietin (EPO), interferons, interleukins, collagen, fibrinogen, insulin, Factor VIII, Factor IX, Enbrel®, Rituxam®, Herceptin®, alpha-glucosidase, Cerazyme / These include, but are not limited to, Ceredose®, vasopressin, ACTH, human serum albumin, gamma globulin, structural proteins, blood product proteins, complex proteins, antigens or antigenic polypeptides, enzymes, antibodies, monoclonal antibodies, and the like; as well as nucleic acid molecules (polymeric forms of two or more nucleotides, polynucleotides, either ribonucleotides (RNA) or deoxyribonucleotides (DNA), including both double-stranded and single-stranded molecules, gene constructs, expression vectors, antisense molecules, and the like), small molecules (e.g., doxorubicin), and other biologically active macromolecules, such as proteins and enzymes. The drug may be a biologically active agent used in medical applications, including veterinary medicine, and in agriculture, such as plants, and other areas. In certain embodiments of the present disclosure, the drug to be delivered may be a mixture of active agents.

[0324] Representative examples of antibiotics include amikacin, amoxicillin, ampicillin, atovaquone, azithromycin, aztreonam, bacitracin, carbenicillin, cefadroxil, cefazolin, cefdinir, cefditoren, cefepime, cefiderocol, cefoperazone, cefotetan, cefoxitin, cefotaxime, cefpodoxime, cefprozil, ceftaroline, ceftazidime, ceftibuten, and cefti Zoxime, ceftriaxone, chloramphenicol, colistimethate, cefuroxime, cephalexin, cephradine, cilastatin, cinoxacin, ciprofloxacin, clarithromycin, clindamycin, dalbavancin, dalfopristin, daptomycin, demeclocycline, dicloxacillin, doripenem, doxycycline, eravacycline, ertapenem, erythromycin, fidaxomi cin, fosfomycin, gatifloxacin, gemifloxacin, gentamicin, imipenem, lefamulin, lincomycin, linezolid, lomefloxacin, loracarbef, meropenem, metronidazole, minocycline, moxifloxacin, nafcillin, nalidixic acid, neomycin, norfloxacin, ofloxacin, omadacycline, oritavancin, oxacillin, oxytetracycline, paroxetine These include momycin, penicillin, pentamidine, piperacillin, plazomycin, quinupristin, rifaximin, sarecycline, secnidazole, sparfloxacin, spectinomycin, sulfamethoxazole, sulfisoxazole, tedizolid, telavancin, telithromycin, ticarcillin, tigecycline, tobramycin, trimethoprim, trovafloxacin, and vancomycin.

[0325] Representative examples of antiviral agents include abacavir, acyclovir, adefovir, amantadine, amprenavir, atazanavir, balavir, baloxavir marboxil, boceprevir, cidofovir, cobicistat, daclatasvir, darunavir, delavirdine, didanosine, docasanol, dolutegravir, doravirine, ecoliever, edoxudine, efavirenz, and elvitegravir. , emtricitabine, enfuvirtide, entecavir, etravirine, famciclovir, fomivirsen, fosamprenavir, forscarnet, fosnonet, famciclovir, favipravir, fomivirsen, foscavir, ganciclovir, ibacitabine, idoxuridine, indinavir, inosine, inosine pranobex, type I interferon, type II interferon, type III interferon , lamivudine, letermovir, letermovir, lopinavir, loviride, maraviroc, methisazone, moroxydine, nelfinavir, nevirapine, nitazoxanide, oseltamivir, peginterferon alfa-2a, peginterferon alfa-2b, penciclovir, peramivir, pleconaril, podophyllotoxin, pyramidine, raltegravir, remdesevir, ribavirin, rilpivirine, rimantadine, lintatrimovir These include, but are not limited to, molnupiravir, ritonavir, saquinavir, simeprevir, sofosbuvir, stavudine, talabivirine, telaprevir, telbivudine, tenofovir alafenamide, tenofovir disoproxil, tenofovir, tipranavir, trifluridine, trizivir, tromantadine, umifenovir, valacyclovir, valganciclovir, vidarabine, zalcitabine, zanamivir, and zidovudine.

[0326] Representative examples of anticoagulants include, but are not limited to, heparin, warfarin, rivaroxaban, dabigatran, apixaban, edoxaban, enoxaparin, and fondaparinux.

[0327] Representative examples of antiplatelet agents include, but are not limited to, clopidogrel, ticagrelor, prasugrel, dipyridamole, dipyridamole / aspirin, ticlopidine, and eptifibatide.

[0328] Representative examples of antifungal agents include, but are not limited to, voriconazole, itraconazole, posaconazole, fluconazole, ketoconazole, clotrimazole, isavuconazonium, miconazole, caspofungin, anidulafungin, micafungin, griseofulvin, terbinafine, flucytosine, terbinafine, nystatin, and amphotericin b.

[0329] Representative examples of steroidal anti-inflammatory drugs include, but are not limited to, hydrocortisone, dexamethasone, prednisolone, prednisone, triamcinolone, methylprednisolone, budesonide, betamethasone, cortisone, and deflazacort. Representative examples of non-steroidal anti-inflammatory drugs include ibuprofen, naproxen, ketoprofen, tolmetin, etodolac, fenoprofen, flurbiprofen, diclofenac, piroxicam, indomethacin, sulindax, meloxicam, nabumetone, oxaprozin, mefenamic acid, and diflunisal.

[0330] Other examples of active agents include chloroquine, hydrochloroquine, pyridoxal phosphate, vitamin D, and vitamin C.

[0331] Representative examples of anti-cytokine or immunomodulatory agents include, but are not limited to, tocilizumab, sarilumab, bevacizumab, fingolimod, imiquimod, and eculizumab.

[0332] Immunotherapeutic agents can 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 (e.g., nivolumab (BMS), pembrolizumab (Merck), pidilizumab (CureTech / Teva), AMP-244 (Amplimmune / GSK), BMS-936559 (BMS), and MEDI4736 (Roche / Genentech)), or combinations thereof.

[0333] Representative examples of contraceptives include, but are not limited to, progestins, estrogens, or any combination thereof.For example, suitable progestins include, but are not limited to, natural and synthetic compounds with progestational activity, such as progesterone, chlormadinone acetate, norethindrone, cyproterone acetate, norethindrone acetate, desogestrel, levonorgestrel, drospirenone, trimegestone, norgestrel, norgestimate, norelgestromin, etonogestrel, gestodene, and other natural and / or synthetic gestagens. For example, suitable estrogens include, but are not limited to, natural and synthetic compounds having estrogenic activity, such as estradiol (17β-estradiol), 17α-estradiol, estriol, estrone, and esters thereof, such as acetate, sulfate, valerate, or benzoate esters 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); mestranol; agonist antiestrogens; and selective estrogen receptor modulators. Other examples of contraceptives include gonodotropin-releasing hormone (GnRh) or its analogs, such as deslorelin, avorelin, leuprolide, triptorelin, nafarelin, goserelin, buserelin, and fertirelin.

[0334] The term "steroid" refers to compounds belonging to or related to the following exemplary families of compounds: corticosteroids, mineralosteroids, and sex steroids (e.g., including potentially androgenic or estrogenic, or antiandrogenic and antiestrogenic molecules). Included among these are, for example, prednisone, prednisolone, methyl-prednisolone, triamcinolone, fluocinolone, aldosterone, spironolactone, danazol (also known as OPTINA), and others. In some embodiments, the therapeutic agent may include a steroid.

[0335] Exemplary cancer drugs or anti-cancer agents can include, but are not limited to, antimetabolic and anti-mitotic anti-cancer agents, and combinations thereof. A variety of such agents, including single antimetabolic and anti-mitotic anti-cancer agents or combinations of such agents, can be used in the methods and compositions described herein.

[0336] Anti-metabolite anti-cancer drugs are typically structurally similar to natural metabolites involved in the normal metabolic processes of cancer cells, such as the synthesis of nucleic acids and proteins.However, antimetabolites are sufficiently different from natural metabolites that they interfere with the metabolic processes of cancer cells.In cells, antimetabolites are mistaken for 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 exert cytotoxic activity by substituting these incorrect nucleotides and entering cellular DNA, thereby interfering with cell division or inhibiting important cellular enzymes that prevent DNA replication.

[0337] Thus, in one embodiment, the anti-cancer agent is a nucleotide or nucleotide analog. In certain embodiments, for example, the anti-metabolite may include a purine (e.g., guanine or adenosine) or analog thereof, or a pyrimidine (cytidine or thymidine) or analog thereof, with or without an attached sugar moiety.

[0338] Suitable antimetabolite anticancer agents for use in the present disclosure can be generally classified according to the metabolic processes they affect and can include, but are not limited to, analogs and derivatives of folic acid, pyrimidines, purines, and cytidine. Thus, in one embodiment, the antimetabolite(s) is selected from the group consisting of cytidine analogs, folic acid analogs, purine analogs, pyrimidine analogs, and combinations thereof.

[0339] In one particular embodiment, for example, the antimetabolite is a cytidine analog. According to this embodiment, for example, the cytidine analog can be selected from the group consisting of cytarabine (cytosine arabinoside), azacytidine (5-azacytidine), and salts, analogs, and derivatives thereof.

[0340] In another specific embodiment, for example, the antimetabolite is a folic acid analogue.Folic acid analogue or antifolate generally functions by inhibiting dihydrofolate reductase (DHFR), which is the enzyme involved in the formation of nucleotides; when this enzyme is blocked, nucleotides are not formed, and DNA replication and cell division are hindered.In a specific embodiment, for example, the folic acid analogue can be selected from the group consisting of denopterin, methotrexate (amethopterin), pemetrexed, pteropterin, raltitrexed, trimetrexate, and their salts, analogues and derivatives.

[0341] In another specific embodiment, for example, antimetabolite is purine analogue.Purine-based antimetabolite works by inhibiting DNA synthesis, for example, by interfering with the production of purine-containing nucleotides, adenine and guanine, thereby stopping DNA synthesis and thereby stopping cell division.In addition, purine analogue can be incorporated into DNA molecule itself during DNA synthesis, which can interfere with cell division. According to certain embodiments, for example, the purine analog may be selected from the group consisting of acyclovir, allopurinol, 2-aminoadenosine, arabinosyladenine (ara-A), azacitidine, azathiprine, 8-aza-adenosine, 8-fluoro-adenosine, 8-methoxy-adenosine, 8-oxo-adenosine, cladribine, deoxycoformycin, fludarabine, gancylovir, 8-aza-guanosine, 8-fluoro-guanosine, 8-methoxy-guanosine, 8-oxo-guanosine, guanosine diphosphate, guanosine diphosphate-beta-L-2-aminofucose, guanosine diphosphate-D-arabinose, guanosine diphosphate-2-fluorofucose, guanosine diphosphate fucose, mercaptopurine (6-MP), pentostatin, thiamiprine, thioguanine (6-TG), and salts, analogs, and derivatives thereof.

[0342] In yet another specific embodiment, for example, antimetabolite is pyrimidine analogue.Similar to the purine analogue discussed above, pyrimidine-based antimetabolite blocks the synthesis of pyrimidine-containing nucleotide (cytosine and thymine in DNA, cytosine and uracil in RNA).By acting as " decoy ", pyrimidine-based compound can prevent the production of nucleotide, and / or can be incorporated into growing DNA chain, causing its termination. According to certain embodiments, for example, the pyrimidine analog is ancitabine, azacitidine, 6-azauridine, bromouracil (e.g., 5-bromouracil), capecitabine, carmofur, chlorouracil (e.g., 5-chlorouracil), cytarabine (cytosine arabinoside), cytosine, dideoxyuridine, 3'-azido-3'-deoxythymidine, 3'-dideoxycytidin-2'-ene, 3'-deoxy-3'-deoxythymidin-2'-ene, dihydrouracil, doxifluridine, enocitabine, floxuridine, 5-fluo The pyrimidine analog may be selected from the group consisting of cytosine, 2-fluorodeoxycytidine, 3-fluoro-3'-deoxythymidine, fluorouracil (e.g., 5-fluorouracil (also known as 5-FU)), gemcitabine, 5-methylcytosine, 5-propynylcytosine, 5-propynylthymine, 5-propynyluracil, thymine, uracil, uridine, and salts, analogs, and derivatives thereof. In one embodiment, the pyrimidine analog is other than 5-fluorouracil. In another embodiment, the pyrimidine analog is gemcitabine or a salt thereof.

[0343] In certain embodiments, the antimetabolite is selected from the group consisting of 5-fluorouracil, capecitabine, 6-mercaptopurine, methotrexate, gemcitabine, cytarabine, fludarabine, pemetrexed, and salts, analogs, derivatives, and combinations thereof. In other embodiments, the antimetabolite is selected from the group consisting of capecitabine, 6-mercaptopurine, methotrexate, gemcitabine, cytarabine, fludarabine, pemetrexed, and salts, analogs, derivatives, and combinations thereof. In a particular embodiment, the antimetabolite is other than 5-fluorouracil. In a particularly preferred embodiment, the antimetabolite is gemcitabine, or a salt thereof (e.g., gemcitabine HCl (Gemzar®)).

[0344] Other antimetabolite anticancer agents include, among others, acantifolic acid, aminothiadiazole, brequinar sodium, Ciba-Geigy CGP-30694, cyclopentylcytosine, cytarabine phosphate stearate, cytarabine conjugate, Lilly DATHF, Merrel Dow DDFC, desaguanine, dideoxycytidine, dideoxyguanosine, didox, Yoshitomi DMDC, Wellcome EHNA, Merck & Co. EX-015, fazarabine, fludarabine phosphate, N-(2'-furanidyl)-5-fluorouracil, Daiichi Seiyaku FO-152, 5-FU-fibrinogen, isopropylpyrrolidine, Lilly LY-188011; Lilly LY-264618, metobenzaprim, Wellcome MZPES, norspermidine, NCI NSC-127716, NCI NSC-264880, NCI NSC-39661, NCI NSC-612567, Warner-Lambert PALA, pentostatin, piritrexim, plicamycin, Asahi Chemical PL-AC, Takeda TAC-788, tiazofurin, Erbamont TIF, tyrosine kinase inhibitors, Taiho UFT, and ulishitin.

[0345] In one embodiment, the antimitotic anticancer drug is a microtubule inhibitor or a microtubule stabilizer. Generally, microtubule stabilizers, such as taxanes and epothilones, bind to the inner surface of beta-microtubule chains and enhance microtubule assembly by promoting the nucleation and elongation phase of polymerization and reducing the concentration of the critical tubulin subunits required for microtubule assembly. Unlike microtubule inhibitors, such as vinca alkaloids, which prevent microtubule assembly, microtubule stabilizers, such as taxanes, reduce the lag time and dramatically shift the dynamic equilibrium between tubulin dimers and microtubule polymers to polymerization. Therefore, in one embodiment, the microtubule stabilizer is a taxane or epothilones. In another embodiment, the microtubule inhibitor is a vinca alkaloid.

[0346] In some embodiments, the anticancer agent may include a taxane, or a derivative or analog thereof. Taxanes may be naturally occurring compounds or related forms, or may be chemically synthesized compounds or derivatives thereof with antitumor properties. Taxanes are a family of terpenes, including, but not limited to, paclitaxel (Taxol®) and docetaxel (Taxotere®), which are derived primarily 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 a preferred taxane and is considered an antimitotic agent, promoting the assembly of microtubules from tubulin dimers and stabilizing microtubules by preventing depolymerization. This stability results in the inhibition of the normal dynamic reorganization of the microtubule network, which is essential for important interphase and mitotic cellular functions.

[0347] Also included are various known taxane derivatives, including both hydrophilic and hydrophobic derivatives.Taxane derivatives include, but are not limited to, the galactose and mannose derivatives described in International Patent Application No. 99 / 18113, the piperazino and other derivatives described in WO99 / 14209, the taxane derivatives described in WO99 / 09021, WO98 / 22451, and U.S. Patent No. 5,869,680, the 6-thio derivatives described in WO98 / 28288, the sulfenamide derivatives described in U.S. Patent No. 5,821,263, the deoxygenated paclitaxel compounds such as those described in U.S. Patent No. 5,440,056, and the taxol derivatives described in U.S. Patent No. 5,415,869. As noted above, taxanes further include 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. Taxanes can also be taxane conjugates, such as paclitaxel-PEG, paclitaxel-dextran, paclitaxel-xylose, docetaxel-PEG, docetaxel-dextran, docetaxel-xylose, and the like. Other derivatives are mentioned, inter alia, in "Synthesis and Anticancer Activity of Taxol Derivatives," D.G.I. Kingston et al., Studies in Organic Chemistry, Vol. 26, entitled "New Trends in Natural Products Chemistry" (1986), edited by Atta-ur-Rabman and P.W.le Quesne (Elsevier, Amsterdam 1986). Each of these references is incorporated herein by reference in its entirety.

[0348] 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 incorporated herein by reference in its entirety, or can be obtained from a variety of commercial sources, including, for example, Sigma-Aldrich Co., St. Louis, Mo.

[0349] Alternatively, antimitotic anticancer drugs can be microtubule inhibitors, and in a preferred embodiment, the microtubule inhibitor is a vinca alkaloid.Generally, vinca alkaloids are spindle poisons.Vinca alkaloids act during mitosis, when chromosomes divide and begin to move along the spindle tube toward one of its poles before cell separation.Under the influence of these spindle poisons, the spindle is disorganized by the dispersion of chromosomes during mitosis, affecting cell proliferation.In a certain embodiment, for example, the vinca alkaloid is selected from the group consisting of vinblastine, vincristine, vindesine, vinorelbine, and their salts, analogs, and derivatives.

[0350] The antimitotic anticancer agent may also be an epothilone. Generally, members of the epothilone class of compounds stabilize microtubule function according to a mechanism similar to that of taxanes. Epothilones can also cause cell cycle arrest at the G2-M transition phase, resulting in cytotoxicity and ultimately apoptosis. Suitable epithilones include epothilone A, epothilone B, epothilone C, epothilone D, epothilone E, and epothilone F, as well as salts, analogs, and derivatives thereof. One particular epothilone analog is ixabepilone (Ixempra™), an epothilone B analog.

[0351] In certain embodiments, the antimitotic anticancer agent is selected from the group consisting of taxanes, epothilones, vinca alkaloids, and salts and combinations thereof. Thus, for example, in one embodiment, the antimitotic agent is a taxane. In this embodiment, the antimitotic agent is more preferably paclitaxel or docetaxel, and even more preferably paclitaxel. In another embodiment, the antimitotic agent is an epothilone (e.g., an epothilone B analog). In another embodiment, the antimitotic agent is a vinca alkaloid.

[0352] Examples of cancer drugs that can be used in the present disclosure include, but are not limited to, thalidomide; platinum coordination compounds, 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 sunitimibe and imatinib.

[0353] Examples of additional cancer drugs include alkylating agents, antimetabolites, natural products, hormones and antagonists, and various drugs. Aliases are shown in parentheses. Examples of alkylating agents include nitrogen mustards, such as mechlorethamine, cyclophosphamide, ifosfamide, melphalan sarcolysin, and chlorambucil; ethyleneimines and methylmelamines, such as hexamethylmelamine and thiotepa; alkyl sulfonates, such as busulfan; nitrosoureas, such as carmustine (BCNU), semustine (methyl-CCNU), lomustine (CCNU), and streptozotocin; 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; taxanes, 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-releasing hormone agonists, such as buserelin; corticosteroids, such as prednisone and related preparations; progestins, such as hydroxyprogesterone caproate, lunedroxyprogesterone acetate, and megestrol acetate; estrogens, such as diethylstilbestrol and ethinylestradiol and related preparations; estrogen antagonists, such as tamoxifen and anastrozole; androgens, such as testosterone propionate and fluoxymesterone and related preparations; androgen antagonists, such as flutamide and bicalutamide; and gonadotropin-releasing hormone analogs, such as leuprolide.These and other examples of cancer drugs have aliases and trade names, and their use methods, including dosage and administration regimens, are known to those skilled in the art.

[0354] In some embodiments, anti-cancer drugs can include chemotherapeutic agents.Suitable chemotherapeutic agents include but are not limited to alkylating agents, antibacterial agents, antimetabolites, hormonal agents, plant-derived drugs and their synthetic derivatives, anti-angiogenic agents, differentiation inducers, cell growth arrest inducers, apoptosis inducers, cytotoxic agents, agents that affect cellular bioenergetics, i.e., agents that affect cellular ATP levels and the molecules / activities that regulate these levels, biological agents, such as monoclonal antibodies, kinase inhibitors, and inhibitors of growth factors and their receptors, gene therapy agents, cell therapy, such as stem cells, or any combination thereof.

[0355] According to these embodiments, the chemotherapeutic agent is selected from the group consisting of cyclophosphamide, chlorambucil, melphalan, mechlorethamine, ifosfamide, busulfan, lomustine, streptozocin, temozolomide, dacarbazine, cisplatin, carboplatin, oxaliplatin, procarbazine, uramustine, 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 a separate embodiment of the present invention.

[0356] Antitumor agents include abiraterone acetate, Abitrexate (methotrexate), Abraxane (paclitaxel albumin-stabilized nanoparticle formulation), ABVD, ABVE, ABVE-PC, AC, AC-T, Adcetris (brentuximab vedotin), ADE, Ado-trastuzumab emtansine, Adriamycin (doxorubicin hydrochloride), Adrucil (fluorouracil), afatinib dimaleate, Afinitor (everolimus), Akynzeo (netupitant and palonosetron hydrochloride), and Aldara (imipenem). Quimod), aldesleukin, alemtuzumab, Alimta (pemetrexed disodium), Aloxi (palonosetron hydrochloride), Ambochlorin (chlorambucil), Aminolevulinic acid, Anastrozole, Aprepitant, Aredia (pamidronate disodium), Arimidex (anastrozole), Aromasin (exemestane), Arranon (nelarabine), Arsenic trioxide, Arzerra (ofatumumab), asparaginase Erwinia chrysanthemumii, Ava bevacizumab, axitinib, azacitidine, BEACOPP, Becenum (carmustine), Beleodaq (belinostat), belinstat, bendamustine hydrochloride, BEP, bevacizumab, bexarotene, Bexxar (tositumomab and iodine-131 tositumomab), bicalutamide, BiCNU (carmustine), bleomycin, blinatumomab, Blincyto (blinatumomab), bortezomib, Bosulif (bosutinib), bosutinib, brentuximab vedotin, busulfan, Busulfex (bustatin) sulfane), cabazitaxel, cabozantinib-S-malate, CAF, Camppath (alemtuzumab), Camptosar (irinotecan hydrochloride), capecitabine, CAPOX, carboplatin, carboplatin-TAXOL, carfilzomib, Carmubris (carmustine), carmustine, carmustine implant, Casodex (bicalutamide), CeeNU (lomustine), ceritinib, Cerubidine (daunorubicin 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), Cyramza (ramucirumab), cytarabine, cytarabine, liposomal, Cytosar-U (cytarabine), Cytox an (cyclophosphamide), dabrafenib, dacarbazine, Dacogen (decitabine), dactinomycin, dasatinib, daunorubicin hydrochloride, decitabine, degarelix, denileukin diftitox, denosumab, DepoCyt (liposomal cytarabine), DepoFoam (liposomal cytarabine), dexrazoxane hydrochloride, dinutuximab, docetaxel, Doxil (doxorubicin hydrochloride liposome), doxorubicin hydrochloride, doxorubicin hydrochloride liposome, Dox-SL (doxorubicin hydrochloride liposome) dm), DTIC-Dome (dacarbazine), Efudex (fluorouracil), Elitek (rasburicase), Ellence (epirubicin hydrochloride), Eloxatin (oxaliplatin), eltrombopag olamine, Emend (aprepitant), enzalutamide, epirubicin hydrochloride, EPOCH, Erbitux (cetuximab), eribulin mesylate, Erivedge (vismodegib), erlotinib hydrochloride, Erwinaze (asparaginase erwinia chrysanthemum), Etopophos (phosphorazine) etoposide acid), etoposide, etoposide phosphate, Evacet (doxorubicin hydrochloride liposomal), everolimus, Evista (raloxifene hydrochloride), exemestane, Fareston (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 nonavalent vaccine), Gazyva (obinutuzumab), gefitinib, gemcitabine hydrochloride, gemcitabine-cisplatin, gemcitabine-oxaliplatin, gemtuzumab ozogamicin, Gemzar (gemcitabine hydrochloride), Gilotrif (afatinib dimaleate), Gleevec (imatinib mesylate), Gliadel (carmustine implant), Gliadel wafer (carmustine implant), glucarpidase, goserelin acetate, Halaven (eribulin mesylate), Herceptin (trastuzumab), HPV bivalent vaccine, recombinant HPV nonavalent 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-131 tositumomab and tositumomab, ipilimumab, Iressa (gefitinib), irinotecan hydrochloride, Istodax (romidepsin), ixabepilone, Ixempra (ixabepilone), Jakafi (ruxolitinib phosphate), Jevtana (cabazitaxel), Kadcyla (Ado-trastuzumab emtansine), Keoxifene (raloxifene hydrochloride), Kepivance (palifermin), Keytruda (pembrolizumab), Kyprolis (carfilzomib), lanreotide acetate, lapatinib ditosylate, lenalidomide, lenvatinib mesylate, Lenvima (lenvatinib mesylate), letrozole, leucovorin calcium,Leukeran (chlorambucil), leuprolide acetate, Levulan (aminolevulinic acid), Linfolizin (chlorambucil), LipoDox (liposomal doxorubicin hydrochloride), liposomal cytarabine, lomustine, Lupron (leuprolide acetate), Lupron Depot (leuprolide acetate), Lupron Depot-Ped (leuprolide acetate), Lupron Depot-3 Month (leuprolide acetate), Lupron Depot-4 Month Month (leuprolide acetate), Lynparza (olaparib), Marqibo (vincristine sulfate liposome), Matulane (procarbazine hydrochloride), mechlorethamine hydrochloride, Megace (megestrol acetate), megestrol acetate, Mekinist (trametinib), mercaptopurine, mesna, Mesnex (mesna), Methazolastone (temozolomide), methotrexate, methotrexate Rexate LPF (methotrexate), Mexate (methotrexate), Mexate-AQ (methotrexate), mitomycin C, mitoxantrone hydrochloride, Mitozytrex (mitomycin C), MOPP, Mozobil (plelixafor), Mustargen (mechlorethamine hydrochloride), Mutamycin (mitomycin C), Myleran (busulfan), Mylosar (azacytidine), M ylotarg (gemtuzumab ozogamicin), nanoparticle paclitaxel (paclitaxel albumin-stabilized nanoparticle formulation), Navelbine (vinorelbine tartrate), nelarabine, Neosar (cyclophosphamide), netupitant and palonosetron hydrochloride, Neupogen (filgrastim), Nexavar (sorafenib tosylate), nilotinib, nivolumab, Nolvadex (tamoxifen citrate) Nplate (romiplostim), obinutuzumab, Odomzo (sonidegib), OEPA, ofatumumab, OFF, olaparib, omacetaxine mepesuccinate, Oncaspar (pegaspargase), ondansetron hydrochloride, Ontak (denileukin diftitox), Opdivo (nivolumab), OPPA, oxaliplatin, paclitaxel, paclitaxel albumin-stabilized nanoparticle formulation, PAD,Palbociclib, Palifermin, Palonosetron Hydrochloride, Palonosetron Hydrochloride and Netupitant, Pamidronate Disodium, Panitumumab, Panobinostat, Paraplat (Carboplatin), Paraplatin (Carboplatin), Pazopanib Hydrochloride, Pegaspargase, Peginterferon alfa-2b, PEG-Intron (Peginterferon alfa-2b), Pembrolizumab, Pemet Rexed 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 olamine), Provenge (sipuleucel-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) nonavalent vaccine, recombinant human papillomavirus (HPV) quadrivalent vaccine, Recombinant interferon alfa-2b, regorafenib, R-EPOCH, Revlimid (lenalidomide), Rheumatrex (methotrexate), Rituxan (rituximab), rituximab, romidepsin, romiplostim, rubidomycin (daunorubicin hydrochloride), ruxolitinib phosphate, Sclerosol intrapleural aerosol (talc), siltuximab, sipuleucel-T, Somatuline Depot (lanreotide acetate), sonidegib, sorafenib tosylate, Sprycel (dasatinib), STANFORD V, sterile talc powder (talc), Steritalc (talc), Stivarga (regorafenib), sunitinib malate, Sutent (sunitinib malate), Sylatron (peginterferon alfa-2b), Sylvant (siltuximab), Synovir (thalidomide), Synribo (omacetaxine mepesuccinate), 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-131 tositumomab, Totect (dexrazoxane hydrochloride), TPF, trametinib, trastuzumab, Treanda (bendamustine hydrochloride), Trisenox (arsenic trioxide), Tykerb (lapatinib ditosylate), Unituxin (dinutuximab), vandetanib, VAMP, Vectibix (panitumumab), VeIP, Velban (vinblastine sulfate), Velcade (bortezomib), Velsar (vinblastine sulfate), vemurafenib, VePesid (etoposide), Viadur (leuprolide acetate), Vidaza (azacitidine), vinblastine sulfate, Vincasar PFS (vincristine sulfate), vincristine sulfate, vincristine sulfate liposome, vinorelbine tartrate, VIP, vismodegib, Voraxaze (glucarpidase), vorinostat, Votrient (pazopanib hydrochloride), Wellcovorin (leucovorin calcium), Xalkori (crizotinib), Xeloda (capecitabine), XELIRI, XELOX, Xgeva (denosumab), Xofigo (radium-223 dichloride), Xtandi (enzalutamide), Yervoy (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 (ceritinib), and Zytiga (abiraterone acetate).

[0357] Growth factors useful as therapeutic agents include transforming growth factor-alpha ("TGF-α"), transforming growth factor ("TGF-β"), platelet-derived growth factor ("PDGF"), fibroblast growth factor ("FGF"), including acidic isoforms 1 and 2, basic form 2, and FGFs 4, 8, 9, and 10, nerve growth factor ("NGF") 2.5s, NGF 7.0s, and NGF, including beta-NGF, as well as neurotrophins, 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 TGF alpha, beta, beta 1, beta 2, beta 3, skeletal growth factor, bone matrix-derived growth factor, and bone-derived growth factor, and mixtures thereof.

[0358] Cytokines useful as anti-cancer agents can include, but are not limited to, cardiotrophins, stromal cell-derived factors, macrophage-derived chemokine (MDC), macrophage inflammatory protein 1 alpha (MIP-1 alpha), 2, 3 alpha, 3 beta, 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 (IFN) (e.g., interferon alpha, interferon alpha A, interferon beta, interferon gamma, interferon 1 alpha), growth factors (e.g., colony-stimulating factors (CSFs), granulocyte colony-stimulating factor (G-CSF) (e.g., filgrastim (Neupogen®; Amgen)), hematopoietic growth factor (HGF)), melanoma growth stimulating activity (MGSA), monoclonal antibodies (mAbs)—anti-CD40 antibodies, anti-PDL1 antibodies ( For example, 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 (e.g., nivolumab (BMS), pembrolizumab (Merck), pidilizumab (CureTech / Teva), AMP-244 (Amplimmune / GSK), BMS-936559 (BMS), and MEDI4736 (Roche / Genentech)), or combinations thereof.

[0359] 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.

[0360] Other molecules useful as anti-cancer agents include, but are not limited to, growth hormone, leptin, leukemia inhibitory factor (LIF), tumor necrosis factor alpha and beta, endostatin, thrombospondin, bone morphogenetic protein-1, bone morphogenetic proteins 2 and 7, osteonectin, somatomedin-like peptide, and osteocalcin.

[0361] Tumor antigens can be based on specific mutations (neoepitopes) and those expressed by cancer germline genes (antigens common to tumors found in multiple patients, referred to herein as "conventional cancer antigens" or "common cancer antigens"). In some embodiments, conventional antigens are those known to be commonly found in cancers or tumors or to be found in specific types of cancers or tumors. In some embodiments, conventional cancer antigens are non-mutated tumor antigens. In some embodiments, conventional cancer antigens are mutated tumor antigens. Tumor antigens include 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, CTLA4, and Cript. o, ED-B, ErbB1, ErbB2, ErbB3, ErbB4, EGFL7, EpCAM, EphA2, EphA3, EphB2, FAP, fibronectin, folate receptor, ganglioside GM3, GD2, glucocorticoid-inducible 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, cellular tumor antigen p53, aberrant products 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 new 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)), cancer-testis antigens (e.g., MAGEA1, MAGEA2, MAGEA3, MAGEA4, MAGEA5, MAGEA6, MAGEA8, MAGEA9, MAGEA10, MAGEA11, MAGEA12, MAGEA13, MAGEA14, MAGEA15, MAGEA16, MAGEA17, MAGEA18, MAGEA19, MAGEA20, MAGEA21, MAGEA22, MAGEA23, MAGEA24, MAGEA25, MAGEA26, MAGEA27, MAGEA28, MAGEA29, MAGEA30, MAGEA31, MAGEA32, MAGEA33, MAGEA34, MAGEA35, MAGEA36, MAGEA37, MAGEA38, MAGEA39, MAGEA40, MAGEA41, MAGEA42, MAGEA43, MAGEA44, MAGEA45, MAGEA46, MAGEA47, MAGEA48, MAGEA49, MAGEA50, MAGEA51, MAGEA52, MAGEA53, MAGEA54, MAGEA55, MAGEA56, MAGEA57, MAGEA58, MAGEA59, MAGEA60, MAGEA61, MAGEA62, MAGEA63, MAGEA64, MAGEA65, M 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, SP ANXN4, 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, LUZ P4, CASC5, TFDP3, JARID1B, LDHC, MORC1, DKKL1, SPO11, CRISP2, FMR1NB, FTHL17, NXF2, TAF7L, TDRD1, TDRD6, TDRD4, TEX15, FATE1, TPTE, CT45A1, CT45A 2, 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、CXorf4 8、THEG、ACTL8、NLRP4、COX6B2、LOC348120、CCDC33、LOC196993、PASD1、 LOC647107、TULP2、CT66 / AA884595、PRSS54、RBM46、CT69 / BC040308、CT 70 / BI818097、SPINLW1、TSSK6、ADAM29、CCDC36、LOC440934、SYCE1、CPXC R1、TSPY3、TSGA10、HIWI、MIWI、PIWI、PIWIL2、ARMC3、AKAP3、Cxorf61、P BK、C21orf99、OIP5、CEP290、CABYR、SPAG9、MPHOSPH1、ROPN1、PLAC1、CA LR3、PRM1、PRM2、CAGE1、TTK、LY6K、IMP-3、AKAP4、DPPA2、KIAA0100、DCA F12、SEMG1、POTED、POTEE、POTEA、POTEG、POTEB、POTEC、POTEH、GOLGAGL2 FA, CDCA1, PEPP2, OTOA, CCDC62, GPATCH2, CEP55, FAM46D, TEX14, CTNNA2, FAM133A, LOC130576, ANKRD45, ELOVL4, IGSSF11, TMEFF1, TMEFF2, ARX, SPEF2, GPAT2 TMEM108、NOL4、PTPN20A、SPAG4、MAEL、RQCD1、PRAME、TEX101、SPATA19、ODF1、O DF2、ODF3、ODF4、ATAD2、ZNF645、MCAK、SPAG1、SPAG6、SPAG8、SPAG17、FBXO39、RG S22、サイクリンA1、C15orf60、CCDC83、TEKT5、NR6A1、TMPRSS12、TPPP2、PRSS55、DMR T1、EDAG、NDR、DNAJB8、CSAG3B、CTAG1A、GAGE12B、GAGE12C、GAGE12D、GAGE12E、G AGE12F、GAGE12G、GAGE12H、GAGE12I、GAGE12J、GAGE13、LOC728137、MAGEA2B、MA GEA9B / LOC728269、NXF2B、SPANXA2、SPANXB2、SPANXE、SSX4B、SSX5、SSX6、SSX7、The antigens may include, but are not limited to, SSX9, TSPY1D, TSPY1E, TSPY1F, TSPY1G, TSPY1H, TSPY1I, TSPY2, XAGE1E, XAGE2B / CTD-2267G17.3, and / or variants thereof), oncogenic viral antigens (e.g., human papillomavirus (HPV) or hepatitis B), oncogenic bacterial antigens (e.g., Helicobacter pylori), and / or variants thereof.

[0362] Diagnostic agents include gases; metals; commercially available imaging agents used in positron emission tomography (PET), computed tomography (CAT), single-photon emission computed tomography, X-ray, fluoroscopy and magnetic resonance imaging (MRI); and contrast agents.Examples of suitable materials for use as contrast agents in MRI include gadolinium chelate, and iron, magnesium, manganese, copper and chromium.Examples of materials useful for CAT and X-ray imaging include iodine-based materials.

[0363] Vaccines can include isolated proteins or peptides, inactivated organisms and viruses, killed organisms and viruses, genetically modified organisms or viruses, cell extracts, and RNA encoding at least one antigenic polypeptide or an immunogenic fragment thereof (e.g., an immunogenic fragment capable of inducing an immune response to the antigenic polypeptide). The active agent can be combined with interleukins, interferons, cytokines, and adjuvants, such as cholera toxin, alum, Freund's adjuvant, and the like. The prophylactic agent may contain antigens of bacterial organisms, such as Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Streptococcus pyrogenes, 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, Leptospirosis interrogans, Borrelia burgdorferi, Camphylobacter jejuni, etc.;Viral antigens, 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, and the like. viruses (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, and the like; antigens of fungal, protozoan, and parasitic organisms, e.g., Cryptococcus Neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia ricketsii, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydial psittaci, Chlamydial 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 combinations thereof;

[0364] The nucleic acids that can be introduced by the methods herein include all types of DNA, cDNA, and RNA sequences. For example, the nucleic acid can be double-stranded DNA, single-stranded DNA, complexed DNA, encapsulated DNA, naked RNA, encapsulated RNA, plasmid RNA, miniRNA, circular RNA, messenger RNA (mRNA), tRNA, small 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, miniDNA, mRNA, self-amplifying RNA, or circular RNA. The nucleic acid can also be a DNA construct, such as an expression vector, encoding a desired gene product (e.g., a gene product homologous or heterologous to the target into which the gene product is introduced).

[0365] In some embodiments, nucleic acids (e.g., mRNA) can be used to induce a balanced immune response to infection. In some embodiments, nucleic acids (e.g., mRNA) can be used to encode antibodies against viruses, such as metapneumoviruses, e.g., human metapneumovirus (hMPV), parainfluenza viruses, e.g., human parainfluenza viruses (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-1, HCOV-2, HCOV-3, HCOV-4, HCOV-5, HCOV-6, HCOV-7, HCOV-8, HCOV-9, HCOV-10, HCOV-11, HCOV-12, HCOV-13, HCOV-14, HCOV-15, HCOV-16, HCOV-17, HCOV-18, HCOV-19, HCOV-20, HCOV-21, HCOV-22, HCOV-23, HCOV-24, HCOV-25, HCOV-26, HCOV-27, HCOV-28, HCOV-29, HCOV-30, HCOV-31, HCOV-32, HCOV-33, HCOV-34, HCOV-35, HCOV-36, HCOV-37, HCOV-38, HCOV-39, HCOV-41, HCOV-42, HCOV-43, HCOV-44, HCOV-45, HCOV-46, HCOV-47, In some embodiments, nucleic acids (e.g., mRNA) can be used to induce a balanced immune response against respiratory viruses. The term "respiratory virus" as used herein refers to viruses that cause respiratory disease. For example, negative-sense single-stranded RNA viruses of the Paramyxoviridae family, 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 coronavirus. Coronaviruses are enveloped viruses with a positive-sense single-stranded RNA genome and a helical symmetric nucleocapsid. Coronaviruses are a species of virus belonging to the subfamily Coronavirinae in the family Coronaviridae in the order Nidovirales.

[0366] Representative examples of betacoronaviruses include, but are not limited to, envecoviruses (e.g., betacoronavirus 1, human coronavirus OC43, Chinese Rattus coronavirus HKU24, human coronavirus HKU1, and murine coronavirus), hibecoviruses (e.g., bat Hp-betacoronavirus Zhejiang 2013), merbecoviruses (e.g., hedgehog coronavirus 1, Middle East respiratory syndrome-associated coronavirus (MERS-CoV), Pipistrellus bat coronavirus HKU5, and Tylonycteris bat coronavirus HKU4), nobecoviruses (e.g., Rousettus bat coronavirus GCCDC1 and Rousettus bat coronavirus HKU9), and sarbecoviruses (e.g., severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)).

[0367] Representative examples of gammacoronaviruses include, but are not limited to, Segakoviruses (eg, beluga coronavirus SQ1) and Igakoviruses (eg, avian coronavirus (IBV)).

[0368] Representative examples of deltacoronaviruses include, but are not limited to, anddecoviruses (e.g., Wigeon coronavirus HKU20), burdecoviruses (e.g., Brown-eared ...

[0369] 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).

[0370] 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 bronchial tubes (bronchi) and lungs (bronchi, bronchioles, and alveoli), as well as the interstitial tissue of the lungs.

[0371] In another embodiment, the coronavirus infection is an infection of the gastrointestinal tract. The "gastrointestinal tract" can include any region of the tract from the mouth to the anus, including the mouth, esophagus, stomach, and intestines. In yet another embodiment, the coronavirus infection is a kidney infection.

[0372] Other indications related to coronavirus infections are described in Gralinski & Baric, 2015, J. Pathol. 235:185-195 and Cavanagh, 2005, "Coronaviridae: a review of coronaviruses and toroviruses," in Coronaviruses with Special Emphasis on First Insights Concerning SARS 1, edited by A. Schmidt, M. H. Wolff and O. Weber, Birkhauser Verlag Baser, Switzerland, each of which is incorporated herein by reference in its entirety.

[0373] In some embodiments, the nucleic acids disclosed herein can comprise at least one chemically modified nucleotide. In some embodiments, the at least one chemically modified nucleotide comprises a chemically modified nucleobase, a chemically modified ribose, a chemically modified phosphodiester linkage, or a combination thereof.

[0374] 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).

[0375] All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. Although the compositions and methods of the present disclosure have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that modifications may be made to the compositions and methods, and to the steps or sequence of steps of the methods described herein, without departing from the concept, spirit, and scope of the present disclosure. More specifically, it will be apparent that certain agents that are chemically related to each other may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutions and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the present disclosure, as defined by the appended claims.

[0376] By way of non-limiting example, examples of specific embodiments of the present disclosure are provided below. [Example]

[0377] To overcome the current challenges posed by cancer vaccines, we utilize lipid nanoparticle (LNP) delivery technology to deliver single-chain trimers (SCTs) consisting of peptides, β2-microglobulin (β2m), and MHC class I heavy chains to bypass antigen processing and result in stable presentation of peptides on the surface of antigen-presenting cells, as well as nucleic acids (DNA or RNA) to express universal MHC II-restricted peptides to activate host memory CD4 T cells and enhance tumor-specific CD8 T cell immunity.

[0378] Example 1: Kb-E6-SCT mRNA induces robust antigen-specific CD8+ T cell immune responses and exhibits potent anti-tumor activity in animal models. Design and validation of Kb-E6-SCT mRNA 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 contains a peptide, β2m, and mouse MHC class I, Kb. Kb-E6-SCT mRNA is shown in Figure 1A, and in vitro efficacy was studied, with the results shown in Figures 1B–1E. A linearized plasmid template is shown in Figure 1B. In vitro transcription of SCT-E6 mRNA is shown in Figure 1C. The physicochemical properties of the LNP formulation and the results for the encapsulation efficacy of L2M6 are shown in Figures 1D–1E. Figure 1D shows particle size and polydispersity index, and Figure 1E shows zeta potential and encapsulation efficiency. Additionally, Figure 1F shows the expression of SCT-E6 in 293T cells after transfection.

[0379] animal research C57BL / 6 mice were immunized intramuscularly with 1 μg of mRNA of the indicated vaccines twice, 2 weeks apart. Seven days after the final immunization, E6-specific T cell populations were determined by ELISPOT and intracellular IFN-gamma staining using the E6-CD8 T cell peptide. The results are shown in Figures 2A-2C.

[0380] The in vivo antitumor efficacy of prophylactic vaccination with mRNA-L2M6 was assayed. Figure 3A shows a schematic representation of five C57BL / c mice immunized intramuscularly with 1 μg of mRNA / SCT-E6 on days 1 and 14, and on day 21 with 1 × 10 mRNA / SCT-E6, a well-established cancer cell line derived from lung epithelial cells transformed with the oncogenic human papillomavirus (HPV) E6 and E7 genes. 5 TC1 tumor cells were injected subcutaneously. Tumor growth and survival were monitored. The results are shown in Figure 3B and Figure 3C.

[0381] The in vivo efficacy of therapeutic administration of mRNA-L2M6 vaccine was assayed. Figure 4A shows a schematic of 10 female C57BL / 6 mice subcutaneously injected with 2 x 10^5 TC-1 tumor cells in the flank 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. The results are shown in Figure 4B and Figure 4C.

[0382] Conclusion: In conclusion, our study demonstrates that mice immunized with the Kb-E6-SCT mRNA vaccine exhibit enhanced E6-specific CD8+ T cell immune responses. More importantly, our findings demonstrate a substantial enhancement of the antitumor effect against E6-expressing tumor cells. This underscores the effectiveness of our strategy to bypass antigen processing and presentation machinery and highlights its potential for significantly enhancing the efficacy of cancer vaccines.

[0383] Example 2: Therapeutic mRNA vaccine induces broad and specific CD8 T cell responses targeting HPV 16 E6 in HLA A2.1 transgenic mice. Design and validation of four mRNAs 1. HPV-E6-E7-S2-mRNA (mRNA-1): This DNA construct contains HPV-16 E6, E7, and the SARS-CoV-2 spike glycoprotein (subunit S2) (SEQ ID NO: 498). It encodes the full-length HPV-16 E6 / E7 protein (SEQ ID NO: 497), in which known oncogenic transforming regions are inactivated, and a truncated S2 protein, intended to induce broad E6- and E7-specific T cell responses. Additionally, it stimulates S2-specific CD4 T cell responses. Given the widespread global exposure to SARS-CoV-2 infection or COVID-19 immunization, these S2-specific CD4 T cells can enhance CD8 T cell antitumor function.

[0384] 2. HLA A0201 mRNA (mRNA-2): This DNA construct encodes the HLA A0201 gene (SEQ ID NO: 500) and facilitates the overexpression of its protein (SEQ ID NO: 499) to enhance T cell immunity.

[0385] 3. HLA A0201-E6-SCT mRNA (mRNA-3): This DNA construct encodes the immunodominant T cell epitope derived from E6 of HPV16, β2 microglobulin, and HLA A0201 (SEQ ID NOs: 17 and 18).

[0386] 4. HLA A0201-E7-SCT mRNA (mRNA-4): The DNA construct encodes the immunodominant T cell epitope derived from E7 of HPV16, β2 microglobulin, and HLA A0201 (SEQ ID NOs: 19 and 20).

[0387] mRNA was transcribed from the indicated constructs and is shown in Figure 5. Figures 6A-6C show in vitro validation of mRNAs 1-4 after transfection into 293T cells.

[0388] animal research HLA-A2.1 transgenic mice (C57BL / 6-Mcph1Tg(HLA-A2.1)1Enge / J, strain number 003475) were purchased from Jackson Laboratory. Mice were immunized intramuscularly with 1 μg of the designated vaccine twice, 2 weeks apart. 14 days after the final immunization, spleens were harvested for analysis (Figure 7A).

[0389] 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-gamma staining. The results are presented in Figures 7B-7D.

[0390] In vitro CTL killing assay: As depicted in Figures 8A-8B, the CD8 T cell responses induced by HPV-16 mRNA were further evaluated through an in vitro CTL killing assay. Fourteen days after two immunizations with mRNA-1 and mRNA-3, splenocytes were harvested from HLA-A2.1 transgenic mice. CD8+ T cells were isolated (Figure 8A) and cocultured with tumor cells (TC1 / A2) expressing HPV-16 E6 / E7 and transduced with HLA A2.1. Cell death was assessed by FACS, as shown in Figure 8B.

[0391] Conclusion: In conclusion, a vaccine utilizing mRNA encoding HPV 16 E6 / E7 / S2 and SCT-E6 / E7 has shown promising results in HLA A2.1 transgenic mice. This vaccine induced robust and specific CD8 T cell responses targeting HPV E6 and E7 antigens. Furthermore, activated CD8 T cells demonstrated the ability to effectively eliminate HLA A2-positive E6 / E7-expressing tumor cells in an ex vivo setting. These findings suggest that the SCT technique used in this vaccine has the potential to enhance the clearance of established HPV-positive cancers in vivo.

Claims

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

2. 2. The single-stranded trimer nucleic acid of claim 1, wherein the nucleic acid comprises a plasmid DNA, a minicircle DNA, a microRNA, an mRNA, a self-amplifying RNA, a circle RNA, a DNA-triggered self-amplifying RNA, or a viral vector.

3. The single-chain trimeric nucleic acid of claim 1 or 2, wherein the T cell epitope comprises a viral, bacterial, parasitic, or cancer T cell epitope.

4. The single-stranded trimer nucleic acid according to any one of claims 1 to 3, 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 trimer nucleic acid of any one of claims 1 to 4, wherein the single-stranded trimer 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-chain trimer nucleic acid of any one of claims 1 to 5, wherein the first T cell epitope comprises an epitope listed in Table 2.

7. The single-chain trimer nucleic acid of any one of claims 1 to 6, 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.

8. 8. The single-chain trimer nucleic acid of claim 1, wherein the single-chain trimer nucleic acid comprises a nucleic acid encoding, in amino to carboxy terminus order, a T cell epitope, the β2 microglobulin, and an MHC heavy chain sequence.

9. The MHC class I heavy chain is HLA-A1:01, HLA-A2:01, HLA-A2:03, HLA-A2:06, HLA-A2:07, HLA-A3:01, HLA-A9, HLA-A10, HLA-A11:

0. 1, 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, HL AB-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, HL A-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, HL 9. The single-chain trimer nucleic acid of claim 1, comprising an 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 any one of claims 1 to 9, further comprising a second T cell epitope.

11. The single-chain trimeric nucleic acid of claim 10, wherein the second T cell epitope is an MHC class II restricted epitope.

12. 12. The single-chain trimer nucleic acid of 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-chain trimeric nucleic acid according to any one of claims 1 to 12, further comprising 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 trimeric nucleic acid encoding a first T cell epitope, β2 microglobulin, and an MHC class I heavy chain sequence.

15. 15. The composition of claim 14, wherein the nucleic acid comprises a plasmid DNA, a minicircle DNA, a microRNA, an mRNA, a self-amplifying RNA, a circle RNA, or a DNA-triggered self-amplifying RNA.

16. 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. 18. The composition of any one of claims 14 to 17, wherein the single-stranded trimer 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. 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. 20. The composition of any one of claims 14 to 19, 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.

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

22. The MHC class I heavy chain is HLA-A1:01, HLA-A2:01, HLA-A2:03, HLA-A2:06, HLA-A2:07, HLA-A3:01, HLA-A9, HLA-A10, HLA-A11:

0. 1, 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, H LA-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, HL A-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, 22. The composition of any one of claims 14 to 21, comprising an 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. 24. The composition of claim 23, wherein the second T cell epitope is an MHC class II restricted epitope.

25. 24. 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 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 the MHC class I heavy chain sequence.

27. 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 vector, or any combination thereof.

28. 28. The composition of any one of claims 14 to 27, wherein the composition is a lipid nanoparticle, and the lipid nanoparticle comprises 20% to 80% of an ionized lipid, a cationic lipid, or any combination thereof, 0% to 5% of a pegylated lipid, 0% to 40% of a helper lipid, and 0% to 80% of a sterol.

29. A lipid nanoparticle, comprising: 20% to 80% ionizable lipids, cationic lipids, or any combination thereof; 0% to 5% PEGylated lipid, 0% to 40% helper lipid, 0% to 80% sterols, and A lipid nanoparticle comprising 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 nanoparticle.

30. The lipid nanoparticle of claim 29, wherein the nucleic acid is plasmid DNA, minicircle DNA, microRNA, mRNA, self-amplifying RNA, circle RNA, or DNA-activated 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-chain trimeric nucleic acid comprises a nucleic acid encoding a T cell epitope, said β2 microglobulin, and an MHC heavy chain sequence in the order from amino to carboxy terminus.

37. The MHC class I heavy chain is 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-A32:0 2, 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 37. The lipid nanoparticle of any one of claims 29 to 36, comprising an 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-chain 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 the MHC class I heavy chain sequence.

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

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

44. 38. 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 expanding antigen-presenting cells, the method comprising co-culturing the antigen-presenting cells with cells comprising the single-stranded trimeric nucleic acid of any one of claims 1 to 13, the composition of any one of claims 14 to 28, or the lipid nanoparticle of any one of claims 29 to 41, or the pharmaceutical composition of claim 42.

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

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

48. 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 the subject that can be used for immunotherapy.

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