Self-assembling peptide nanoparticles and uses thereof

Abstract

Self-assembled nanoparticle compositions are disclosed that include multiple cationic cell-penetrating peptides, each covalently linked to a hydrophobic therapeutic molecule (e.g., an antigenic peptide, mRNA, siRNA, DNA, etc.) and, optionally, non-covalently linked to at least one Toll-like receptor (TLR) ligand. Methods of using the nanoparticle compositions in the treatment, prevention, and / or amelioration of one or more symptoms of mammalian disease, including, but not limited to, cancer, infection, inflammation, and related diseases and abnormal conditions, are also disclosed.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 791,795, filed January 12, 2019, the entire contents of which are specifically incorporated herein by reference. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] Not applicable. Name of the organization under the joint research agreement

[0003] Not applicable. Technical Field

[0004] The present disclosure relates to nanoparticles comprising multiple cationic cell-penetrating peptides (CPPs), each covalently linked to a hydrophobic therapeutic peptide (e.g., an antigenic peptide), and optionally at least one or more TLR (Toll-like receptor) ligands non-covalently linked to the CPP-linked therapeutic peptides. The amphiphilic nature of the CPP-linked therapeutic peptides allows them to self-assemble with negatively charged nucleic acids (such as CpG, poly(I:C), mRNA, siRNA, and DNA) and hydrophobic MPLA to form nanoparticles under neutral conditions (pH=7.0), but the nanoparticles are destroyed under acidic conditions (pH<5). The resulting self-assembled nanoparticles containing the CPP-linked therapeutic peptides and TLR ligands or mRNAs can be co-delivered into antigen-presenting cells (APCs) for effective presentation for T cell activation, resulting in potent immunogenicity against cancer and other diseases. Thus, the present disclosure also provides methods for treating and / or preventing cancer (including various tumors), or infectious diseases by utilizing CPP-T cell peptide / TLR ligand assembled nanoparticles. [Background technology]

[0005] 1. Cancer immunotherapy Cancer is the leading cause of death in the United States and worldwide, posing a major public health problem. Cancer immunotherapy has become a promising approach to cancer therapy (Di Lorenzo et al., 2011; Lesterhuis et al., 2011; Rosenberg, 2011; Wang and Wang, 2017). Several immunotherapy-based checkpoint blockade drugs, such as the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) monoclonal antibody (Ab), ipilimumab (Yervoy), and the programmed cell death (PD)-1 antibody, pembrolizumab (Keytruda), have been approved by the U.S. Food and Drug Administration (FDA) for the treatment of many types of cancer (Bagcchi, 2014; Hodi, 2010; Kantoff et al., 2010; Bender, 2017). Furthermore, cell-based immunotherapy using T cell receptor (TCR) or chimeric antigen receptor (CAR) engineered T cells has shown promising clinical responses in hematological cancers (e.g., leukemia and lymphoma).

[0006] Despite these rapid advances, the majority of cancer patients generally do not respond to checkpoint blockade therapy. For example, approximately 20% of lung cancer patients respond to immune checkpoint therapy. Only 13–18% of breast and prostate cancer patients respond to immune checkpoint therapy (Nanda et al., 2016; Kwon et al., 2014). CAR-T cell immunotherapy techniques are effective in hematological cancers (Sadelain et al., 2017; Johnson and June, 2017), but are not effective in solid tumors, likely due to immune suppression in the tumor microenvironment. Recent studies have shown that the clinical efficacy of immune checkpoint blockade therapy depends on the presence of tumor-reactive T cells in tumor tissue and is associated with tumor-infiltrating T cells, PD-L1 expression, and mutation load (Sharma et al., 2017). Cancer patients who lack tumor-infiltrating antigen-specific T cells in tumor tissue generally do not respond to immune checkpoint therapy. To overcome these issues, cancer vaccines may increase tumor-specific T cells to control tumors. An alternative strategy is the adoptive transfer of tumor-specific T cells, either derived from cancer patients or engineered to express tumor antigen-specific TCRs or CARs on the T cells.

[0007] Cancer vaccine immunotherapy offers the potential for highly tumor-specific cytotoxicity and is therefore a very attractive approach for cancer treatment. Indeed, the first therapeutic cancer vaccine (sipuleucel-T; PROVENGE©, Dendreon) was approved by the FDA in 2010 for the treatment of metastatic prostate cancer (Kantoff et al., 2010). However, cancer vaccines have generally achieved only limited clinical success. Even the FDA-approved sipuleucel-T (PROVENGE©) vaccine extends patient survival by 4.1 months compared to the control group, but does not show a significant clinical response. Vaccination with antigen peptides or dendritic cells (DCs) pulsed with antigen peptides can generate antitumor immunity, but fails to generate sufficient immune responses to obtain significant clinical efficacy in some types of cancers that have been tested (Melero et al., 2014; Rosenberg et al., 2004). DC / peptide or protein vaccines alone may not be potent enough to generate strong and durable anti-tumor responses (Rosenberg et al., 2004).

[0008] Previous studies have shown that intracellular delivery of cancer antigen peptides, such as tyrosinase-related protein-2 (TRP-2), into DCs via cell-penetrating peptide (CPP) covalent linkages enhances antigen-specific T cell responses and antitumor immunity against cancer, mainly due to the extended antigen presentation time of DCs to T cells (Wang and Wang, 2002; Wang et al., 2002). Based on these preclinical studies, clinical trials with the TAT-NY-ESO-1 peptide were initiated and found that this peptide vaccine was safe and capable of inducing antigen-specific T cell responses in six of nine prostate cancer patients evaluated, which correlated with an increased PSA doubling time in vaccinated patients (Sonpavde et al., 2014). However, all immune responses are too weak and transient to induce cancer regression. Thus, new strategies are urgently needed to develop more potent vaccines against cancer and other diseases.

[0009] Toll-like receptors (TLRs) have recently been found as essential components of the innate immune system, where TLRs activate DC maturation programs to detect microbial infections and induce adaptive immune responses (Iwasaki and Medzhitov, 2004; Akira and Takeda; 2016). In DCs, triggering of innate immune receptors (e.g., TLRs, Nod-like receptors (NLRs), and RIG-like receptors (RLRs)) with their corresponding ligands activates nuclear factor kappa B (NFkB), type I interferon (IFN), and inflammatory responses. These signaling pathways lead to the production of proinflammatory cytokines and induce strong innate and adaptive immune responses. By administering an antigen with a TLR ligand, the immunogenicity of that antigen can be increased and the ability of DCs to stimulate T cell responses can be enhanced (Blander and Medzhitov; 2006; Blander and Medzhitov; 2006). Loading DCs with antigenic peptides together with TLR ligands can be effective to generate strong T cell responses using peptides and TLR ligands (Palucka and Banchereau, 2013).

[0010] By using nanotechnology such as multi-stage vectors (MSV), more antigens could be loaded into nanoliposomes or nanoparticles, which could generate stronger antitumor immunity against breast cancer compared to traditional DC vaccines (Xia et al., 2015). In melanoma, it was found that the cancer antigen peptide TRP-2 must be loaded into MSV together with TLR ligands (CpG and MPLA) and then taken up as a vaccine by the same DCs (Zhu et al., 2018). Vaccination with a mixture of MSV / TRP-2-loaded DCs and MSVTLR ligand-loaded DCs did not generate strong antitumor immunity, suggesting that the co-delivery of peptides and TLR ligands is crucial (Zhu et al., 2018). However, despite the advances made by MSV technology, the survival time of mice that could be extended by DC / MSV-based vaccination was limited (10 days), further suggesting that DC / MSV-based vaccination could only delay tumor growth and could not generate sufficient antitumor immunity for complete elimination of tumor cells.

[0011] Based on these studies, it was reasoned that current vaccine strategies could not generate sufficient immunity to completely eliminate cancer cells. Instead, immune suppression in the tumor microenvironment inhibits antitumor immunity induced by peptide vaccines. To understand why DCs loaded with CPP-conjugated therapeutic peptides are unable to induce antitumor immunity to eradicate cancer, CPP-conjugated therapeutic peptides were found to promote intracellular delivery of antigen peptides into DCs. Similarly, CPPs have been used to deliver various cargoes, including proteins, DNA, siRNA, and mRNA, intracellularly (into target cells). However, the CPP TAT-NY-ESO-1 peptide proved difficult to generate a stable emulsion with the vaccine adjuvant Montanide ISA-51. Emulsion droplets of TAT-NY-ESO-1 and Montanide ISA-51 were unstable and spread in water for a short time, which may affect the efficacy of the vaccine. This unstable property of TAT-NY-ESO-1 and Montanide-ISA-51 prompted us to further investigate how to solve this problem. One potential problem is that the positive charge (hydrophilicity) of the CPP (i.e., TAT) may be destroying the emulsion of CPP-NY-ESO-1 and Montanide-ISA-51.

[0012] A potent vaccine must contain innate immune signaling components. Recent studies by the present inventors and collaborators have found that co-delivery of antigen peptides and TLR ligands into the same DCs is necessary to generate a strong and effective immune response (Zhu et al., 2018). The MSV-based approach improves the co-delivery of antigen peptides and nucleic acid-based TLR ligands into the same DCs, which enhances antitumor immunity. However, this approach does not solve the fundamental problem mentioned above, i.e., antigen peptides and nucleic acid-based TLR3 and TLR9 ligands do not form complexes due to the hydrophobic nature of peptides and the negative charge of nucleic acids.

[0013] In view of the above, there is a need for improved delivery methods for cancer therapeutic molecules, including T cell epitopes, B cell epitopes, therapeutic nucleic acid molecules, and adjuvants. 2. Self-assembled peptide-based nanostructures

[0014] Molecular self-organization is the spontaneous formation of ordered structures that occurs under certain thermodynamic and kinetic conditions due to specific and local molecular interactions. Hydrogen bonds, hydrophobic interactions, electrostatic interactions, and van der Waals forces hold the molecules together and maintain them in a stable low-energy state. These minimum energies are achieved by self-association to form hierarchical structures at both the nano- and / or microscales (Han et al., 2010).

[0015] Self-assembly occurs spontaneously in nature during protein folding, DNA double helix formation, and cell membrane formation (Korolkov et al., 2013). Self-assembled nanostructures fabricated from natural biomolecular building blocks such as amino acids are biocompatible and easy to fabricate "bottom-up" (Yan et al., 2010), making them far preferable over their synthetic self-assembled monolayer (SAM) alternatives (Tayebe Zohrabi et al., 2015). 3. Cell-penetrating peptides

[0016] Cell-penetrating peptides (CPPs) are generally described as short peptides of 8-30 amino acids that can penetrate biological membranes to trigger the translocation of various biomolecules across the cell membrane into the cytoplasm and improve their intracellular pathways, thereby facilitating their interaction with targets (see, for example, U.S. Pat. No. 9,598,465). CPPs are either protein- or chimeric-sequence derived, usually amphipathic, and have a positive net charge (Morris et al., 2008; Hansen et al., 2008; Heitz et al., 2009). From proteins, several CPPs have been identified, including the Tat protein of human immunodeficiency virus (HIV) (Frankel and Pabo, 1988), the VP22 protein of herpes simplex virus (Elliott and O'Hare; 1997; Phelan et al., 1998), and fibroblast growth factors (Lin et al., 1995; Rojas et al., 1998). Tat peptide and membrane-translocating sequences (MTS) have been used to introduce proteins into cells both in vitro and in vivo (Farwell et al., 1994; Kim et al., 1997; Schwarz et al., 1999; Lindgren et al., 2000).

[0017] CPPs can be subdivided into two broad classes, the first of which requires chemical conjugation with the cargo, and the second of which involves the formation of a stable non-covalent complex. CPPs have been used to deliver a wide variety of cargoes (plasmid DNA, oligonucleotides, siRNA, PNA, proteins, peptides, liposomes, nanoparticles) into a wide variety of cell types and in vivo models (Morris et al., 2008; Beggars and Sagan, 2013; Huang et al., 2015; Marcus et al., 2016; Gungor et al., 2014). In these cases, CPPs transport the cargo into the cells primarily through their membrane trafficking ability (Figure 1). In these applications, there are no therapeutic T cell epitopes covalently linked to the CPP. Thus, CPPs are not amphipathic, which is different from CPP-T cell peptides, which are amphipathic to self-assemble into nanoparticles with negatively charged molecules. Summary of the Invention [Means for solving the problem]

[0018] The present invention overcomes these limitations and others inherent in the prior art by providing a new vaccine with a targeted delivery system. Disclosed are self-assembled nanoparticles composed of a population of cationic cell-penetrating peptides (CPPs) conjugated to one or more hydrophobic therapeutic peptide ligands, including TLRs (toll-like receptors) and antigenic peptides. The amphiphilic nature of the resulting nanoparticles (i.e., containing both hydrophilic and hydrophobic portions) additionally facilitates the incorporation of one or more therapeutic mRNA, siRNA, and / or DNA molecules. The resulting particles are self-assembled at neutral pH and can be delivered into antigen-presenting cells (APCs), such as dendritic cells, for presentation to T cells, triggering immune system activation, or the particles may be delivered directly as a vaccine.

[0019] In certain embodiments, we demonstrate that multiple cationic CPPs, each covalently linked to a specific therapeutic peptide (e.g., an antigenic peptide, which is preferably hydrophobic), can form dense, small-sized (50-100 nm) self-assembled nanoparticles that can be used to achieve effective intracellular delivery of therapeutic peptides. To facilitate nanoparticle formation and delivery across cell membranes, other components such as negatively charged molecules (DNA, dsRNA, siRNA, or mRNA) may be incorporated into the nanoparticles (Figure 2). The nanoparticles include (i) a corona comprising a CPP with a positively charged peptide covalently linked to a therapeutic peptide with preferred hydrophobicity, and (ii) a hydrophobic molecule (such as MPLA) in addition to the negatively charged molecule (DNA, dsRNA, siRNA, or mRNA). Based on charge and hydrophobicity, we designed and developed a novel technique of self-assembling CPP-T cell peptide nanoparticles containing TLR ligands [CpG and MPLA (abbreviated as CM); CpG and MPLA and poly(I:C) (abbreviated as CMI)], as shown in Figure 2A and Figure 2B. Amphiphobic or amphiphilic CPP therapeutic peptides, consisting of CPPs (such as TAT) with positively charged peptides and covalently linked to therapeutic peptides (e.g., NY-ESO-1 [SLLMWITQCFLPV (SEQ ID NO: 1)] and TRP-2 [SYVDFFVWL (SEQ ID NO: 2)] (generally hydrophobic)), form nanoparticles with negatively charged CpG and / or poly(I:C) on the one hand through electrical interaction, and with MPLA on the other hand through hydrophobicity inside the particle. [Brief description of the drawings]

[0020] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0021] The following drawings form part of this application and are included to illustrate certain aspects of the present invention. In order to promote an understanding of the principles of the invention, reference will be made to the embodiments or examples shown in the drawings and specific terms will be used to describe them. It will nevertheless be understood that this is not intended to limit the scope of the invention. Any changes and further modifications in the described embodiments, and any further applications of the principles of the invention described herein, as would normally occur to one skilled in the art to which the invention pertains, are contemplated.

[0022] The present invention is better understood by reference to the following description taken in conjunction with the accompanying drawings, in which like reference numerals identify like elements and in which:

[0023] [Figure 1] FIG. 1 illustrates that CPPs act as carriers to deliver cargo into cells.

[0024] [Figure 2-1]Figures 2A and 2B show that cationic CPP-T cell epitope peptides have amphopathetic properties, form nanoparticles, and deliver them into endosomes. Figure 2A: Cationic CPP (positive charge) is covalently linked to T cell epitope peptide (hydrophobic) to give it amphopathic properties. CpG and poly(I:C) are negatively charged, whereas MPLA is hydrophobic. CPP-T cell peptides form nanoparticles with CpG and / or poly(I:C) on the one hand through electrical interactions, and with MPLA on the other hand through hydrophobic interactions inside the particle. Figure 2B: CPP peptide / TLR ligand nanoparticles are taken up by DCs or macrophages and delivered into endosomes, where the CPP peptide / TLR ligand particles are destroyed at pH 4.0. CPP peptides are processed and presented to T cells by MHC class I or II molecules, while TLR ligands bind to TLR3, TLR4, and TLR9 to trigger innate immune responses and cytokine production, thereby enhancing the efficiency and quality of T cell responses. [Figure 2-2] Same as above.

[0025] [Figure 3-1] Figures 3A-1, 3A-2, 3B-1, 3B-2, 3C-1, and 3C-2 show the self-assembled nanoparticles of TAT-TRP2 with CpG and MPLA (abbreviated as CM). TRP2 itself could not form particles with CpG and MPLA. AFM analysis shows the cross-sectional size of the nanoparticles. [Figure 3-2] Same as above.

[0026] [Figure 4] Figure 4 shows DLS measurements of size distribution of self-assembled nanoparticles of TAT-TRP2 with various ratios of TLR ligands. The combinations of TAT-TRP2 and TLR ligands (different ratios) are listed in Table 3. Grey bars indicate unstable / polydisperse complexes with large PDI (PDI>0.5).

[0027] [Diagram 5] FIG. 5 shows the zeta potential of the TAT-TRP2-CM complex composed of TAT-TRP2, CpG, and MPLA at various nitrogen (+) to phosphate (-) (N / P) ratios.

[0028] [Figure 6-1] Figures 6A and 6B show characterization of TAT-TRP2-CM complexes. DLS measurements of size of TAT-TRP2-CM complexes in HO (Figure 6A). Zeta potential of TAT-TRP2 (also known as TRP2TAT), CpG, MPLA, and TAT-TRP2-CM complexes in HO (Figure 6B). PDI is the polydispersity index. [Figure 6-2] Same as above.

[0029] [Figure 7-1] 7A, 7B, 7C, 7D, 7E, and 7F show nanoparticles of TAT-TRP2 and TAT-ESO-1 with CpG and MPLA (TAT-TRP2-CM, TAT-ESO-1-CM), and nanoparticles of TAT-TRP2 and TAT-ESO-1 with CpG, MPLA, and poly(I:C) (TAT-TRP2-CMI and TAT-ESO-1-CMI). Schematic of TAT-TRP2 and TAT-ESO-1 nanoparticles (FIG. 7A). Nanoparticle size of TAT-TRP2 and TAT-ESO-1 with CM (FIG. 7B), or nanoparticle size of TAT-TRP2 and TAT-ESO-1 with CMI (FIG. 7C and FIG. 7D). FIG. 7C discloses SEQ ID NO:30. Figure 7E and Figure 7F: Zeta potential of TAT-TRP2-CM, TAT-TRP2-CMI, TAT-ESO-l-CM, and TAT-ESO-1-CMI. [Figure 7-2] Same as above. [Figure 7-3] Same as above.

[0030] [Figure 8]Figures 8A, 8B, and 8C show characterization of TAT-TRP2-CM complexes at various pH values. (Figures 8A and 8B) DLS measurements of the size of TAT-TRP2-CM complexes in potassium phosphate buffers at pH 7.0 (Figure 8A) and pH 4.0 (Figure 8C). (Figure 8C) Zeta potential of TAT-TRP2 (also known as TRP2TAT), CpG, MPLA, and TAT-TRP2-CM complexes in potassium phosphate buffers at pH 7.0 and pH 4.0. PDI is the polydispersity index.

[0031] [Figure 9-1] Figures 9A, 9B, 9C, and 9D show that the assembly and nanoparticles of TAT-TRP2 / TLR and TAT-ESO-1 / TLR are pH-dependent. Figure 9A shows DLS measurements of the size change of the TAT-TRP2-CM complex in potassium phosphate buffers of pH 4 to 7. Figure 9B is a schematic diagram of the uptake of TAT-TRP2 or TAT-TRP2-CM complexes in DCs and the pH-dependent complex disassembly process. Figures 9C and 9D show the DLS measurements of the size change of the TAT-TRP2 / TLR and TAT-ESO-1 / TLR nanoparticles in potassium phosphate buffers of pH 7 and pH 4. [Figure 9-2] Same as above.

[0032] [Figure 10-1] Figure 10 shows the combination of TLR ligands to stimulate innate immune responses and cytokine production. Bone marrow-derived DCs were isolated and then treated with various TLR ligands (single type only), two-type combinations, or three-type combinations. Cytokine (TNF-α, IL-6, IFN-α, and IFN-β) production in cell supernatants was determined by ELISA. Poly(I:C) / CpG, two-type combination of CpG / MPLA, and three-type combination of CpG / Poly(I:C) / MPLA were more potent than other groups in triggering innate immune cytokine production. [Figure 10-2] Same as above.

[0033] [Figure 11] Figure 11 shows a lung metastasis model of B16 tumor in C57BL / 6 mice vaccinated with DC / TAT-TRP2-CM and DC / TRP2-CM. C57BL / C mice were injected (intravenously) with B16 tumor cells (0.2x106 cells / mouse) on day 0 and vaccinated with DC / TAT-TRP2-CM, DC / TRP2-CM, or DC / β-gal-CM (5x106 cells / mouse) on day 5. Mice were sacrificed on day 18. The number of lung metastases was counted.

[0034] [Figure 12-1] Figure 12A, Figure 12B, and Figure 12C show lung metastasis and survival of B16-bearing C57BL / 6 mice after inoculation with different vaccines. Figure 12A shows the tumor model and vaccination schedule. In Figure 12B, C57BL / C mice were injected (intravenously) with B16 tumor cells (0.2x106 cells / mouse) on day 0, and five different groups of vaccinations were performed on day 5 (5x106 cells / mouse). All mice were sacrificed on day 18. The number of lung metastases was counted. Figure 12C shows that B16 injection and vaccination were the same as Figure 12B. Different groups of vaccinated B16-bearing mice were monitored for survival for 55 days. Error bars show standard deviation. *P<0.05, **P<0.01, ***P<0.001. [Figure 12-2] Same as above.

[0035] [Figure 13-1] Figure 13A and Figure 13B show that DC / TAT-ESO-CM and TAT-ESO-CMI vaccines generate strong anti-tumor immunity. RM1 / A2-ESO-1 tumor cells were injected into HLA-A2 Tg mice on day 0. Tumor-bearing mice were treated with vaccines (DC / control, DC / TAT-ESO-CM, or DC / TAT-ESO-CMI). Tumor growth was monitored every 2 days. Figure 13A shows the tumor size on day 15. Figure 13B shows the tumor growth curve. P values ​​between different groups are indicated. [Figure 13-2] Same as above.

[0036] [Figure 14-1] Figures 14A and 14B show T cell responses in mice vaccinated with DC / control, DC / TAT-ESO-CM, or DC / TAT-ESO-CMI (Figure 14A). Figure 14B shows % IFN-γ (INFγ) in CD8+ T cells (cT cells). [Figure 14-2] Same as above.

[0037] [Figure 15] FIG. 15 shows significant inhibition of breast cancer growth after DC / TAT-ESO-CM vaccination. E0771 / A2-ESO-1 tumor cells were injected into HLA-A2 Tg mice on day 0. Tumor-bearing mice were treated with DC / control or DC / TAT-ESO-CM. Tumor growth was monitored every 2 days. Vaccination with DC / TAT-ESO-CM significantly inhibited breast cancer growth.

[0038] [Figure 16] Figure 16A, Figure 16B, and Figure 16C show that direct immunization with TAT-ESO-CMI generated strong and therapeutic antitumor immunity compared with DC / TAT-ESO-CMI vaccination. In Figure 16A, RM1 / A2-ESO tumor cells were injected into HLA-A2 Tg mice on day 0, followed by three injections of TAT-ESO-CMI on days 10, 13, and 18, or DC / TAT-ESO-CMI vaccination on day 10. Tumor growth was monitored every 2 days. In Figure 16B, tumor size is shown for each group. In Figure 16C, tumor growth of the three groups after vaccination was recorded. P values ​​for significant differences between groups are shown.

[0039] [Figure 17]Figure 17A, Figure 17B, Figure 17C, and Figure 17D show CT83 expression in breast cancer samples and cell lines. Figure 17A shows RF-PCR analysis of CT83-expressing breast cancer cell lines. Figure 17B shows CT83 expression in breast cancer samples using RT-PCR analysis. NY-ESO-1 served as a positive control. Figure 17C is a Western blot analysis of breast cancer cell lines using anti-CT83 antibody. Antibody staining of normal and breast cancer tissues for CT83 expression is shown in Figure 17D.

[0040] [Figure 18] Figure 18A and Figure 18B show CT83 expression in lung cancer samples and cell lines. Figure 18A shows RF-PCR analysis of CT83-expressing lung cancer cell lines and cancer samples. Figure 18B shows Western blot analysis of CT83 expression in lung cancer cell lines using anti-CT83 antibody. MDA-468 served as a positive control.

[0041] [Figure 19-1] Figure 19A, Figure 19B, Figure 19C, and Figure 19D show the generation of CD83-specific T cells using self-assembled TAT-CT83 peptide nanoparticles containing CMI. Figure 19A shows the intracellular staining of IFN-γ released from splenocytes of TAT-CT83-CMI immunized mice. Figure 19B shows the IFN-γ release assay by ELISA. Figure 19C shows the T cell response to CT83 peptide after one cycle of culture. Figure 19D shows the establishment of CT83-A2 restricted peptide T cell clones. [Figure 19-2] Same as above.

[0042] [Figure 20-1]Figure 20A, Figure 20B, Figure 20C, and Figure 20D show that TAT-CT83-CMI vaccine generates strong anti-tumor immunity against E0771-A2-CT83 breast cancer. (Figure 20A) Establishment of E0771-A2-CT83 breast cancer model in HLA-A2 transgenic mice and scheme of vaccination schedule. (Figure 20B) Images of E0771-A2-CT83 breast tumor masses after two immunizations (10 mg / kg body weight, intraperitoneally) with the indicated vaccine formulations alone or with anti-PD1 blockade therapy. (Figure 20C) Tumor weights excised and measured on day 16 from mice vaccinated with the indicated formulations with or without anti-PD1 antibody. (Figure 20D) Immunohistochemical staining of CD3+ T cell infiltration in tumor sites with or without TAT-CT83-CMI vaccination. [Figure 20-2] Same as above.

[0043] [Figure 21] Figure 21A, Figure 21B, Figure 21C, and Figure 21D show that vaccination with TAT-ESO-CMI generates strong therapeutic immunity against breast cancer. (Figure 21A) Schematic of the vaccination experiment design. (Figure 21B) Tumor growth of each group in HLA-A2 Tg mice. (Figure 21C) Images of mice and tumor size. (Figure 21D) Vaccination with TAT-ESO-CMI without DC significantly inhibited tumor growth. *P<0.05, **P<0.01.

[0044] [Figure 22] Figures 22A, 22B, and 22C show vaccination with TAT-TRP2-CMI alone or in combination with anti-PD-1 therapy. (Figures 22A and 22B) Lung images and lung metastasis counts for mice vaccinated with TAT-TRP-2 / CMI alone or in combination with anti-PD-1 treatment. (Figure 22C) Survival time of mice after vaccination with TAT-TRP-2 / CMI alone or in combination with anti-PD-1 therapy.

[0045] [Diagram 23] Figures 23A and 23B show vaccination with TAT-ESO-CMI alone or in combination with anti-PD-1 therapy. (Figure 23A) Tumor images of RM1 / A2-ESO tumor cells in HLA-A2-Tg mice vaccinated with TAT-ESO-CMI alone or in combination with anti-PD-1 treatment, compared to the control group. (Figure 23B) Tumor growth curves after vaccination with TAT-ESO-CMI alone or in combination with anti-PD-1 therapy, compared to the control group.

[0046] [Figure 24] Figures 24A and 24B show that SAPNANO vaccination after TCR-T cell transfer increases tumor-infiltrating T cells and significantly inhibits tumor growth. (Figure 24A) Tumor growth between different treatment groups. *P value < 0.05. (Figure 24B) Percentage increase in tumor-infiltrating A2-ESO TCR-T cells after vaccination with A2-ESO TCR-T cells alone or in combination with TAT-ESO-CMI. A2-ESO TCR-T cells were detected using anti-TCR human Vβ13 antibody encountered on anti-CD3 positive T cells. Endogenous T cells induced by TAT-ESO-CMI vaccine could not be detected by anti-TCR human Vβ13 antibody.

[0047] [Diagram 25]Figure 25A, Figure 25B, Figure 25C, Figure 25D, and Figure 25E show that the combination of NY-ESO TCR-T therapy and TAT-ESO-CMI vaccination generated a strong anti-tumor response in a humanized NSG mouse model. (Figure 25A) Schematic of the animal experiment. 3-4 week old NSG mice were humanized by intravenous injection of 1x107 human PBMCs to reconstitute the human immune system 3-4 weeks prior to tumor incubation. HLA-A2 / NY-ESO positive human breast cancer cells (MDA-MB-231-A2-ESO) were injected subcutaneously into the fat pad of humanized NSG mice (1 million / mouse). Tumor-bearing mice were treated with NY-ESO TCR-T cells on day 5. Three doses of human IL2 (50000IU) and four doses of TAT-ESO-CMI vaccine were administered by intravenous injection. Mice were sacrificed on day 30. (Figure 25B) Three weeks after humanization, human lymphocytes were detected in NSG mice by FACS. (Figure 25C) After treatment, tumor growth was monitored. Data represent mean ± SEM. PBS control group (N=4), other groups (N=5), 2-way ANOVA test was used for statistical analysis. *p<0.05, **p<0.01. (Figure 25D and Figure 25E) After excision from mice, tumor weight was imaged and weighed. (Mean ± SEM, T-test was used for statistical analysis. *p<0.05, **p<0.01, ***p<0.001) DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0048] A quick explanation of the sequence: SEQ ID NO:1 is an exemplary therapeutic NY-ESO-1 specific peptide for use according to one embodiment of the present disclosure.

[0049] SEQ ID NO:2 is an exemplary therapeutic TRP-2-specific peptide for use according to one embodiment of the present disclosure.

[0050] SEQ ID NO:3 is an exemplary HIV Tat47-57 specific cell-penetrating peptide sequence for use according to one embodiment of the present disclosure.

[0051] SEQ ID NO:4 is an exemplary TAT-PTD-4 specific cell-penetrating peptide sequence for use according to one embodiment of the present disclosure.

[0052] SEQ ID NO:5 is an exemplary TAT-PTD-5 specific cell-penetrating peptide sequence for use according to one embodiment of the present disclosure.

[0053] SEQ ID NO:6 is an exemplary DPV3-specific cell-penetrating peptide sequence for use according to one embodiment of the present disclosure.

[0054] SEQ ID NO:7 is an exemplary DPV6-specific cell-penetrating peptide sequence for use according to one embodiment of the present disclosure.

[0055] SEQ ID NO:8 is an exemplary DPV7-specific cell-penetrating peptide sequence for use according to one embodiment of the present disclosure.

[0056] SEQ ID NO:9 is an exemplary nine-residue polyarginine cell-penetrating peptide sequence for use according to one embodiment of the present disclosure.

[0057] SEQ ID NO:10 is an exemplary 9-residue polylysine cell-penetrating peptide sequence for use according to one embodiment of the present disclosure.

[0058] SEQ ID NO:11 is an exemplary FHV coat-specific cell-penetrating peptide sequence for use according to one embodiment of the present disclosure.

[0059] SEQ ID NO:12 is an exemplary signal peptide II-specific cell-penetrating peptide sequence for use according to one embodiment of the present disclosure.

[0060] SEQ ID NO: 13 is an exemplary amphipathic model peptide-specific cell-penetrating peptide sequence for use according to one embodiment of the present disclosure.

[0061] SEQ ID NO:14 is an exemplary HSV VP22-specific cell-penetrating peptide sequence for use according to one embodiment of the present disclosure.

[0062] SEQ ID NO:15 is an exemplary peptide carrier-specific cell-penetrating peptide sequence for use according to one embodiment of the present disclosure.

[0063] SEQ ID NO:16 is an exemplary CL22-specific cell-penetrating peptide sequence for use according to one embodiment of the present disclosure.

[0064] SEQ ID NO:17 is an exemplary TRP-2-specific peptide sequence for use according to one embodiment of the present disclosure.

[0065] SEQ ID NO:18 is an exemplary TAT-binding peptide for use according to one embodiment of the present disclosure.

[0066] SEQ ID NO:19 is an exemplary TAT-binding peptide for use according to one embodiment of the present disclosure.

[0067] SEQ ID NO:20 is an exemplary TAT-binding peptide for use according to one embodiment of the present disclosure.

[0068] SEQ ID NO:21 is an exemplary TAT-binding peptide for use according to one embodiment of the present disclosure.

[0069] SEQ ID NO:22 is an exemplary TAT-binding peptide for use according to one embodiment of the present disclosure.

[0070] SEQ ID NO:23 is an exemplary TAT-binding peptide for use according to one embodiment of the present disclosure.

[0071] SEQ ID NO:24 is an exemplary TAT-binding peptide for use according to one embodiment of the present disclosure.

[0072] SEQ ID NO:25 is an exemplary TAT-binding peptide for use according to one embodiment of the present disclosure.

[0073] SEQ ID NO:26 is an exemplary TAT-binding peptide for use according to one embodiment of the present disclosure.

[0074] SEQ ID NO:27 is an exemplary TAT-binding peptide for use according to one embodiment of the present disclosure.

[0075] SEQ ID NO:28 is an exemplary TAT-binding peptide for use according to one embodiment of the present disclosure. Description of illustrative embodiments

[0076] Illustrative embodiments of the present invention are described below. To provide clarity, not all features of an actual implementation are described herein. It will, of course, be understood that the development of any such actual embodiment will require numerous implementation-specific decisions to be made to achieve the developer's particular goals, such as compatibility with system-related and business-related constraints, which will vary from implementation to implementation. Moreover, it will be appreciated that such a development effort may be complex and time-consuming, but would be a routine undertaking for those of ordinary skill in the art having the benefit of this disclosure.

[0077] Thus, in a first aspect, the present invention provides nanoparticles comprising a multiplicity of cationic CPPs, each covalently linked to a hydrophobic therapeutic peptide, which self-assemble under neutral conditions, e.g., pH 7.0, and dissociate under acidic conditions, e.g., pH 4.5 (Figures 2A and 2B).

[0078] In some embodiments, the cationic CPP is selected from the group consisting of TAT, TAT-PTD-4, TAT-PTD-5, DVP3, DVP6, DVP7, polyarginine (R9), polylysine (K9), FHV coat, signal peptide I, signal peptide II, PRES, transportan, amphipathic model peptide, HSV VP22, and CL22. In some embodiments, the cationic CPP consists of 8-30 amino acids. In one embodiment, the cationic CPP is Tat.

[0079] In some embodiments, the therapeutic peptide is an antigenic peptide or a non-immunogenic peptide that contains a T cell epitope. In some embodiments, the T cell epitope is a tumor-specific epitope or a pathogen-specific epitope. In some embodiments, the therapeutic peptide consists of 9 to 25 amino acids. The antigenic peptide or the non-immunogenic peptide that contains a T cell epitope is directed against a specific disease, such as a tumor, and an infectious disease.

[0080] The nanoparticles of the present invention may further comprise at least one negatively charged molecule, preferably a negatively charged TLR ligand, non-covalently bound to the CPP. In some embodiments, the negatively charged molecule is a CpG oligodeoxynucleotide, poly(I:C), or a combination thereof. In some embodiments, the CpG oligodeoxynucleotide is 20-24 bp in length. In some embodiments, the poly(I:C) is 0.2-1 kb in length.

[0081] The nanoparticles described herein may also carry at least one hydrophobic molecule, preferably a hydrophobic TLR ligand, non-covalently attached to the therapeutic peptide. In some embodiments, the hydrophobic molecule is monophosphoryl lipid A (MPLA), R848, or a combination thereof.

[0082] The nanoparticles disclosed herein can be taken up by cells. In some embodiments, nanoparticles containing antigenic peptides or non-immunogenic peptides with T cell epitopes can be taken up by APCs (particularly DCs or macrophages) both in vitro and in vivo. In some embodiments, TLR ligands activate one or more TLR signaling pathways.

[0083] In another aspect, the present disclosure provides a pharmaceutical composition comprising the nanoparticles of the present invention and a pharma- ceutically acceptable carrier, preferably one having a pH of about 7.0.

[0084] In a third aspect, the disclosure also provides a composition for producing the above-described nanoparticles comprising a plurality of cationic CPPs, each of which is covalently linked to a hydrophobic therapeutic peptide, and optionally comprising at least one negatively charged molecule and / or at least one hydrophobic molecule.

[0085] The components in the composition may be mixed in a medium at about pH 7.0 prior to administration of the nanoparticles. The nanoparticles self-assemble in the medium and are taken up by APCs by endocytosis in vitro, and then used as a vaccine. Alternatively, the self-assembled nanoparticles may be prepared and delivered to an animal, where they are taken up by DCs or macrophages in vivo. Regardless of whether the nanoparticles are taken up by DCs or macrophages in vitro or in vivo, the self-assembled nanoparticles enter endosomes or lysosomes, where they are disrupted at pH 4.5; CPP-bound peptides and TLR ligands are released. Endosome-localized TLR3, TLR7, TLR8, and TLR9 bind to TLR ligands to trigger innate immune signaling, while CPP-bound peptides bind to MHC class II molecules in endosomes for loading and presentation, or cross the endosomal membrane into the cytoplasm, ER, and Golgi for antigen processing and presentation to T cells by MHC class I molecules.

[0086] In some embodiments, the cationic CPP is selected from the group consisting of TAT, TAT-PTD-4, TAT-PTD-5, DVP3, DVP6, DVP7, polyarginine (R9), polylysine (K9), FHV coat, signal peptide I, signal peptide II, PRES, transportan, amphipathic model peptide, HSV VP22, and CL22. In some embodiments, the cationic CPP consists of 8-30 amino acids. In one embodiment, the cationic CPP is Tat. In some embodiments, the therapeutic peptide is an antigenic peptide or a non-immunogenic peptide that includes a T cell epitope. In some embodiments, the T cell epitope is a tumor-specific epitope or a pathogen-specific epitope. In some embodiments, the therapeutic peptide consists of 8, 9, 10, or 11 amino acids that are presented by MHC class I molecules, or 9-25 amino acids that are presented by MHC class II molecules. Antigenic or non-immunogenic peptides containing T cell epitopes are directed against certain diseases, such as tumors, and infectious diseases.

[0087] In some embodiments, the negatively charged molecule is a negatively charged TLR ligand. In some embodiments, the negatively charged molecule is a CpG oligodeoxynucleotide, poly(I:C), or a combination thereof. In some embodiments, the CpG oligodeoxynucleotide is 15-24 bp in length. In some embodiments, the poly(I:C) is 0.2-1 kb in length. In some embodiments, the non-covalently bound hydrophobic molecule is a hydrophobic TLR ligand. In some embodiments, the hydrophobic molecule is monophosphoryl lipid A (MPLA), R848, or a combination thereof.

[0088] In a fourth aspect, the present disclosure provides a method for treating, preventing, and / or ameliorating at least one symptom of cancer or infectious disease. In a comprehensive and general sense, the method typically comprises providing a therapeutically effective amount of a pharmaceutical formulation comprising the nanoparticles disclosed herein to a subject in need thereof.

[0089] In some embodiments, the cancer is a dangerous tumor, and such tumors can be structurally solid or non-solid, depending on the specific disease. In certain embodiments, the cancerous tumor to be treated is a primary tumor or a metastatic tumor (for example, but not limited to, one or more melanomas or lung cancers).

[0090] In other embodiments, the treatment of disease may be specifically contemplated, for example, in the treatment of one or more viral, fungal, and / or bacterial infections.

[0091] Antigenic or non-immunogenic peptides containing T cell epitopes in nanoparticles are processed by APCs (especially DCs or macrophages) and presented to T cells by newly synthesized MHC class II molecules for a long time. TLR ligands contained within the nanoparticles stimulate DCs or immune cells to generate natural immune responses (e.g., release of type I interferon cytokines), presenting epitopes to T cells and enhancing the ability of DCs to co-stimulate T cells for activation, as well as cytokine stimulation for T cell growth and expansion.

[0092] The nanoparticles described herein allow for enhanced co-delivery of antigenic or non-immunogenic peptides containing T cell epitopes and two or more TLR ligands into the same antigen-presenting cells (e.g., DCs or macrophages), thereby generating a strong and effective immune response, the resulting effect (e.g., anti-tumor or anti-pathogen effect) may be superior to some other delivery platforms (e.g., MSV).

[0093] Other objects, features, and advantages of the present disclosure will become apparent from the following detailed description. However, this detailed description and specific examples, while indicating preferred embodiments of the present invention, are given for illustrative purposes only, since various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description. Pharmaceutical preparations

[0094] In certain embodiments, the present disclosure relates to self-assembled nanoparticle compositions prepared in a pharma- ceutically acceptable formulation for administration to one or more cells or tissues of an animal, alone or in combination with one or more other modes of diagnosis, prophylaxis, and / or treatment. The formulation of pharma-ceutically acceptable excipients and carrier solutions is well known to those skilled in the art, as is the development of appropriate dosing and treatment regimens for using the self-assembled nanoparticle compositions described herein in various therapeutic, prophylactic, diagnostic, and prognostic regimens.

[0095] In certain circumstances, it may be desirable to deliver the self-assembled nanoparticle compositions of the present disclosure in an appropriately formulated pharmaceutical vehicle to one or more cells, tissues, or organs in or around an animal by one or more standard delivery devices, including but not limited to subcutaneous, parenteral, intravenous, intramuscular, intrathecal, intratumoral, intraperitoneal, transdermal, topical, oral or nasal inhalation, or by direct injection.

[0096] Methods of administration may also include those described in U.S. Patent No. 5,543,158; U.S. Patent No. 5,641,515; and U.S. Patent No. 5,399,363, each of which is specifically incorporated herein in its entirety by express reference. Solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in sterile water, suitably mixed with one or more surfactants, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycol, oil, or mixtures thereof. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0097] For administration of injectable aqueous solutions, including but not limited to, the solutions may be appropriately buffered if necessary, and the liquid diluent may first be made isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intramuscular, subcutaneous, transdermal, subdermal, and / or intraperitoneal administration. In this regard, the compositions of the present invention may be formulated in one or more pharma-ceutically acceptable vehicles, including, for example, sterile aqueous media, buffers, diluents, and the like. For example, a given dose of active ingredient(s) may be dissolved in a particular volume of isotonic solution (e.g., isotonic NaCl-based solution) and then administered to the intended administration site, or may be further diluted with a vehicle suitable for intravenous infusion (see, for example, "REMINGTON'S PHARMACEUTICAL SCIENCES'", 15th edition, pp. 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated, the extent of treatment, and the site of administration; nevertheless, the person responsible for administration will be able to use ordinary knowledge in the medical and pharmaceutical arts to determine the correct dosage regimen appropriate for the individual subject.

[0098] Sterile injectable compositions can be prepared by mixing the self-assembled nanoparticle composition of the present disclosure in the required amount with some of the other ingredients listed above as necessary in a suitable solvent, and then sterilizing by filtration.In general, dispersions can be prepared by incorporating the selected sterilized active ingredient(s) into a sterile vehicle that contains a basic dispersion medium and the other ingredients listed above as required.The self-assembled nanoparticle composition disclosed herein can also be formulated in neutral or salt form.

[0099] Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of proteins) formed with inorganic acids (such as, but not limited to, hydrochloric or phosphoric acid) or organic acids (such as, but not limited to, acetic acid, oxalic acid, tartaric acid, mandelic acid, etc.). Salts formed with free carboxyl groups can also be derived from inorganic bases (such as, but not limited to, sodium, potassium, ammonium, calcium, or ferric hydroxides) and organic bases (such as, but not limited to, isopropylamine, trimethylamine, histidine, procaine, etc.). Once formulated, solutions are administered in a manner compatible with the dosage formulation and in an amount effective for the intended use. Formulations of the compounds of the present invention can be administered in a variety of dosage forms, such as injectable solutions, topical preparations, oral formulations (including sustained release capsules), hydrogels, colloids, viscous gels, transdermal agents, intranasal and inhalation formulations.

[0100] The amount, dosage regimen, formulation, and administration of the self-assembled nanoparticle composition disclosed herein are within the scope of the skilled artisan having the benefit of the present teachings. However, it is likely that administration of a diagnostically effective (i.e., medicamentously effective) amount of one or more compositions of the present disclosure can be achieved by a single administration, for example, but not limited to, by a single injection of a delivery agent in an amount sufficient to provide the desired benefit to the patient in need thereof. Alternatively, in some circumstances, multiple or continuous administrations of the self-assembled nanoparticle composition of the present disclosure over a relatively short period of time or even a relatively long period of time (as may be determined by the physician supervising the administration of such compositions to the selected individual undergoing such procedure(s), treatment, therapy, or diagnosis).

[0101] Typically, a formulation of one or more self-assembled nanoparticle compositions described herein will contain at least an effective amount of a first active agent. Preferably, the formulation will contain at least about 0.001% of each active ingredient, preferably at least about 0.01%, although of course the percentage of the active ingredient(s) may vary and may conveniently be present in an amount of about 0.01 to about 90% by weight or volume, or about 0.1 to about 80% by weight or volume, or preferably about 0.2 to about 60% by weight or volume, based on the total formulation. Of course, the amount of active compound(s) in each composition may be prepared in such a way that an appropriate dosage is achieved in any given unit dose of the compound. Solubility, bioavailability, biological activity, and the like may be considered. 1 / 2 Factors such as the dosage, route of administration, shelf life of the product, and other pharmacological considerations will be contemplated by those skilled in the art of preparing such pharmaceutical formulations, and as such, various dosages and treatment regimens may be desirable.

[0102] In many embodiments of the present invention, systemic administration is expected to be effective, but it is also expected that the formulations disclosed herein are suitable for direct injection into one or more organs, tissues, or cell types in the body.Direct injection of the nanoparticles of the present disclosure into specific, discreet sites in the body, or directly into tumors, tumor stem cells, cancerous tissues, and / or cancer stem cells, may be performed, for example, using any suitable method known to those skilled in the art of oncology.

[0103] Pharmaceutical formulations comprising one or more self-assembled nanoparticle compositions disclosed herein may further comprise one or more excipients, buffers, or diluents specifically formulated for contact with mammalian cells (and, in particular, human cells) and / or for administration to a mammalian subject (e.g., a human patient). The compositions may optionally further comprise one or more diagnostic or prognostic agents, and / or may be formulated with additional population(s) of microspheres, microparticles, nanospheres, or nanoparticles, or may be formulated to include one or more additional therapeutic agent(s) and / or diagnostic agent(s) useful for administration to one or more cells, tissues, organs, or bodies of a mammalian patient (and, in particular, a human patient).

[0104] The formulation of pharma- ceutically acceptable excipients and carrier solutions is well known to those of skill in the art, as is the development of appropriate administration, diagnostic, and / or treatment regimens for using the particular self-assembled nanoparticle compositions described herein in various modalities (e.g., including, but not limited to, oral, parenteral, intravenous, intranasal, intratumoral, and intramuscular routes of administration).

[0105] The specific amount of self-assembled nanoparticle composition employed and the specific administration time or dosage regimen for the composition employing the formulation of the present disclosure are within the knowledge of the person skilled in the art who has the benefit of the present teaching.However, it is likely that administration of the formulation of the present disclosure can be achieved by administration of one or more doses of the formulation for a period of time effective to bring about desired benefits to the patient undergoing such treatment.Such administration regimen can be determined by the physician supervising the administration of the compound according to the specific condition or patient, the degree or duration of the treatment being administered, etc.

[0106] Pharmaceutical preparations comprising one or more self-assembled nanoparticle compositions disclosed herein are in no way limited to use only in humans, or even primates or mammals. In certain embodiments, the methods and compositions disclosed herein may be used with birds, amphibians, reptiles, or other animal species. However, in preferred embodiments, the compositions of the present disclosure are appropriately formulated for administration to mammals (particularly humans) in various regimens for diagnosing, ameliorating, and / or treating one or more diseases in the patient's body, and in particular for treating one or more types of tumors or cancer cells, or for treating one or more infections. As mentioned above, such compositions are not limited to use only in humans, but may also be formulated for veterinary administration, including but not limited to administration to selected livestock, exotic or domesticated animals, companion animals (including pets, etc.), non-human primates, and zoological or otherwise captured specimens, etc. Compositions for the preparation of medicines

[0107] Another important aspect of the present invention relates to methods for using the self-assembled nanoparticle compositions of the present disclosure (as well as formulations comprising same) in the preparation of a medicament for preventing, diagnosing, treating, and / or ameliorating one or more symptoms of one or more diseases, dysfunctions, abnormal conditions, or disorders in an animal (including, for example, a vertebrate mammal). Use of the self-assembled nanoparticle compositions of the present disclosure is particularly contemplated for use in the diagnosis and / or prognosis of cancer, in the detection and / or prediction of, or for monitoring the extent of, cancer metastasis, and / or for the treatment of one or more abnormal conditions (e.g., treatment of one or more cancer cell types in vivo, ex vivo, and / or in situ).

[0108] Such uses generally involve administering to a mammal in need thereof one or more self-assembled nanoparticle compositions of the present disclosure comprising at least a first active agent in an amount and for a period of time sufficient to diagnose, treat, alleviate or ameliorate one or more symptoms of tumor formation, or cancer growth and / or metastasis in the affected mammal. Pharmaceutical formulations comprising one or more self-assembled nanoparticle compositions of the present disclosure also form part of the present disclosure, particularly those compositions further comprising at least a first pharma- ceutical acceptable excipient for use in treating and / or ameliorating one or more symptoms of cancer in an affected mammal. Self-assembled nanoparticles

[0109] The present disclosure describes the use of cationic cell-penetrating peptides, each covalently linked to a hydrophobic therapeutic peptide and, optionally, at least one negatively charged molecule and / or at least one hydrophobic molecule, to form nanoparticles. The resulting nanoparticles have (i) a core comprising the hydrophobic therapeutic peptide and, optionally, a hydrophobic molecule, and (ii) a corona comprising the CPP and, optionally, a negatively charged molecule.

[0110] The negatively charged molecule may be a TLR ligand, such as CpG oligodeoxynucleotide, poly(I:C), DNA, and RNA (mRNA or siRNA). The hydrophobic molecule may preferably be a hydrophobic TLR ligand, which may be monophosphoryl lipid A (MPLA), or R848.

[0111] Without wishing to be bound by any theory, it is believed that self-assembly occurs in aqueous solution at about pH 7.0 due to the hydrophobic nature of the therapeutic peptides (and, if desired, hydrophobic molecules) and the hydrophilic nature of the CPPs, as well as electrical binding by the positively and negatively charged molecules.

[0112] The amount of components used to form the nanoparticles of the present invention can be determined by those skilled in the art.The more negatively charged molecules used, the more CPPs are involved in nanoparticle formation.In addition, changing the amount of some components will change the size and shape of nanoparticles and in vivo biodistribution due to the difference in zeta potential.

[0113] In one embodiment, cationic CPP (positively charged)-antigenic peptides or weakly immunogenic peptides (containing T cell epitopes (generally hydrophobic)) are designed and synthesized and mixed with CpG oligonucleotides (negatively charged) and monophosphoryl lipid A (MPLA, hydrophobic) in phosphate buffered saline (PBS). Both CPP antigen peptides (10 mM) and CpG (10 mM) are soluble in PBS, but when mixed (1:1), precipitation or aggregates were observed. These aggregates were round nanoparticles with a size of 100 nM in diameter. This self-assembly was thought to occur due to the electrical interaction of the positively charged CPP and negatively charged molecules, as well as the hydrophobic interaction between the peptide itself and monophosphoryl lipid A (MPLA). Further experiments with different ratios (positive charge:negative charge, or molar concentration) found that different ratios of CPP therapeutic peptides, CpG, and MPLA at pH 7 could produce nanoparticles of 100 nM in size but with different zeta potentials (surface charge) and assembly efficiencies of each component.

[0114] As described above, the nanoparticles of the present invention self-assemble under neutral conditions (e.g., pH 7.0) and are destroyed under acidic conditions (e.g., pH 4.5). Thus, the nanoparticles are delivered to dendritic cells or macrophages as dense, small-sized particles, and then are destroyed inside endosomes where the pH is 4.5, releasing CPPs into the cytoplasm along with therapeutic peptides (preferably antigenic peptides or non-immunogenic peptides containing T cell epitopes) and other molecules (preferably TLR ligands). Then, the antigenic peptides or non-immunogenic peptides containing T cell epitopes bind to MHC class I or II molecules, and the epitopes are presented to T cells, while the TLR ligands bind to TLRs to trigger TLR-mediated signaling pathways (NF-κB, and type I interferon), producing proinflammatory cytokines and inducing strong innate and adaptive immune responses. Chemotherapeutic methods and uses

[0115] An important aspect of the present disclosure relates to methods for using the self-assembled nanoparticle formulations of the present disclosure to treat or ameliorate one or more symptoms of cancer, including, for example, tumors or metastatic cancer (including, but not limited to, melanoma metastasis to the lungs of a mammal). Such methods generally involve administering to a mammal (and particularly to a human in need thereof) one or more self-assembled nanoparticle compositions of the present disclosure comprising at least a first anti-cancer therapeutic, in an amount and for a period of time sufficient to treat (or alternatively, ameliorate one or more symptoms of) the cancer in the affected mammal.

[0116] In certain embodiments, the self-assembled nanoparticle compositions described herein may be provided to an animal in a treatment modality (either as a single dose, or alternatively, multiple doses over a period of hours (hrs) to days (or even weeks or months)) as needed to treat a particular disease, disorder, dysfunction, or abnormal condition. Alternatively, in some embodiments, it may be desirable to continue the treatment or incorporate the treatment in combination with one or more additional modes of therapy for a period of months or more. In other embodiments, it may be desirable to provide the treatment in combination with one or more conventional treatment regimens.

[0117] The present disclosure also provides for the use of one or more self-assembled nanoparticle compositions of the present disclosure in the manufacture of a medicament for the therapy and / or amelioration of one or more symptoms of an infection or cancer, and in particular for the use in the manufacture of a medicament for treating and / or ameliorating one or more symptoms of a mammalian infection or cancer (e.g., including human infections, cancerous tumors, and ligans).

[0118] The present invention also provides the use of one or more self-assembled nanoparticle compositions of the present disclosure in the manufacture of a medicament for the treatment of a disease or disorder in a mammal, and in particular for the treatment of one or more human diseases, such as, for example, infection and / or cellular hyperproliferation (i.e., cancer). Medical kit

[0119] Therapeutic kits comprising one or more of the self-assembled nanoparticle compositions of the present disclosure, and instructions for using the kit in a particular treatment modality, also represent preferred embodiments of the present disclosure. These kits may, optionally, further comprise one or more additional therapeutic compounds, one or more diagnostic reagents, or any combination thereof.

[0120] The kits of the present invention may be packaged for commercial distribution and may further include one or more delivery devices (e.g., syringes, injections, etc.) adapted for delivering the self-assembled nanoparticle composition(s) to an animal, if necessary. Such kits typically include at least one vial, test tube, flask, bottle, syringe, or other container in which the self-assembled nanoparticle composition(s) may be placed, preferably appropriately aliquotted. If a second pharmaceutical product is also provided, the kit may also include a second separate container in which the second composition may be placed. Alternatively, multiple self-assembled nanoparticles as disclosed herein may be prepared in a single mixture (e.g., a suspension or solution) and packaged in a single container (e.g., a vial, flask, syringe, catheter, cannula, bottle, or other suitable single container).

[0121] The kits of the present invention may also typically include a retention mechanism adapted to contain or hold in close confinement the vial(s) or other container(s) for commercial sale, such as, for example, an injection- or blow-molded plastic container or the like within which the desired vial(s) or other container(s) can be retained to reduce or prevent breakage, exposure to sunlight, or other undesirable factors, or to allow ready use of the composition(s) included in the kit. Cell-penetrating peptides

[0122] Cell-penetrating amphipathic peptides, such as HIV-Tat-based peptides and chimeric cell-penetrating peptides, have also been applied to deliver therapeutic cargoes to their targets (Magzoub et al., 2004).

[0100] CPPs have long been used as drug delivery vehicles because they can translocate cell membranes (Gupta et al., 2005). CPPs, which are short cationic peptides of less than 30 amino acids, and polyarginine-based CPPs (8-10 arginine residues in length) have shown the most efficient cell membrane permeability (Fuchs et al., 2006).

[0123] According to the classical mechanism (Fuchs et al., 2006), membrane penetration of CPPs is based on hydrogen-bonding interactions between the guanidinium groups of arginine residues and the carboxyl, phosphoryl or sulfuryl groups of cell surface carbohydrates and phospholipids. The pathways of CPP translocation across membranes were initially explained by receptor / endocytosis-independent mechanisms, but now new mechanisms of CPP internalization have been demonstrated.

[0124] Several CPPs have been identified from proteins including the Tat protein of human immunodeficiency virus (HIV), the VP22 protein of herpes simplex virus, and fibroblast growth factors.

[0125] Some examples of cell penetrating peptides include, but are not limited to, the following: 1. YGRKKRRQRRR (HIV Tat(47-57)) (SEQ ID NO:3); 2. YARAAARQARA (TAT-PTD-4) (SEQ ID NO: 4); 3. YARAARRARR (TAT-PTD-5) (SEQ ID NO:5); 4. RKKRRRESRKKRRRES (DPV3) (SEQ ID NO: 6); 5. GRPRESGKKRKRKRLKP (DPV6) (SEQ ID NO: 7); 6. GKRKKKGKLGKKRDP (DPV7) (SEQ ID NO: 8); 7. RRRRRRRRR (polyarginine, R9) (SEQ ID NO:9); 8. KKKKKKKKK (polylysine, K9) (SEQ ID NO: 10); 8. RRRRNRTRRNRRRVR (FHV coat) (SEQ ID NO: 11); 9. GALFLGWLGAAGSTMGAWSQPKKKRKV (signal peptide II) (SEQ ID NO: 12); 10. KLALKLALKALKAALKLA (amphipathic model peptide) (SEQ ID NO: 13); 11. DAATATRGRSAASRPTERPRAPARSASRPRRPVE (HSV VP22) (SEQ ID NO: 14); 12. KETWWETWWTEWSQPKKKRKV (peptide carrier) (SEQ ID NO: 15); and 13. KKKKKKGGFLGFWRGENGRKTRSAYERMCNILKGK (CL22) (SEQ ID NO: 16).

[0126] A comprehensive list of known CPPs can be found online at the publicly available website, CPPsite 2.0, which is an updated version of the Database of Cell-Penetrating Peptides (CPPsite).

[0127] In the present disclosure, cationic CPPs are covalently linked to T cell epitope peptides and are present in the corona portion of the nanoparticles. The positively charged or neutral corona allows the nanoparticles to attach to negatively charged cell surfaces and then be taken up by cells such as DCs or macrophages with improved uptake efficiency.

[0128] CPPs also help antigenic peptides containing T cell epitopes or weakly immunogenic peptides (to which the T cell epitope is attached) to bind directly to MHC class II molecules in endosomes for antigen presentation at the cell surface, or help antigenic peptides containing T cell epitopes or non-immunogenic peptides to escape from endosomes and then enter the ER and Golgi, where they bind to newly synthesized MHC class I molecules for presentation at the cell surface for T cell activation.

[0129] Cationic CPP is necessary in the present invention as described above.However, non-cationic CPP can be modified by adding or attaching some amino acids such as Lys, Arg and His to the backbone chain, as known to those skilled in the art.For example, poly-Lys or Arg peptide is synthesized as cationic CPP. Antigenic peptides

[0130] In the context of the present disclosure, the term "antigenic peptide" refers to a peptide antigen that is common to a particular tumor or pathogen and that binds to an MHC molecule.

[0131] The tumor antigens of the present disclosure are preferably derived from cancers, including, but not limited to, primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, leukemia, uterine cancer, cervical cancer, bladder cancer, renal cancer, and adenocarcinoma (e.g., breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, etc.), etc. In one embodiment, the tumor antigens of the present disclosure comprise one or more antigenic cancer epitopes immunologically recognized by tumor infiltrating lymphocytes (TILs) derived from a mammalian cancerous tumor.

[0132] Malignant tumors express numerous peptides that can serve as target antigens for immune attack. These molecules include, but are not limited to, tissue-specific antigens such as MART-1, tyrosinase, and GP100 in melanoma, and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. 4 Other target molecules belong to the group of transformation-associated molecules, such as the oncogene HER-2 / Neu / ErbB-2. Yet another group of target antigens are the onco-fetal antigens, such as carcino-embryonic antigen (CEA).

[0133] Tumor antigens, such as cancer-testis antigens and neoantigens derived from mutations, include, but are not limited to, NY-ESO-1, CT83, MAGE gene family, and neoantigens. 4 .

[0134] Similarly, oncogene product peptide antigens common to certain tumor types have been identified. These polypeptides are used in the polypeptide complexes of the present invention as reagents that can be used generally to stimulate effective T cell responses to react with tumors bearing such antigens. Oncogene product peptide antigens include, but are not limited to, HER-2 / neu, which is associated with human breast and gynecological cancers, and carcinoembryonic antigen (CEA), which is associated with cancer of the pancreas.

[0135] Tumor antigens and their antigenic cancer epitopes may be purified and isolated from natural sources, such as primary clinical isolates, cell lines, etc. Cancer peptides and their antigenic epitopes may also be obtained by chemical synthesis or recombinant DNA techniques known in the art. Techniques for chemical synthesis are described in Steward et al. (1969); Bodansky et al. (1976); Meienhofer (1983); and Schroder et al. (1965).

[0136] In addition, there are numerous antigens known in the art, as described in Renkvist et al. (2001). + or helper CD4 + Various T cell determined epitopes recognized by T cell antigens (either T cell antigens or T cell antigens) are listed in PCT International Patent Application Publication No. WO 02 / 064057, which is specifically incorporated herein in its entirety by express reference.

[0137] Analogs or artificially modified epitopes are not listed, but the skilled artisan will know how to obtain or generate them by standard methods in the art. Other antigens (such as those identified by antibodies and detected by the SEREX method [see Sahin et al. (1997) and Chen et al. (2000)]) are identified in the Ludwig Institute for Cancer Research database, which the skilled artisan can easily find on the World Wide Web.

[0138] The antigen peptide of the present invention needs to be hydrophobic so that it can be incorporated into nanoparticles and delivered to endosomes.An antigen peptide that is not hydrophobic may be modified by adding or attaching one or more amino acids such as Gly, Ala, Val, Leu, Ile, Pro, Phe, Met and Trp to the backbone chain to enhance the antigenicity of the peptide, as is well known to those skilled in the art. T cell epitopes

[0139] The immunogenic peptide that forms the core of the nanoparticles of the invention comprises a T-cell epitope.

[0140] T cell epitopes do not need to be presented on the surface of the carrier to elicit immunity and may therefore be incorporated into the core of the nanoparticle.

[0141] T cell epitopes may be selected from different sources. For example, T cell epitopes may be determined by experimental methods. Such epitopes may be known from the literature, or may be predicted by algorithms based on existing protein sequences of specific pathogens or cancer antigens, or may be newly designed.

[0142] There is a wealth of known T cell epitopes available through the scientific literature. These T cell epitopes may be selected from specific pathogens, cancer-specific peptide sequences, or newly designed peptides with specific characteristics, such as PADRE peptides that bind to many different MHC II molecules (thereby becoming so-called promiscuous T cell epitopes) (see, for example, U.S. Patent No. 5,736,142, which is specifically incorporated herein in its entirety by express reference). There are publicly available databases that contain thousands of different T cell epitopes, such as the MHC database "MHCBN VERSION 4.0", or the PDB database "Protein Data Bank", or other databases.

[0143] By incorporating helper T cell (HTL) epitopes into peptide sequences that are not originally immunogenic or by attaching them to non-peptide antigens, it is possible to make them highly immunogenic. The Pan-DR binding peptide HTL epitope PADRE has been used extensively in vaccine design for malaria, Alzheimer's disease, and many other vaccines.

[0144] As defined by the MHCBN database (see above), T cell epitopes are characterized by their binding affinity (IC 50 MHC binders are peptides with a molecular weight (MHC binding affinity) of less than 50,000 nM. Such peptides are considered as MHC binders. According to this definition, up to August 2006, the following data are available in the MHCBN database version 4.0: 20717 MHC binders and 4022 MHC non-binders.

[0145] Suitable T cell epitopes can also be obtained by using prediction algorithms. These prediction algorithms can search existing protein sequences from pathogens or cancer antigens for putative T cell epitopes, or can predict whether newly designed peptides bind to specific MHC molecules. Many such prediction algorithms are publicly accessible on the Internet. Examples include SVRMHCdb (Wan et al., 2006), SYFPEITHI, MHCPred, Motif Scanner or NetMHCIIpan for MHC II binding molecules, and NetMHCpan for MHC I binding epitopes.

[0146] As described herein, preferred HTL epitopes for design have a binding affinity (IC) of greater than 500 nM for any MHC II molecule, either measured by biophysical methods or predicted by NetMHCIIpan. 50These are considered to be weak binders. Preferably, these epitopes have an IC value of greater than 50 nM for MHC II molecules, as measured by biophysical methods or predicted by NetMHCIIpan. 50 Bind by value. These are considered to be strong binders.

[0147] T cell epitopes can be incorporated at several positions in non-immunogenic peptides. To achieve this, the specific sequence with T cell epitopes must comply with the rules of MHC binding. The rules of binding to MHC molecules are incorporated into the MHC binding prediction program, which uses advanced algorithms to predict MHC binding peptides.

[0148] There are many different HLA molecules, each with restrictions on the amino acids in the sequence that best bind to it. The binding motifs are summarized in Table 3 of U.S. Patent No. 8,546,337, which is specifically incorporated herein in its entirety by express reference. In this table, positions that may be any amino acid are indicated with an x, and within the brackets is a (list of) amino acids that may only be present at certain positions in the binding motif.

[0149] Generally, in the present invention, the non-immunogenic peptide that comprises T cell epitopes must be hydrophobic so that it can be incorporated into nanoparticles and delivered to endosomes.The non-immunogenic peptide that comprises T cell epitopes that is not hydrophobic can be modified by adding or attaching some amino acids such as Gly, Ala, Val, Leu, Ile, Pro, Phe, Met and Trp to the backbone chain, as is well known to those skilled in the art. Toll-like receptors and TLR signaling

[0150] Toll-like receptors (TLRs) are evolutionarily conserved receptors and homologs of the Drosophila Toll protein that have been found to be important for defense against microbial infections. TLRs recognize highly conserved structural motifs known as pathogen-associated microbial patterns (PAMPs), which are exclusively expressed by pathogenic microorganisms, or damage-associated molecular patterns (DAMPs), which are endogenous molecules released from necrotic or dying cells.

[0151] TLRs include TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, and TLR13, although the last two have not been identified in humans.

[0152] TLRs are expressed in innate immune cells such as dendritic cells (DCs) and macrophages, as well as non-immune cells such as fibroblasts and epithelial cells. Based on their localization, TLRs are broadly classified into two subfamilies: cell surface TLRs and intracellular TLRs. Cell surface TLRs include TLR1, TLR2, TLR4, TLR5, TLR6, and TLR10, while intracellular TLRs are localized in endosomes and include TLR3, TLR7, TLR8, TLR9, TLR11, TLR12, and TLR13.

[0153] Stimulation of TLRs by their corresponding PAMPs or DAMPs initiates a signaling cascade that leads to the activation of transcription factors such as AP-1, NF-κB, and interferon regulatory factors (IRFs). Signaling by TLRs leads to a variety of cellular responses, including the production of interferons (IFNs), proinflammatory cytokines, and effector cytokines that induce adaptive immune responses.

[0154] TLR signaling consists of at least two distinct pathways: a MyD88-dependent pathway that leads to the production of inflammatory cytokines, and a TRIF-dependent pathway that is involved in the stimulation of IFN-β and maturation of dendritic cells. TLR Ligands

[0155] Due to their specificity, TLRs (and other innate immune receptors) cannot easily change over the course of evolution; these receptors recognize molecules that are often associated with threats (e.g., pathogens or cellular stress) and are highly specific for these threats.

[0156] Pathogen-associated molecules that fulfill this requirement are critical to the function of the pathogen and are not easily altered by mutations; they are said to be evolutionarily conserved. Features that are conserved to some degree in pathogens include lipopolysaccharides (LPS), lipoproteins, lipopeptides, and lipoarabinomannans on the cell surface of bacteria; proteins such as flagellin from bacterial flagella; double-stranded RNA of viruses; or unmethylated CpG islands in bacterial and viral DNA; and CpG islands found in promoters of eukaryotic DNA; and certain other RNA and DNA molecules. CpG-A and CpG-B

[0157] Table 1 lists some of the well-known TLRs and their common ligands. Table 1 Commonly known TLRs and their ligands [Table 1-1] [Table 1-2] [Table 1-3]

[0158] The nanoparticles of the present invention are taken up by antigen-presenting cells (APCs), particularly dendritic cells (DCs) or macrophages, which have a wide variety of TLRs. Thus, the ligands in Table 1, and other ligands described herein or elsewhere, may be fashioned into nanoparticles to trigger robust T cell responses. For various diseases to be treated or prevented, TLRs may be selected to form nanoparticles according to the major types of antigen-presenting cells involved in the immune response.

[0159] One or more, preferably two or more, TLR ligands may be included in one nanoparticle. The hydrophobic TLR ligand may be located in the core of the nanoparticle together with the hydrophobic therapeutic peptide, while the negatively charged TLR ligand may be present in the corona of the nanoparticle together with the cationic CPP. It is believed that the electrical interaction of the positively charged CPP with the negatively charged TLR ligand leads to a more stable and dense nanostructure. In one embodiment, two negatively charged TLR ligands are included in the corona of the nanoparticle. In another embodiment, one hydrophobic TLR ligand is included in the core of the nanoparticle and two negatively charged TLR ligands are included in the corona of the nanoparticle. The presence of many types of TLR ligands in the nanoparticles clearly improves the T cell response. The TLR ligands may also be modified to have desired properties, as known to those skilled in the art. Illustrative Definitions

[0160] In accordance with the present invention, polynucleotides, nucleic acid segments, nucleic acid sequences, and the like include, but are not limited to, DNA (including but not limited to genomic or extragenomic DNA), genes, peptide nucleic acids (PNAs), RNA (including but not limited to rRNA, mRNA, and tRNA), nucleosides, and suitable nucleic acid segments obtained from natural sources, chemically synthesized, modified, or otherwise prepared or synthesized in whole or in part by the hand of man.

[0161] Unless otherwise defined, all technical and scientific terms used herein have the meanings that are commonly understood by those skilled in the art to which the present invention belongs.The following references provide those skilled in the art with the general definitions of many terms used in the present invention: Dictionary of Biochemistry and Molecular Biology (2nd Edition), J.Stenesh (ed.), Wiley-Interscience (1989); Dictionary of Microbiology and Molecular Biology (3rd Edition), P.Singleton and D.Sainsbury (ed.), Wiley-Interscience (2007); Chambers Dictionary of Science and Technology (2nd Edition), P.Walker (ed.), Chambers (2007); Glossary of Genetics (5th Edition), R.Rieger et al. (ed.), Springer-Verlag (1991); and The HarperCollins Dictionary of Biology, WGHale and JPMargham (ed.), HarperCollins (1991).

[0162] Although any methods and compositions similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and compositions are described herein. For purposes of the present invention, and for ease of clarity and reference, the following terms are defined below.

[0163] Following long-standing patent law convention, the words "a" and "an" mean "one or more" as used throughout this specification and in the claims.

[0164] The terms "about" and "approximately" as used herein are interchangeable and should generally be understood to refer to a range of numbers around a given number, as well as all numbers within the range of numbers recited (e.g., unless otherwise specified, "about 5 to 15" means "about 5 to about 15"). Additionally, all numerical ranges herein should be understood to include each and every integer within that range.

[0165] "Biocompatible" refers to a material that, when exposed to a living cell, maintains proper cellular activity of the cell without causing undesirable effects in the cell, such as alterations in the cell's life cycle, alterations in the cell's proliferation rate, or cytotoxic effects.

[0166] The term "biologically functional equivalent" is well understood in the art and is further defined herein. Thus, sequences having about 85% to about 90%; or, more preferably, about 91% to about 95%; or, even more preferably, about 96% to about 99% of nucleotides identical or functionally equivalent to one or more nucleotide sequences provided herein are expected to be particularly useful in the practice of the methods and compositions provided herein.

[0167] As used herein, "biomimetic" is intended to mean a synthetic material analogue to a substance that occurs naturally in the human body, which analogue is not rejected by the human body (e.g., does not cause an adverse reaction in the human body).

[0168] As used herein, the term "buffer" includes one or more compositions, or aqueous solutions thereof, that resist changes in pH when an acid or alkali is added to the solution or composition containing the buffer. This resistance to pH change is due to the buffering properties of such a solution and may be a function of one or more specific compounds contained in the composition. Thus, a solution or other composition that exhibits buffering activity is referred to as a buffer or buffer solution. Buffers generally do not have an infinite ability to maintain the pH of a solution or composition, but rather are typically capable of maintaining the pH within a certain range, e.g., a pH of about 5 to 7.

[0169] As used herein, the term "carrier" is intended to include any solvent(s), dispersion medium, coating(s), diluent(s), buffer(s), isotonicity agent(s), solution(s), suspension(s), colloid(s), inert(s), etc., or combinations thereof, that are pharma- ceutically acceptable for administration to the relevant animal. The use of one or more delivery vehicles for chemical compounds in general, and chemotherapeutic agents in particular, is well known to those skilled in the art of medicine. Any conventional media or agent is contemplated for use in diagnostic, prophylactic, and therapeutic compositions, except insofar as it is incompatible with the active ingredient. One or more supplementary active ingredient(s) may also be incorporated into or administered together with one or more chemotherapeutic compositions of the present disclosure.

[0170] As used herein, the term "DNA segment" refers to a DNA molecule that is isolated from and free of contamination by the total genomic DNA of a particular species. Thus, a DNA segment obtained from a biological sample using one of the compositions disclosed herein refers to one or more DNA segments that are isolated or purified from the total genomic DNA of the particular species from which the DNA segment is obtained. The term "DNA segment" includes DNA segments and smaller fragments of such DNA segments, as well as recombinant vectors (including, for example, plasmids, cosmids, phages, viruses, etc.).

[0171] As used herein, the term "effective amount" refers to an amount that can treat or ameliorate a disease or condition, or otherwise produce the intended therapeutic effect.

[0172] As used herein, the terms "for example" or "eg" are not intended to be limiting and are used for illustrative purposes only and should not be construed as referring only to items explicitly listed herein.

[0173] As used herein, "heterologous" sequence is defined in relation to a predefined reference sequence, e.g., a polynucleotide or polypeptide sequence.For example, in relation to a structural gene sequence, a heterologous promoter is defined as a promoter that does not naturally occur adjacent to the referenced structural gene, but is placed by experimental manipulation.Similarly, a heterologous gene or nucleic acid segment is defined as a gene or segment that does not naturally occur adjacent to the referenced promoter and / or enhancer element.

[0174] As used herein, "homologous" when referring to polynucleotides means sequences that have the same basic nucleotide sequence, although originating from different sources. Typically, homologous nucleic acid sequences are derived from closely related genes or organisms with one or more substantially similar genomic sequences. In contrast, "analogous" polynucleotides are polynucleotides that share the same function as polynucleotides from different species or organisms, but may have significantly different primary nucleotide sequences (nucleotide sequences that encode one or more proteins or polypeptides that perform a similar function or have similar biological activity). Often, analogous polynucleotides may be derived from two or more organisms that are not closely related (e.g., genetically or phylogenetically).

[0175] As used herein, the term "homology" refers to the degree of complementarity between two or more polynucleotide or polypeptide sequences. When a first nucleic acid or amino acid sequence has an identical primary sequence to a second nucleic acid or amino acid sequence, the term "identity" can be used instead of the term "homology". Sequence homology and sequence identity can be determined by analyzing two or more sequences using algorithms and computer programs known in the art. Such methods can be used to assess whether a given sequence is identical or homologous to another selected sequence.

[0176] The terms "identical" or percent "identity," in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that are identical, or to two or more sequences or subsequences that have a certain percentage of identical amino acid residues or nucleotides, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (or other algorithms available to those of skill in the art) or by visual observation.

[0177] As used herein, the phrase "in need of treatment" refers to a judgment made by a health care provider, such as a physician or veterinarian, that a patient needs treatment (or will benefit in one or more ways from treatment). Such a judgment may be made based on a variety of factors within the health care provider's area of ​​expertise, which may include knowledge that the patient is ill as a result of a disease condition treatable by one or more compounds or pharmaceutical compositions, such as those defined herein.

[0178] The phrases "isolated" or "biologically pure" refer to material that is substantially or essentially free from components that normally accompany it when found in the natural state.

[0179] As used herein, the term "kit" may be used to refer to a portable, self-contained, multi-compartment containing at least one set of reagents, components, or pharma- ceutical formulated compositions for carrying out one or more assay methods of the invention. Optionally, such kits may also include one or more sets of instructions for using the enclosed reagents (e.g., in a laboratory or clinical application, etc.).

[0180] "Link" or "join" refers to any method known in the art for operatively linking one or more proteins, peptides, nucleic acids, or polynucleotides, including, but not limited to, recombinant fusion, covalent bonding, disulfide bonding, ionic bonding, hydrogen bonding, electrostatic bonding, and the like.

[0181] As used herein, the term "naturally occurring" when applied to an object refers to an object that can be found in nature.For example, a polypeptide or polynucleotide sequence that exists in an organism (including viruses), can be isolated from a natural source, and has not been intentionally modified by human hands in a laboratory is naturally occurring.As used herein, a laboratory strain of rodent that may have been selectively bred according to classical genetics is considered to be a naturally occurring animal.

[0182] As used herein, the term "nucleic acid" includes one or more types of polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), and any other type of polynucleotide that is an N-glycoside of a purine or pyrimidine base, or a modified purine or pyrimidine base (including abasic sites). As used herein, the term "nucleic acid" also includes polymers of ribonucleosides or deoxyribonucleosides, typically linked by phosphodiester bonds between the subunits, but in some cases covalently linked by phosphorothioates, methylphosphonates, etc. "Nucleic acid" includes single- and double-stranded DNA, as well as single- and double-stranded RNA. Exemplary nucleic acids include, but are not limited to, gDNA; hnRNA; mRNA; rRNA, tRNA, microRNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA (snORNA), small nuclear RNA (snRNA), and small transient RNA (stRNA), and the like, and any combination thereof.

[0183] As used herein, the terms "operably linked" and "operably linked" refer to the union of multiple nucleic acid sequences that are linked in such a way that the coding regions are adjacent and in the correct reading frame. Such multiple sequences are typically adjacent or substantially adjacent. However, since enhancers generally function when separated by several kilobases from the promoter, and intron sequences can vary in length, some polynucleotide elements may not be adjacent even though they are operably linked.

[0184] As used herein, the term "patient" (also interchangeably referred to as "host" or "subject") refers to any host that may receive one or more pharmaceutical compositions disclosed herein. Preferably, the subject is a vertebrate, which is intended to mean any animal species (preferably a mammalian species, such as a human). In certain embodiments, "patient" refers to any animal host, including, but not limited to, any mammalian host. Preferably, the term refers to any mammalian host, the latter including, but not limited to, humans and non-human primates, cows, dogs, caprines, caveines, crows, epines, horses, cats, hircines, lapins, leporines, wolves, murines, sheep, pigs, frogs, racines, foxes, etc. (including livestock, zoological specimens, exotic animals, as well as companion animals, pets, and any animals under veterinary care). Patients may be of any age capable of responding to inoculation with the vaccine by developing an immune response. In certain embodiments, the mammalian patient is preferably human.

[0185] The phrase "pharmaceutical acceptable" refers to molecular entities and compositions that preferably do not produce allergic or similar undesirable reactions when administered to mammals, particularly humans.

[0186] As used herein, "pharmaceutical acceptable salt" refers to the salt that preferably retains the desired biological activity of the compound and does not give any undesired toxic effects.Such salts include, but are not limited to, the acid addition salts formed with inorganic acids (e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, etc.); and the salts formed with organic acids (including but not limited to, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid (embonic acid), alginic acid, naphthoic acid, polyglutamic acid, naphthalenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid); for example, the salts with polyvalent metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, etc.; the salts formed with organic cations formed from N,N' dibenzylethylenediamine or ethylenediamine; and combinations thereof.

[0187] As used herein, the term "plasmid" or "vector" refers to a genetic construct composed of genetic material (i.e., nucleic acid). Typically, a plasmid or vector contains an origin of replication that functions in a bacterial host cell (e.g., E. coli) and a selection marker for detecting bacterial host cells that contain the plasmid. The plasmids and vectors of the present invention may contain one or more genetic elements described herein arranged such that the inserted coding sequence can be transcribed and translated in an appropriate expression cell. In addition, a plasmid or vector may contain one or more nucleic acid segments, genes, promoters, enhancers, activators, multiple cloning sites, or any combination thereof (including segments obtained or derived from one or more natural and / or artificial sources).

[0188] As used herein, "polymer" means a chemical compound or mixture of compounds formed by polymerization and contains repeating structural units. Polymers can be configured into a number of forms and compositions or combinations of compositions.

[0189] As used herein, the term "polypeptide" is intended to encompass the singular "polypeptide" as well as the plural "polypeptides", including any chain or chains of two or more amino acids. Thus, as used herein, terms including, but not limited to, "peptide", "dipeptide", "tripeptide", "protein", "enzyme", "amino acid chain", and "contiguous amino acid sequence" are all included within the definition of "polypeptide", and the term "polypeptide" can be used in place of or interchangeably with any of these terms. The term also further includes polypeptides that have undergone one or more post-translational modification(s), including, for example, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization, proteolytic cleavage, post-translational processing, or modification by incorporation of one or more non-naturally occurring amino acids. Conventional nomenclature exists in the art for polynucleotide and polypeptide structures.

[0190] For example, one-letter and three-letter abbreviations are widely used to describe amino acids: alanine (A; Ala), arginine (R; Arg), asparagine (N; Asn), aspartic acid (D; Asp), cysteine ​​(C; Cys), glutamine (Q; Gin), glutamic acid (E; Glu), glycine (G; Gly), histidine (H; His), isoleucine (I; Ile), leucine (L; Leu), methionine (M; Met), phenylalanine (F; Phe), proline (P; Pro), serine (S; Ser), threonine (T; Thr), tryptophan (W; Trp), tyrosine (Y; Tyr), valine (V; Val), and lysine (K; Lys). The amino acid residues described herein are preferably present in the "L" isomer. However, residues in the "D" isomeric form can be substituted for any L-amino acid residue, as long as the desired properties of the polypeptide are retained.

[0191] As used herein, the terms "prevent", "preventing", "prevention", "inhibit", "inhibiting", and "inhibition" as used herein refer to the administration of a compound, alone or in a pharmaceutical composition, prior to the onset of clinical symptoms of a disease state, such that any symptom, aspect, or characteristic of the disease state is prevented. Such prevention and inhibition need not be absolute to be considered medically useful.

[0192] "Protein" is used interchangeably herein with "peptide" and "polypeptide" and includes both synthetically, recombinantly, or in vitro produced peptides and polypeptides, as well as peptides and polypeptides expressed in vivo following administration of a nucleic acid sequence to a host animal or human subject. The term "polypeptide" is preferably intended to refer to any amino acid chain length, including short peptide polypeptides of about 2 to about 20 amino acid residues in length, oligopeptides of about 10 to about 100 amino acid residues in length, and longer polypeptides of about 100 amino acid residues or more in length. Additionally, the term is also intended to include enzymes, i.e., functional biomolecules that include at least one amino acid polymer. The polypeptides and proteins of the present invention also include polypeptides and proteins that are or have been post-translationally modified, and also include any sugar or other derivative(s) or conjugate(s) that are added to the backbone amino acid chain.

[0193] As used herein, "purified" means separated from many other compounds or entities. A compound or entity may be partially purified, substantially purified, or pure. A compound or entity is considered pure when it is removed from substantially all other compounds or entities, i.e., it is preferably at least about 90% pure, more preferably at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more than 99% pure. A partially or substantially purified compound or entity may be removed from at least 50%, at least 60%, at least 70%, or at least 80% of the materials with which it is naturally found, e.g., cellular materials such as cellular proteins and / or nucleic acids.

[0194] The term "recombinant" refers to a material (e.g., a polynucleotide or polypeptide) that has been artificially or synthetically (non-naturally) modified by human intervention. Modifications may be made to materials in their natural environment or state, or to materials removed from their natural environment or state. Specifically, for example, a promoter sequence is "recombinant" when produced by expression of a nucleic acid segment that has been manipulated by the hand of man. For example, a "recombinant nucleic acid" is a nucleic acid that is produced by recombining nucleic acids, for example, during cloning, DNA shuffling, or other procedures, or by chemical or other mutagenesis; a "recombinant polypeptide" or "recombinant protein" is a polypeptide or protein produced by expression of a recombinant nucleic acid; and a "recombinant virus", e.g., a recombinant AAV virus, is produced by expression of a recombinant nucleic acid.

[0195] As used herein, the term "regulatory element" refers to a region or regions of a nucleic acid sequence that regulate transcription. Exemplary regulatory elements include, but are not limited to, enhancers, post-transcriptional elements, transcriptional control sequences, and the like.

[0196] The term "RNA segment" refers to an isolated RNA molecule that is free of total cellular RNA of a particular species. Thus, an RNA segment may refer to one or more RNA segments (natural or synthetic in origin) that have been isolated or purified from other RNA. The term "RNA segment" includes RNA segments and smaller fragments of such segments.

[0197] The term "a sequence essentially as set forth in SEQ ID NO:X" means that the sequence corresponds substantially to a portion of SEQ ID NO:X and contains a relatively small number of nucleotides (amino acids) that are not identical to the nucleotides (or amino acids, in the case of a polypeptide sequence) of SEQ ID NO:X, or is biologically functionally equivalent to the nucleotides (amino acids) of SEQ ID NO:X. The term "biologically functionally equivalent" is well understood in the art and is further defined in detail herein. Thus, sequences having about 85% to about 90%; more preferably about 91% to about 95%; or even more preferably about 96% to about 99% of the nucleotides that are identical or functionally equivalent to one or more nucleotide sequences provided herein are expected to be particularly useful in the practice of the invention.

[0198] Suitable standard hybridization conditions for nucleic acids for use in the present invention include, for example, hybridization in 50% formamide, 5x Denhardt's solution, 5x SSC, 25 mM sodium phosphate, 0.1% SDS, and 100 μg / mL denatured salmon sperm DNA at 42° C. for 16 hours, followed by sequential washing with 0.1x SSC, 0.1% SDS solution at 60° C. for 1 hour to remove a desired amount of background signal. Lower stringency hybridization conditions for the present invention include, for example, hybridization in 35% formamide, 5x Denhardt's solution, 5x SSC, 25 mM sodium phosphate, 0.1% SDS, and 100 μg / mL denatured salmon sperm DNA or E. coli DNA at 42° C. for 16 hours, followed by sequential washing with 0.8x SSC, 0.1% SDS at 55° C. It will be understood by one of skill in the art that such hybridization conditions may be readily adjusted to achieve the desired level of stringency for a particular application.

[0199] As used herein, the term "structural gene" is intended to generally describe a polynucleotide (e.g., a gene) that is expressed to produce an encoded peptide, polypeptide, protein, ribozyme, catalytic RNA molecule, or antisense molecule.

[0200] As used herein, the term "subject" describes an organism (including mammals such as primates) that can be provided with treatment by the composition according to the present invention. Mammalian species that can benefit from the treatment method of the present disclosure include, but are not limited to, apes; chimpanzees; orangutans; humans; monkeys; domesticated animals such as dogs and cats; livestock animals such as horses, cows, pigs, sheep, goats, and chickens; and other animals such as mice, rats, guinea pigs, and hamsters.

[0201] The term "substantially complementary", when used to define either an amino acid or a nucleic acid sequence, means that a particular subject sequence (e.g., an oligonucleotide sequence) is substantially complementary to all or a portion of a selected sequence and therefore specifically binds to a portion of the mRNA encoding the selected sequence. Thus, typically, the sequence is highly complementary to the mRNA "target" sequence, with no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 base mismatches throughout the complementary portion of the sequence. In many instances, it may be desirable for the sequence to be a perfect match, i.e., completely complementary to the sequence to which the oligonucleotide specifically binds, and thus with zero mismatches along the complementary stretch. Thus, highly complementary sequences typically bind very specifically to the target sequence region of the mRNA, and are therefore highly efficient in reducing and / or even inhibiting the translation of the target mRNA sequence into a polypeptide product.

[0202] A substantially complementary nucleic acid sequence has a complementarity (or "percent perfect match") of greater than about 80 percent to the corresponding nucleic acid target sequence to which the nucleic acid specifically binds, and more preferably, has a complementarity of greater than about 85 percent to the corresponding target sequence to which the nucleic acid specifically binds. In certain embodiments, as described above, it is desirable to have even more substantially complementary nucleic acid sequences for use in the practice of the present invention, and in such instances, the nucleic acid sequence may have a complementarity of greater than about 90 percent to the corresponding target sequence to which the nucleic acid specifically binds, and in certain embodiments, may have a complementarity of greater than about 95 percent to the corresponding target sequence to which the nucleic acid specifically binds, or may even have a complementarity of up to (inclusive of) about 96%, about 97%, about 98%, about 99%, or even about 100% perfect match to the whole or part of the target sequence to which the designed nucleic acid specifically binds.

[0203] The percent similarity or percent complementarity of any of the nucleic acid sequences of the present disclosure can be determined by comparing sequence information, for example, using the GAP computer program, version 6.0, available from University of Wisconsin Genetics Computer Group (UWGCG). The GAP program utilizes the alignment method of Needleman and Wunsch (1970). Briefly, the GAP program defines similarity as the number of aligned similar symbols (i.e., nucleotides or amino acids) divided by the total number of symbols in the shorter sequence of the two sequences. Preferred initial parameters for the GAP program include: (1) a unary comparison matrix (containing a value of 1 for match and 0 for non-match) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess (1986); (2) a penalty of 3.0 for each gap, and an additional penalty of 0.10 for each symbol in each gap; and (3) no penalty for terminal gaps.

[0204] As used herein, the terms "substantially free" or "essentially free" in reference to the amount of a component preferably refer to a composition containing less than about 10 weight percent of a compound, preferably less than about 5 weight percent, more preferably less than about 1 weight percent. In preferred embodiments, these terms refer to less than about 0.5 weight percent, less than about 0.1 weight percent, or less than about 0.01 weight percent.

[0205] As used herein, the term "structural gene" is intended to generally describe a polynucleotide (e.g., a gene) that is expressed to produce an encoded peptide, polypeptide, protein, ribozyme, catalytic RNA molecule, or antisense molecule.

[0206] As used herein, the term "subject" describes an organism (including mammals such as primates) that can be provided with treatment by the composition according to the present invention. Mammalian species that can benefit from the treatment method of the present disclosure include, but are not limited to, humans, non-human primates (such as apes; chimpanzees; monkeys, and orangutans), domesticated animals (including dogs and cats), and livestock such as horses, cows, pigs, sheep, and goats, or other mammalian species (including, but are not limited to, mice, rats, guinea pigs, rabbits, hamsters), etc.

[0207] As used herein, the terms "substantially corresponding", "substantially homologous" or "substantial identity" refer to a characteristic of a nucleic acid or amino acid sequence, where a selected nucleic acid sequence or a selected amino acid sequence has at least about 70 or about 75 percent sequence identity compared to a selected reference nucleic acid or amino acid sequence. More typically, the selected sequence and the reference sequence have at least about 76, 77, 78, 79, 80, 81, 82, 83, 84, or even 85 percent sequence identity, more preferably at least about 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 percent sequence identity. More preferably, more homologous sequences often share at least about 96, 97, 98, or more than 99 percent sequence identity between the selected sequence and the reference sequence to which it is compared.

[0208] As used herein, "synthetic" is intended to mean that the material is not of human or animal origin.

[0209] A "targeting moiety" is any agent that can facilitate targeting of a particle to a specific site. For example, a targeting moiety can be a chemical targeting moiety, a physical targeting moiety, a geometric targeting moiety, or a combination thereof. A chemical targeting moiety can be a chemical group or molecule on the surface of the particle; a physical targeting moiety can be a specific physical property of the particle, such as the hydrophobicity of the surface; a geometric targeting moiety includes the size and shape of the particle. In addition, a chemical targeting moiety can be a dendrimer, an antibody, an aptamer (which can be a thioaptamer), a ligand, an antibody, or a biomolecule that binds to a specific receptor on the targeting site. A physical targeting moiety can be a surface charge. The charge can be introduced during the manufacture of the particle using a chemical treatment, such as a specific wash. For example, immersing a porous silica or silicon oxide surface in water can result in a negative charge on the surface. A surface charge can also be imparted by an additional layer on the particle surface, or by chemical chains (such as polymer chains). For example, polyethylene glycol chains can be the source of the negative charge on the surface. The polyethylene glycol chains may be coated or covalently attached to the surface using methods known to those skilled in the art.

[0210] The term "therapeutically effective period" refers to the period of time necessary for one or more active agents to be therapeutically effective. The term "therapeutically effective" refers to reducing the severity and / or frequency of one or more symptoms, eliminating one or more symptoms and / or their underlying causes, preventing the onset of symptoms and / or their underlying causes, and ameliorating or reducing damage.

[0211] A "therapeutic agent" may be any physiologically or pharmacologically active substance that can produce a desired biological effect at a target site in a subject. Therapeutic agents may be chemotherapeutic agents, immunosuppressants, cytokines, cytotoxic agents, nucleolytic compounds, radioisotopes, receptors, and prodrug-activating enzymes, which may be natural, synthetic, or recombinantly produced, or combinations thereof. Drugs affected by classical multidrug resistance, such as vinca alkaloids (e.g., vinblastine and vincristine), anthracyclines (e.g., doxorubicin and daunorubicin), RNA transcription inhibitors (e.g., actinomycin D), and microtubule stabilizing agents (e.g., paclitaxel), may have particular utility as therapeutic agents. Cytokines may also be used as therapeutic agents. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Cancer chemotherapeutic agents may be preferred therapeutic agents. For a more detailed description of anti-cancer and other therapeutic agents, those of skill in the art are referred to any number of texts, including, but not limited to, the Physician's Desk Reference, and Hardman and Limbird (2001).

[0212] As used herein, "transcription factor recognition site" and "transcription factor binding site" refer to a polynucleotide sequence(s) or sequence motif(s) that are identified as sites for sequence-specific interaction of one or more transcription factors, often in the form of direct protein-DNA binding. Typically, transcription factor binding sites can be identified by DNA footprinting, gel mobility shift assays, etc., and / or predicted based on known consensus sequence motifs, or by other methods known to those skilled in the art.

[0213] "Transcriptional regulatory element" refers to a polynucleotide sequence that alone or in combination with one or more other nucleic acid sequences activates transcription. Transcriptional regulatory elements may include, for example, one or more promoters, one or more response elements, one or more negative regulatory elements, and / or one or more enhancers.

[0214] "Transcription unit" refers to a polynucleotide sequence comprising at least a first structural gene operably linked to at least a first cis-acting promoter sequence, and optionally, one or more other cis-acting nucleic acid sequences necessary for efficient transcription of the structural gene sequence, and at least a first distal regulatory element that may be required for proper tissue-specific developmental transcription of the structural gene sequence operably placed under the control of the promoter and / or enhancer element, as well as any additional cis sequences required for efficient transcription and translation (e.g., polyadenylation site(s), mRNA stability control sequence(s), etc.).

[0215] As used herein, the term "transformation" is intended to generally describe the process of introducing an exogenous polynucleotide sequence (e.g., a viral vector, a plasmid, or a recombinant DNA or RNA molecule) into a host cell or protoplast, where the exogenous polynucleotide is integrated into at least a first chromosome or is capable of autonomously replicating within the transformed host cell. Transfection, electroporation, and uptake of "naked" nucleic acid all represent examples of techniques used to transform a host cell with one or more polynucleotides.

[0216] As used herein, the term "transformed cell" is intended to mean a host cell whose nucleic acid complement has been altered by the introduction of one or more exogenous polynucleotides into the cell.

[0217] As used herein, "treating" or "treatment of" refers to providing any type of medical or surgical management to a subject. Treating may include, but is not limited to, administering a composition containing a therapeutic agent to a subject. "Treatment" includes any administration or application of a compound or composition of the present invention to a subject for the purpose of curing, reversing, alleviating, reducing the severity of, inhibiting the progression of, or reducing the likelihood of a disease, disorder, or condition, or one or more symptoms or signs of a disease, disorder, or condition. In certain embodiments, the compositions of the present invention may also be administered prophylactically before any symptoms or signs of a condition occur, where prevention is warranted. Typically, in such cases, the subject is one who has been diagnosed as "at risk" of developing such a disease or disorder, either as a result of family history, medical records, or one or more diagnostic or prognostic tests that suggest a propensity for subsequent development of such a disease or disorder.

[0218] As used herein, the term "vector" refers to a nucleic acid molecule (typically composed of DNA) capable of replicating in a host cell and / or to which another nucleic acid segment may be operatively linked so as to bring about the replication of the linked segment. Plasmids, cosmids, or viruses are exemplary vectors.

[0219] In certain embodiments, one or more nucleic acid segments of the present invention are advantageously utilized in combination with a suitable detectable marker (i.e., "label"), such as in the case of utilizing labeled polynucleotide probes when determining the presence of a given target sequence in a hybridization assay. A wide variety of suitable indicator compounds and compositions for labeling oligonucleotide probes are known in the art, including, but not limited to, fluorescent, radioactive, enzymatic, or other ligands (e.g., avidin / biotin), which can be detected in a suitable assay. In certain embodiments, one or more fluorescent labels or enzyme tags, such as urease, alkaline phosphatase, or peroxidase, may also be utilized in place of radioactive or other less environmentally desirable reagents. In the case of enzyme tags, it is known that colorimetric, chromogenic, or fluorogenic indicator substrates can be utilized to provide a method of detection of samples visible to the human eye, or can be utilized in analytical methods, such as scintigraphy, fluorimetry, spectrophotometry, etc., to identify specific hybridization with samples containing one or more complementary or substantially complementary nucleic acid sequences. In the case of so-called "multiplexed" assays (where two or more labeled probes are detected simultaneously or sequentially), it may be desirable to label a first oligonucleotide probe with a first label having a first detection property or parameter (e.g., emission and / or excitation spectral maximum) and to further label a second oligonucleotide probe with a second oligonucleotide probe having a second detection property or parameter that is different (i.e., discrete or distinguishable) from the first label. The use of multiplexed assays is well known to those skilled in the art of molecular genetics, particularly in the context of gene amplification / detection protocols. biological functional equivalence

[0220] It is possible to modify and change the structure of nucleic acid, or vector containing nucleic acid, and mRNA, polypeptide, or therapeutic agent encoded therein, and still obtain a functional system containing one or more therapeutic agents with desired properties.As mentioned above, it is often desirable to introduce one or more mutations into a particular polynucleotide sequence.In certain circumstances, the resulting encoded polypeptide sequence is altered by this mutation, or in other cases, the sequence of this polypeptide is unchanged by one or more mutations in the encoding polynucleotide.

[0221] Where it is desirable to alter the amino acid sequence of a polypeptide to produce an equivalent, or even improved, second generation molecule, this can be accomplished by altering one or more codons in the encoding DNA sequence in accordance with Table 2.

[0222] For example, certain amino acids can be substituted for other amino acids in a protein structure without significantly impairing its interactive binding ability to structures such as, for example, the antigen-binding region of an antibody or the binding site on a substrate molecule. Because it is the interactive ability and properties of a protein that define its biological functional activity, it is possible to make certain amino acid sequence substitutions in a protein sequence (and, of course, in the DNA coding sequence on which it is based) and still obtain a protein with similar properties. Thus, the inventors contemplate that various changes may be made to the peptide sequences of the compositions of the present disclosure, or the corresponding DNA sequences encoding the peptides, without significantly impairing their biological usefulness or activity.

[0223] Table 2 [Table 2]

[0224] When making such changes, the hydropathic index of amino acids may be taken into consideration. The importance of the hydropathic amino acid index in imparting interactive biological function to proteins is generally recognized in the art (Kyte and Doolittle, 1982, incorporated herein by reference). It is accepted that the relative hydropathic properties of amino acids contribute to the secondary structure of a resulting protein, which in turn determines the interaction of the protein with other molecules (e.g., enzymes, substrates, receptors, DNA, antibodies, antigens, etc.). Each amino acid is assigned a hydropathic index based on its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982). The values ​​are as follows: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0225] It is known in the art that certain amino acids can be replaced by other amino acids with similar hydropathic index or score and still result in proteins with similar biological activity, i.e., proteins with equivalent biological functions can still be obtained. When making such changes, substitution of amino acids with hydropathic index within ±2 is preferred, substitution within ±1 is particularly preferred, and substitution within ±0.5 is especially preferred. It is also understood in the art that substitution of similar amino acids can be effectively made based on hydrophilicity. U.S. Patent No. 4,554,101 (specifically incorporated herein in its entirety by express reference) states that the maximum value of the local average hydrophilicity of a protein (determined by the hydrophilicity of adjacent amino acids) correlates with the biological properties of the protein.

[0226] As detailed in U.S. Patent No. 4,554,101, the following hydrophilicity values ​​are assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0±1); glutamic acid (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that one amino acid may be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent, protein. In the case of such changes, substitution of amino acids whose hydrophilicity values ​​are within ±2 is preferred, particularly within ±1, and particularly within ±0.5.

[0227] Thus, as outlined above, amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, e.g., hydrophobicity, hydrophilicity, charge, size, etc. Examples of substitutions that take into account one or more of the above characteristics are well known to those of skill in the art and include arginine for lysine, glutamic acid for aspartic acid, serine for threonine, glutamine for asparagine, and valine, leucine for isoleucine.

[0228] Section headings used throughout are for organizational purposes only and should not be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including but not limited to patents, patent applications, articles, books, and papers, are expressly incorporated herein in their entirety by express reference. In the event that one or more of the incorporated documents or similar materials defines a term in a manner that contradicts the definition of the term in this application, this application controls. EXAMPLES

[0229] The following examples are included to illustrate illustrative embodiments of the present invention. Those skilled in the art should understand that the techniques disclosed in these examples are techniques that have been found to work well in the practice of the present invention, and therefore can be considered to constitute preferred modes for the practice of the present invention. However, those skilled in the art should understand, in light of this disclosure, that many changes can be made to the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the present invention. Example 1

[0230] Although many clinical trials have been reported using NY-ESO-1 peptide and Montanide ISA-51 plus CpG or poly(I:C), the clinical and immune responses are generally weak or moderate. One plausible reason is that NY-ESO-1 peptide and TLR ligands [CpG or poly(I:C)] do not interact with each other to form complexes or particles, which results in a weak immune response. Previously, it was demonstrated that DC / TAT-TRP-2 vaccine could generate strong protective (but not therapeutic) immunity (Wang et al., 2002). In a phase 1 clinical trial, TAT-ESO-1 peptide was mixed with Montanide ISA-51 for the vaccine, and the vaccine only generated a weak T cell response. During the course of this study, we found two potential problems: 1) TAT-ESO-1 could not form a stable complex with Montanide ISA-51, possibly due to the positive charge at the N-terminus; 2) TAT-ESO-1 formed a precipitate when mixed with CpG. Based on the charge and hydrophobicity, we designed and developed a novel technique of self-assembling CPP-T cell peptide nanoparticles (PEP-NANO) with TLR ligands [CpG, MPLA, and poly(I:C) (abbreviated as CMI)], as shown diagrammatically in Figure 2A and Figure 2B. Amphiphobic or amphiphilic CPP therapeutic peptides, consisting of a CPP such as TAT, which has a positively charged peptide and is covalently linked to a therapeutic peptide (generally hydrophobic) such as NY-ESO-1 (SLLMWITQCFLPV) (SEQ ID NO: 1) and TRP-2 (SYVDFFVWL) (SEQ ID NO: 2), form nanoparticles with negatively charged CpG and / or poly(I:C) on the one hand through electrical interactions and with MPLA on the other hand through the hydrophobicity of the particle interior. Example 2 Self-assembly and characterization of TAT-TRP2 peptide nanoparticles with TLR ligands or their combinations

[0231] TAT-TRP2, TAT-ESO-1, and CpG were first completely dissolved in sterile ultrapure water at a concentration of 10 mg / mL as stock solutions. MPLA and poly I:C were completely dissolved in sterile ultrapure water at a concentration of 1 mg / mL. To prepare TAT-peptide vaccine nanoparticles, the specified volumes of CpG, MPLA, and poly I:C (shown in Table 3) were thoroughly mixed by vigorous vortexing in ultrapure water or buffer.

[0232] Table 3 TLR Ligand Concentrations and Combinations [Table 3]

[0233] Self-assembly of TAT-peptide nanoparticles was triggered by dropwise addition of TAT-peptide within 1 min while sonicating in an ice-cold water bath. TAT-TRP2 exhibits hydrophilic / positive charge and hydrophobic properties and promotes electrostatic interactions with CpG ODN. The hydrophobic C-terminus of TRP2 peptide forms a hydrophobic core with MPLA through hydrophobic interactions. Electrostatic and hydrophobic interactions drive the self-assembly of TAT-TRP2 and double or triple TLR agonists to form spherical TAT-TRP2-CM nanoparticles as shown by AFM analysis of both height and DMT modulus distribution across a representative cross section (red line). In contrast, TRP2 peptide without TAT modification failed to complex with CpG-MPLA and formed long fibers.

[0234] To formulate stable and highly effective peptide vaccine nanoparticles, we optimized the formulation using various different ratios of TAT-TRP2 peptide:TLR ligands as shown in Table 3, and analyzed the size of each nanoparticle (80-150 nm) of TAT-TRP2 and different combinations of TLR ligands (Figure 4). Grey bars indicate unstable / polydisperse complexes with large PDI (PDI>0.5). Example 3 Zeta potential of CPP peptide nanoparticles

[0235] To determine the surface charge of the nanoparticles, the zeta potential was measured for complexes composed of different nitrogen (+) to phosphate (-) (N / P) ratios (N = nitrogen from amino acid residues; P = CpG ODN phosphate group). The zeta potential of TAT-TRP2-CM nanoparticles varied with different N / P ratios (Figure 5). For the following studies, condition #2 was applied for TAT-TRP2-CM and condition #18 for TAT-TRP2-CMI (Table 3 and Figure 4). Figures 6A and 6B show the nanoparticle size of TAT-TRP2-CM and the zeta potential of TAT-TRP2, CpG, MPLA, and TAT-TRP2-CM. Similar results were obtained using TAT-ESO-1 with CpG and MPLA (CM), and CpG, MPLA and poly(I:C) (CMI) (Figures 7A, 7B, 7C, 7D, 7E, and 7F). Example 4 Both the formation and size of CPP peptide nanoparticles are pH dependent

[0236] It was demonstrated that the nanoparticles and zeta potential of TAT-TRP2-CM at pH 7.0 were destroyed and changed at pH 4.0 (Figures 8A, 8B, and 8C).

[0237] To further characterize the self-assembly and nanoparticle size at different pHs from pH 4 to 7, we found that the self-assembly and nanoparticles were disrupted at different pH values. The nanoparticles of TAT-TRP2-CM were dense and 100 nm in size at pH 7.0, but increased in size at pH 6.0 and decreased in size at pH 5.0. The nanoparticles of TAT-TRP2-CM were completely disrupted and separated (Figure 9A). Based on these results, we inferred that the TAT-TRP2-CM nanoparticles were phagocytosed by APCs into endosomes / lysosomes, where the nanoparticles were disrupted at pH 4 to 5. Acidification in the endosomal / lysosomal compartment increases the positive charge of TAT-TRP2 and neutralizes the CpG ODN. There, TAT-TRP2 peptides are released into the cytoplasm and presented by MHC class I or II molecules in the ER, while TLR ligands bind to TLRs and trigger innate immune responses and cytokine production, which enhances antigen presentation and T cell activation (Figure 9B). In contrast, TAT-TRP2 alone enters APCs by cell permeability and is presented to T cells by APCs, without innate immune responses and cytokine production. This pH-dependent property was further shown to be true for TAT-TRP2-CM, TAT-TRP2-CMI, TAT-ESO-l-CM, and TAT-ESO-l-CMI (Figure 9C). Example 5 CPP peptide nanoparticles trigger innate immune responses and cytokine production with different TLR ligand combinations

[0238] To identify the optimal TLR ligand combinations that stimulate innate immune responses, we freshly isolated bone marrow-derived DCs and then treated them with different TLR ligands (alone), double combinations, or triple combinations (Figure 9A, Figure 9B, Figure 9C, and Figure 9D). After treatment with different TLR ligands or their combinations, cytokine (TNF-α, IL-6, IFN-α, and IFN-β) production was determined by ELISA of cell supernatants. Poly(I:C) / CpG, CpG / MPLA double combinations, and CpG / Poly(I:C) / MPLA triple combinations were found to be superior to other groups in triggering innate immune cytokine production. CpG / Poly(I:C) / MPLA triple combination was the strongest activator for inducing cytokine production (Figure 10). Example 6 Formation and size of CPP peptide nanoparticles are pH dependent

[0239] Although many clinical trials have been reported using NY-ESO-1 peptide and Montanide ISA-51 plus CpG or poly(I:C), the clinical and immune responses are generally weak or moderate. One plausible reason is that NY-ESO-1 peptide and TLR ligands [CpG or poly(I:C)] do not interact with each other to form complexes or particles, which results in a weak immune response. Previously, it was demonstrated that DC / TAT-TRP-2 vaccines could generate strong protective (but not therapeutic) immunity (Wang et al., 2002). In a phase 1 clinical trial, TAT-ESO-1 peptide was mixed with Montanide ISA-51 for vaccination, and this vaccine only generated a weak T cell response. Overall, mouse and human clinical trials have shown that current vaccine approaches do not result in strong immune and clinical responses. The major problem is that the cancer antigen peptide / protein and the TLR ligand are not co-delivered to the same APCs. In many cases, only one TLR ligand is used, rather than two or three ligands being co-delivered. In the following examples, we demonstrate that SAPEP-NANO technology, which utilizes amphipathic CPP therapeutic peptides such as TAT-TRP2 or TAT-ESO-1 to form nanoparticles with negatively charged CpG and / or poly(I:C) on the one hand through electrical interactions and MPLA on the other hand through hydrophobicity, can generate strong anti-tumor immunity in mouse models. Example 7 Generation of robust antitumor immunity by DCs loaded with TAT-TRP2 and TLR ligand nanoparticles

[0240] To enhance antitumor immunity, we hypothesized that TAT-TRP-2 peptide would form a complex with TLR ligands such as CpG and MPLA due to its physical properties (positive / negative charge, hydrophilicity, and hydrophobicity) to induce therapeutic immunity. To test this prediction, we used the B16 mouse model and tyrosinase-related protein-2 (TRP-2) peptide as an experimental system. TAT-TRP-2 (YGRKKRRQRRRSYVDFFVWL) (SEQ ID NO: 17) peptide formed a tight complex with CpG / MPLA (TAT-TRP2-CM), but TRP2 peptide could not form a complex with CpG / MPLA (TRP2-CM) (Figure 3A-1, Figure 3A-2, Figure 3B-1, Figure 3B-2, Figure 3C-1, and Figure 3C-2). DCs loaded with TAT-TRP2-CM or TRP2-CM were prepared and injected intravenously into B16 tumor-bearing mice. Sixteen days later, lung metastasis was examined in these treated mice, and it was confirmed that DC / TAT-TRP2-CM significantly suppressed the number of lung metastases, whereas DC / TRP2-CM failed to suppress the number of lung metastases compared to the DC / β-gal-CM control group (Figure 11).

[0241] Multi-stage vehicle (MSV) nanotechnology has recently been shown to be capable of loading peptides, CpG, and MPLA into silicon particles and inducing a strong immune response against B16 tumor cells (Zhu et al., 2018). To compare DC / MSV and DC / PEP-NANO vaccines for their ability to induce antitumor immunity and survival, we prepared DC / control peptide (group #1), DC / TRP2 / CpG / MPLA (#2), DC / TAT-TRP-2 / CpG / MPLA (group #3), DC / MSV-TRP2 / CpG / MPLA (#4), and DC / MSV-TAT-TRP-2 / CpG / MPLA (#5) and injected them into tumor-bearing mice (Figure 12A). Apparently, DC / TAT-TRP2 / CpG / MPLA group (#3) and DC / MSV-TAT-TRP2 / CpG / MPLA group (#5) could induce stronger therapeutic immunity and suppress B16 lung metastasis independent of MSV compared with DC / TRP-2 / CpG / MPLA (#2) and DC / MSV / TRP-2 / CpG / MPLA (#4) (Fig. 12B), suggesting that TAT sequence, not MSV, is crucially required to generate the strongest immune response. More importantly, we showed that mice immunized with DC / TAT-TRP-2 / CpG / MPLA survived much longer than DC / MSV-TAT-TRP-2 / CpG / MPLA group (all mice died within 35 days after B16 tumor injection) (Fig. 12C). The other vaccine groups (DC / β-Gal / CpG / MPLA, DC / TRP2 / CpG / MPLA, and DC / MSV / TRP-2 / CpG / MPLA) died within 25 days after B16 vaccination. These results suggested that the TAT-TRP-2 / CpG / MPLA vaccine was the best among the different vaccine groups tested. Example 8 Generation of robust antitumor immunity by DCs loaded with TAT-ESO-1 and TLR ligand nanoparticles

[0242] To confirm whether DCs loaded with TAT-ESO-l-CM or TAT-ESO-CMI nanoparticles could induce potent antitumor immunity against RM1 / A2-ESO-1 tumor cells, experiments were performed with vaccination of DCs / control peptide, DCs / TAT-ESO-CM, or DCs / TAT-ESO-CMI. Tumor growth was monitored every 2 days. It was observed that DCs / TAT-ESO-CM vaccination significantly suppressed RM1 / HLA-A2-NY-ESO-1 tumor growth compared to the control group (Figures 13A and 13B). Importantly, DCs / TAT-ESO-CMI showed significantly stronger immunity than DCs / TAT-ESO-CM vaccine (Figures 13A and 13B). Further analysis of immune cell responses confirmed that the antigen-specific responses in DCs / TAT-ESO-CMI were superior to those in DCs / TAT-ESO-CM (Figures 14A and 14B). These results suggested that both the DC / TAT-ESO-CM and DC / TAT-ESO-CMI vaccines elicited potent antitumor immunity.

[0243] To further demonstrate whether DC / TAT-ESO-CM can induce therapeutic antitumor immunity in other tumor models, breast cancer E0771 / A2-ESO tumor cells were used as a tumor model. DC / TAT-ESO-CM vaccination was shown to completely suppress tumor growth compared to the control (Figure 15). Example 9 Direct immunization with TAT-ESO-l / TLR nanoparticles without DCs induces robust therapeutic antitumor immunity

[0244] Most studies for vaccines use antigen peptide-loaded DCs, MSV particles, or a combination of CPP peptides and TLR nanoparticles, but such processes are complicated and very labor-intensive, especially for clinical trials. Therefore, we hypothesized whether CPP peptide / TLR nanoparticles could be directly used for vaccination to generate strong antitumor immunity. To test this possibility, tumor-bearing mice were immunized three times with TAT-ESO-CMI and compared with mice immunized once with DC / TAT-ESO-CMI. The results are shown in Figure 16A. Direct vaccination with TAT-ESO-CMI significantly suppressed tumor growth, and the extent was much higher than that of DC / TAT-ESO-CMI (Figures 16B and 16C). In contrast, rapid tumor growth was observed in the control group, as expected. Example 10 TAT-CT83 peptide vaccine

[0245] CT83 (also known as CXORF61 and KKLC1) has been shown to be highly expressed in human lung and breast cancer (Figures S17A-S17D, S18A, and S18B), consistent with previous reports (Fukuyama et al., 2006; Paret et al., 2015). Thus, CT83 is expected to serve as an immune target for cancer vaccines and immunotherapy.

[0246] To test this possibility, a series of TAT-conjugated CT83 peptides containing potential HLA-A2 binding motifs were synthesized (Table 4). Using in vivo immunization of HLA-A2 transgenic mice, it was shown that self-assembled TAT-CT83 peptide nanoparticles containing CMIs generated strong T cell responses against the CT83-A2 peptide (Figures 19A, 19B, 19C, 19D, and 20A-D). HLA-DR13 and HLA-DP4 restricted T cells were generated after in vitro peptide stimulation.

[0247] To confirm whether TAT-CT83-CMI could generate strong anti-tumor immunity, TAT-CT83 peptide vaccine was prepared by mixing 100 μg of TAT-CT83 peptide mixture (including equal amounts of TAT-CT83-A2-1, -5, -6, and -7, see Table 4 below), 20 μg of CpG, 4 μg of MPLA, and 10 μg of poly(I:C) for each mouse under sonication. The experimental design using HLA-A2 transgenic mice is shown in Figure 20A. It was shown that TAT-CT83-CMI vaccine could strongly induce strong anti-tumor immunity against mouse breast cancer E0771 / A2 / CT83 cells (Figure 20B and Figure 20C). Moreover, such anti-tumor immunity could be further enhanced by anti-PD-1 blockade therapy (Figure 20B and Figure 20C). Importantly, vaccine-induced T cells infiltrated into tumor tissue compared to unvaccinated mice (Figure 20D).

[0248] Table 4 TAT-binding CT83 peptide [Table 4] 1. TAT-ESO-CMI generates potent antitumor immunity in breast cancer

[0249] Similarly, experiments were performed in HLA-A2 Tg mice using E0771 / A2-ESO breast cancer cells. A single vaccination with DC / TAT-ESO-CMI was performed after tumor injection (1 × 10 6 Ten days after vaccination (cells / mouse), complete inhibition of tumor growth was confirmed compared to the control treatment group (Figures 21A, 21B, and 21C). Furthermore, vaccination with TAT-ESO-CMI without DCs was shown to generate strong antitumor immunity and inhibit tumor cell growth in a therapeutic model (Figure 21D). 2. Combination therapy with TAT-TRP2-CMI or TAT-ESO-CMI vaccine with anti-PD-1 blockade

[0250] To verify whether SAPNANO vaccines can be combined with immune checkpoint therapy, we combined TAT-TRP-2-CMI SAPNANO vaccination with anti-PD-1 therapy, which showed that it could further enhance antitumor immunity and extend mouse survival time compared with TAT-TRP-2-CMI SAPNANO alone (Figure 22A, Figure 22B, and Figure 22C). In particular, when TAT-TRP2-CMI was combined with anti-PD-1, the survival time of mice was significantly extended (Figure 22C).

[0251] To further validate this concept, RM1-A2-ESO tumor-bearing HLA-A2 transgenic mice were treated with TAT-ESO-CMI vaccination alone or in combination with anti-PD-1 therapy (Figures 23A and 23B). The SAPNANO vaccine alone was shown to significantly suppress tumor growth (Figures 22A and 22B). The anti-tumor immunity induced by the SAPNANO vaccine could be further enhanced by anti-PD-1 blockade therapy (Figures 23A and 23B). 3. Combination therapy of TAT-ESO-CMI vaccine with TCR-T cell immunotherapy

[0252] To test whether our novel SAPNANO vaccine can boost A2-ESO TCR-T cell-mediated immunity against breast cancer, we performed experiments with E0771 / A2-ESO tumor cells, and the adoptive transfer of A2-ESO TCR-T cells followed by TAT-ESO-CMI vaccination suppressed E0771 / A2-ESO tumor growth better than either vaccine alone (Figure 24A). Notably, the TAT-ESO-CMI vaccine induces stronger antitumor immunity than the adoptive transfer of TCR-T cells (Figure 24A). Consistent with this, the TAT-ESO-CMI vaccine increased tumor-infiltrating A2-ESO TCR-T cells (25.9%) compared to A2-ESO TCR-T cells alone (5.5%) (Figure 24B). These results suggest that A2-ESO TCR-T cells can be expanded in vivo by the TAT-ESO-CMI vaccine.

[0253] To further validate this combination therapy in humanized mice, human PBMCs were injected into NSG mice and allowed to reconstitute the human immune system for 3–4 weeks. These humanized NSG mice were then injected with MDA-MB-231-A2-ESO tumor cells, followed by SAPNANO vaccine, NY-ESO-1 TCR-T cell therapy, or both. Although the TAT-ESO-CMI vaccine alone did not significantly inhibit tumor growth due to limited immune cells such as T cells and DCs after immune reconstitution, combining the TAT-ESO-CMI vaccine with ESO-specific TCR-recombinant T cell therapy was able to produce stronger antitumor effects than either alone in the MDA-MB-231-A2-ESO breast cancer model compared to the TCR-T cell alone group (Figure 25A, Figure 25B, Figure 25C, Figure 25D, and Figure 25E). [Table A-1] [Table A-2] [Table A-3] [Table A-4] [Table A-5] [Table A-6] [Table A-7] [Table A-8] [Table A-9] [Table A-10]

Table A-11

Table A-12

Table A-13

Table A-14

Table A-15

Table A-16

Table A-17

Table A-18

Table A-19

Table A-20

Table A-21

Table A-22

Table A-23

Table A-24

Table A-25

Table A-26

Table A-27

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[0328] It should be understood that the examples and embodiments described herein are merely illustrative, and various modifications or changes in consideration thereof will be suggested to those skilled in the art, and are within the spirit and scope of this application and the scope of the appended claims. All references cited herein, including publications, patent applications, and patents, are incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein. The description of ranges of values ​​herein is a shorthand way of individually referring to each separate value falling within the range, unless otherwise specified, and each separate value is incorporated herein as if it were individually set forth herein.

[0329] Description herein of any aspect or embodiment of the invention using terms such as "comprising," "having," "including," or "containing" in connection with one or more elements is intended to provide support for similar aspects or embodiments of the invention that "consists of," "consists essentially of," or "substantially comprises" that particular element or elements, unless otherwise indicated or clearly contradicted by context. (For example, a composition described herein as comprising a particular element should be understood to also describe a composition consisting of that element, unless otherwise indicated or clearly contradicted by context.)

[0330] All of the compositions and methods described and claimed herein can be made or executed without undue experimentation in light of the present disclosure.Although the compositions and methods of the present invention 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 in the steps or sequence of steps of the methods described herein, without departing from the concept, spirit and scope of the present invention.More specifically, it will be apparent that certain agents that are chemically and / or physiologically related may be substituted for the agents described herein while still achieving the same or similar results.

[0331] All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. The present invention provides, for example, the following items. (Item 1) (a) A population of nanoparticles that are self-assembling at neutral pH and are composed of multiple cationic cell-penetrating peptides covalently linked to hydrophobic therapeutic peptide ligands; (b) a pharma- ceutically acceptable buffer, diluent, carrier, or vehicle; A composition comprising: (Item 2) 2. The composition of claim 1, wherein the nanoparticles further comprise a negatively charged molecule such as mRNA, siRNA, dsRNA, RNA, DNA, or any combination thereof, or a hydrophobic peptide such as MPLA. (Item 3) The composition of claim 1 or 2, further comprising an amphiphobic or amphipathic peptide, such as CPP-TAT, covalently linked to a therapeutic peptide, such as NY-ESO-1 (SEQ ID NO: 1) or TRP-2 (SEQ ID NO: 2). (Item 4) The composition of any of the preceding items, adapted or configured to increase IFN-I expression when introduced into a suitable mammalian cell; preferably adapted or configured to increase expression of IFN-α4 or IFN-β. (Item 5) The composition of any of the preceding items, contained in an isolated population of mammalian cells, such as tumor cells or dendritic cells. (Item 6) The composition of any of the preceding items, comprising one or more cationic cell-penetrating peptides disclosed in any one of SEQ ID NO:3-SEQ ID NO:8 or SEQ ID NO:11-SEQ ID NO:16. (Item 7) (c) The composition of any of the preceding items, further comprising a chemotherapeutic agent, an immunomodulatory agent, a neuroactive agent, an anti-inflammatory agent, an antilipidemic agent, a hormone, a receptor agonist, a receptor antagonist, an anti-infective agent, an antibody, an antigen-binding fragment of an antibody, a ribozyme, a cofactor, a steroid, or any combination thereof. (Item 8) 8. The composition of item 7, wherein the chemotherapeutic agent is selected from the group consisting of cyclophosphamide, doxorubicin, 5-fluorouracil, docetaxel, paclitaxel, trastuzumab, methotrexate, epirubicin, cisplatin, carboplatin, vinorelbine, capecitabine, gemcitabine, mitoxantrone, isabepilone, eribulin, lapatinib, carmustine, nitrogen mustard, sulfur mustard, platin tetranitrate, vinblastine, etoposide, camptothecin, and any combination thereof. (Item 9) The composition of any of the preceding items, further comprising an adjuvant. (Item 10) The composition of any of the preceding items, further comprising an antigen, antigenic polypeptide, or antigenic peptide fragment thereof. (Item 11) The composition of any of the preceding items, wherein: 1) is formulated with a population of liposomes, nanoparticles, or microparticles; or 2) is mixed with one or more surfactants, niosomes, ethosomes, transferosomes, phospholipids, or sphingosomes. (Item 12) The composition of any of the preceding items, in admixture with one or more pharma- ceutically acceptable carriers, buffers, diluents, vehicles, or excipients. (Item 13) The composition of any of the preceding items, formulated for systemic administration to a mammal, preferably for intravenous administration to a human. (Item 14) The composition of any of the preceding items, adapted or configured as part of a therapeutic kit comprising the composition and at least a first set of instructions for administering the composition to a human in need thereof. (Item 15) The composition of any of the preceding items for use in the treatment, prevention, or amelioration of one or more symptoms of a disease, disorder, dysfunction, deficiency, defect, trauma, injury, or abnormal condition in a mammal. (Item 16) 13. The composition of any of the preceding items for use in the treatment, prevention, or amelioration of one or more symptoms of cancer or infection in a human. (Item 17) 2. An isolated population of mammalian cells comprising the composition of any of the preceding items. (Item 18) 18. The isolated population of mammalian cells according to item 17, characterized in that they are human dendritic cells. (Item 19) 17. Use of a composition according to any one of items 1 to 16 in the manufacture of a medicament for treating or ameliorating at least one symptom of cancer or infection in a mammalian subject. (Item 20) 20. The use of item 19, wherein the mammalian subject is a human, a non-human primate, a companion animal, an exotic animal, or a livestock animal. (Item 21) 1) a composition according to any one of items 1 to 16; and 2) instructions for administering the composition to a mammal in need thereof as part of a regimen for the prevention, diagnosis, treatment, or amelioration of one or more symptoms of a disease, dysfunction, abnormal condition, or trauma in said mammal. (Item 22) 17. A method of treating or ameliorating one or more symptoms of cancer or infection in an animal in need thereof, comprising administering to said animal an effective amount of the composition according to any one of items 1 to 16 for a time sufficient to treat or ameliorate said one or more symptoms of said cancer or said infection in said animal. (Item 23) 23. The method of claim 22, wherein the cancer has been diagnosed or identified as a refractory, metastatic, recurrent, or treatment-resistant cancer. (Item 24) 24. The method of claim 22 or 23, wherein the animal is a human. (Item 25) 25. The method of any one of items 22 to 24, wherein the composition is administered systemically to the animal in a single dose or in a series of multiple doses over a period of one or more days, over a period of one or more weeks, or over a period of one or more months or longer. (Item 26) 26. The method of any one of items 22-25, wherein the composition further comprises a second distinct chemotherapeutic agent or a second distinct population of self-assembled nanoparticles comprising a second distinct therapeutic agent. (Item 27) 17. A method of administering a diagnostic, therapeutic or prophylactic agent to one or more cells, tissues, organs or the whole body of a mammalian subject in need thereof, comprising administering to said subject an effective amount of a composition according to any one of items 1 to 16. (Item 28) 28. The method of claim 27, wherein the one or more cells are human dendritic cells. (Item 29) 28. The method of claim 27, wherein the one or more tissues are neoplastic. (Item 30) A method for providing a therapeutic composition to at least one cell, at least one tissue, or at least one organ of a patient in need thereof, comprising administering to a patient in need thereof an amount of the composition according to any one of items 1 to 16 or an isolated population of mammalian cells according to item 17 or item 18 for a period of time effective to provide said therapeutic composition to at least one cell, at least one tissue, or at least one organ of said patient.

Claims

1. (a) a population of nanoparticles that are self-assembling at neutral pH and are composed of MPLA and a plurality of cationic cell-penetrating peptides covalently linked to hydrophobic therapeutic peptide ligands; and (b) a pharma- ceutically acceptable buffer, diluent, carrier, or vehicle; A composition comprising: the nanoparticles further comprise negatively charged molecules selected from CpG, poly(I:C), or a combination of CpG and poly(I:C); A composition, wherein said cationic cell-penetrating peptide interacts with said negatively charged molecule through electrical interactions and said hydrophobic therapeutic peptide ligand interacts with said MPLA through hydrophobic interactions.

2. 2. The composition of claim 1, wherein the hydrophobic therapeutic peptide ligand is selected from NY-ESO-1 (SEQ ID NO:1), a CT83 peptide selected from SEQ ID NOs:18-28, or TRP-2 (SEQ ID NO:2).

3. The composition of claim 1 or claim 2, wherein the nanoparticles are adapted and configured to increase IFN-I expression when introduced into a suitable mammalian cell; preferably adapted and configured to increase expression of IFN-α and / or IFN-β.

4. The composition of any one of claims 1 to 3, which is comprised in an isolated population of mammalian cells, such as tumor cells or dendritic cells.

5. 2. The composition of claim 1, comprising one or more cationic cell-penetrating peptides disclosed in any one of SEQ ID NO:3 to SEQ ID NO:16, or TAT.

6. 6. The composition of any of claims 1-5, further comprising (c) a chemotherapeutic agent, an immunomodulatory agent, a neuroactive agent, an anti-inflammatory agent, an antilipidemic agent, a hormone, a receptor agonist, a receptor antagonist, an anti-infective agent, an antibody, an antigen-binding fragment of an antibody, a ribozyme, a cofactor, a steroid, or any combination thereof.

7. 7. The composition of claim 6, wherein the chemotherapeutic agent is selected from the group consisting of cyclophosphamide, doxorubicin, 5-fluorouracil, docetaxel, paclitaxel, trastuzumab, methotrexate, epirubicin, cisplatin, carboplatin, vinorelbine, capecitabine, gemcitabine, mitoxantrone, isabepilone, eribulin, lapatinib, carmustine, nitrogen mustard, sulfur mustard, platin tetranitrate, vinblastine, etoposide, camptothecin, and any combination thereof.

8. The composition of any one of claims 1 to 7, further comprising an adjuvant.

9. The composition of any one of claims 1 to 8, further comprising an antigen, an antigenic polypeptide, or an antigenic peptide fragment thereof.

10. 10. The composition of any one of claims 1 to 9, which is 1) formulated with a population of liposomes, nanoparticles, or microparticles; or 2) mixed with one or more surfactants, niosomes, ethosomes, transferosomes, phospholipids, or sphingosomes.

11. The composition of any one of claims 1 to 10, in admixture with one or more pharma- ceutically acceptable carriers, buffers, diluents, vehicles, or excipients.

12. The composition of any one of claims 1 to 11, formulated for systemic administration to a mammal, preferably for intravenous administration to a human.

13. 13. The composition of any one of claims 1 to 12, adapted and configured as part of a treatment kit comprising said composition and at least a first set of instructions for administering said composition to a human in need thereof.

14. 14. The composition of any one of claims 1 to 13 for use in the treatment, prevention, or amelioration of one or more symptoms of a disease, disorder, dysfunction, deficiency, defect, trauma, injury, or abnormal condition in a mammal.

15. A composition according to any one of claims 1 to 14 for use in the treatment, prevention or amelioration of one or more symptoms of cancer or infection in a human.

16. An isolated population of mammalian cells comprising the composition of any one of claims 1 to 15.

17. 17. The isolated population of mammalian cells according to claim 16, characterized in that they are human dendritic cells.

18. 19. A kit comprising: 1) a composition according to any one of claims 1 to 15; and 2) instructions for administering the composition to a mammal in need thereof as part of a regimen for the prevention, diagnosis, treatment, or amelioration of one or more symptoms of a disease, dysfunction, abnormal condition, or trauma in said mammal.

19. 16. A composition according to any one of claims 1 to 15 for use in a method of treating or ameliorating one or more symptoms of cancer or infection in an animal in need thereof, the method comprising: administering said composition to said animal for a time sufficient to treat or ameliorate said one or more symptoms of said cancer or said infection in said animal.

20. 20. The composition of claim 19, wherein the cancer has been diagnosed or identified as a refractory, metastatic, recurrent, or treatment-resistant cancer.

21. The composition of claim 19 or 20, wherein the animal is a human.

22. 22. The composition of any one of claims 19 to 21, wherein the composition is administered systemically to the animal in a single dose or in a series of multiple doses over a period of one or more days, over a period of one or more weeks, or over a period of one or more months or longer.

23. 23. The composition of any one of claims 19-22, wherein the composition further comprises a second distinct population of self-assembled nanoparticles comprising a second distinct chemotherapeutic agent or a second distinct therapeutic agent.

24. 16. The composition of any one of claims 1 to 15 for use in a method of administering a diagnostic, therapeutic or prophylactic agent to one or more cells, tissues, organs or the entire body of a mammalian subject in need thereof.

25. 25. The composition of claim 24, wherein the one or more cells are human dendritic cells.

26. 25. The composition of claim 24, wherein the one or more tissues are neoplastic.

27. 18. A composition comprising the composition of any one of claims 1 to 15 or the isolated population of mammalian cells of claim 16 or claim 17 for use in a method of providing a therapeutic composition to at least one cell, at least one tissue or at least one organ of a patient in need thereof, said method comprising: administering said composition to a patient in need thereof for a time effective to provide said therapeutic composition to at least one cell, at least one tissue, or at least one organ of said patient.

28. The composition of any one of claims 1 to 15 for treating or ameliorating at least one symptom of cancer or infection in a mammalian subject.

29. 30. The composition of claim 28, wherein the mammalian subject is a human, a non-human primate, a companion animal, an exotic animal, or a livestock animal.

30. The composition of claim 1, wherein the hydrophobic therapeutic peptide ligand consists of 8-11 amino acids presented by MHC class I molecules or 9-25 amino acids presented by MHC class II molecules.

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