Self-assembling peptide nanoparticle and uses thereof

Abstract

To provide a self-assembling nanoparticle, and to provide uses thereof.SOLUTION: Disclosed are self-assembling nanoparticle compositions that comprise a plurality of cationic cell penetrating peptides, each covalently linked to a hydrophobic therapeutic molecule (e.g., an antigenic peptide, mRNA, siRNA, DNA, or the like), and optionally, non-covalently bound to at least one TLR (Toll-like receptor) ligand. Also disclosed are methods for use of the nanoparticle compositions in the treatment, prophylaxis, and / or the amelioration of one or more symptoms of a mammalian disease, including, without limitation, cancer, infection, inflammation, and related diseases and abnormal conditions.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - reference to related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 791,795, filed on Jan. 12, 2019, the entire content of which is specifically incorporated herein by reference as if fully set forth herein. Statement regarding federally - sponsored research or development

[0002] Not applicable. Name of the consortium for the joint research contract

[0003] Not applicable. Technical Field

[0004] The present disclosure relates to nanoparticles that each contain a number of cationic cell - penetrating peptides (CPPs) covalently linked to a hydrophobic therapeutic peptide (e.g., an antigen peptide), and optionally contain at least one or more TLR (Toll - like receptor) ligands non - covalently bound to the CPP - bound therapeutic peptide. The amphiphilicity of this CPP - bound therapeutic peptide has the ability to self - organize to form nanoparticles under neutral conditions (pH = 7.0) together with negatively charged nucleic acids (such as CpG, poly(I:C), mRNA, siRNA, and DNA) and hydrophobic MPLA, but these nanoparticles are disrupted under acidic conditions (pH < 5). The resulting self - organized nanoparticles containing the CPP - bound therapeutic peptide and TLR ligands or mRNA enable co - delivery into antigen - presenting cells (APCs) for effective presentation for T - cell activation, resulting in strong 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 nanoparticles organized by CPP - T - cell peptides / TLR ligands.

Background Art

[0005] 1. Cancer immunotherapy Cancer is a leading cause of death in the United States and worldwide, presenting a major public health problem. Cancer immunotherapy has emerged as a promising approach to cancer treatment (Di Lorenzo et al., 2011; Lesterhuis et al., 2011; Rosenberg, 2011; Wang and Wang, 2017). Several checkpoint blockade drugs based on immunotherapy, such as cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) monoclonal antibody (Ab), ipilimumab (Yervoy), programmed cell death (PD)-1 antibody, pembrolizumab (Keytruda), have been approved by the US 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) recombinant T cells has shown promising clinical responses in blood cancers (e.g., leukemia and lymphoma).

[0006] Despite such rapid progress, the majority of cancer patients generally do not respond to checkpoint blockade therapy. For example, only about 20% of lung cancer patients respond to immune checkpoint therapy. Only 13 - 18% of breast cancer and prostate cancer patients respond to immune checkpoint therapy (Nanda et al., 2016; Kwon et al., 2014). CAR-T cell immunotherapy technology is effective in blood cancers (Sadelain et al., 2017; Johnson and June, 2017), but is likely not effective in solid cancers due to immunosuppression 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 related to tumor-infiltrating T cells, PD-L1 expression, and mutation burden (Sharma et al., 2017). Cancer patients lacking tumor-infiltrating antigen-specific T cells in tumor tissue generally do not respond to immune checkpoint therapy. To overcome these problems, cancer vaccines can increase tumor-specific T cells to control tumors. An alternative strategy is adoptive transfer of tumor-specific T cells that are either derived from cancer patients or engineered to express tumor antigen-specific TCRs or CARs on T cells.

[0007] Cancer vaccination-based immunotherapy offers the potential for highly tumor-specific cytotoxicity and is thus a very attractive approach for cancer treatment. In fact, 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 prolongs patient survival by 4.1 months compared to the control group, but does not show a significant clinical response. Vaccination with antigenic peptides or dendritic cells (DCs) pulsed with antigenic peptides can generate anti-tumor immunity, but in several types of cancer under investigation, it is not possible to generate an immune response sufficient to achieve significant clinical efficacy (Melero et al., 2014; Rosenberg et al., 2004). DC / peptide or protein vaccines alone may not be potent enough to generate a strong and sustained anti-tumor response (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 dendritic cells (DCs) via cell-permeable peptide (CPP) covalent linkage mainly prolongs the antigen presentation time of DCs to T cells, thus enhancing antigen-specific T cell responses and anti-tumor immunity against cancer (Wang and Wang, 2002; Wang et al., 2002). Based on these preclinical trials, a clinical trial using the TAT-NY-ESO-1 peptide was initiated, and it was found that this peptide vaccine was safe and could induce antigen-specific T cell responses in 6 out of 9 prostate cancer patients evaluated, which correlated with an increased PSA doubling time in vaccinated patients (Sonpavde et al., 2014). However, all immune responses were too weak and transient to induce cancer regression. Therefore, new strategies are urgently needed to develop more potent vaccines against cancer and other diseases.

[0009] Toll-like receptors (TLRs) have recently been identified as essential components of the innate immune system. TLRs detect microbial infections and activate the DC maturation program to induce an acquired immune response (Iwasaki and Medzhitov, 2004; Akira and Takeda, 2016). In DCs, activation of the innate immune receptors (e.g., TLRs, Nod-like receptors (NLRs), and RIG-like receptors (RLRs)) together with their corresponding ligands of the receptors activates nuclear factor κB (NFκB), type I interferon (IFN), and inflammatory responses. These signaling pathways produce pro-inflammatory cytokines and induce strong innate and acquired immune responses. Administration of an antigen together with a TLR ligand can increase the immunogenicity of the antigen and enhance the ability of DCs to stimulate T cell responses (Blander and Medzhitov, 2006; Blander and Medzhitov, 2006). Loading DCs with antigenic peptides together with a TLR ligand can be effective in generating strong T cell responses using the peptides and TLR ligands (Palucka and Banchereau, 2013).

[0010] By using nanotechnology such as multi-stage vectors (MSVs), more antigens can be loaded into nanoliposomes or nanoparticles, and stronger anti-tumor immunity against breast cancer can be generated compared to conventional DC vaccines (Xia et al., 2015). In melanoma, the cancer antigen peptide TRP-2 was found to be loaded into MSVs together with TLR ligands (CpG and MPLA) and then must be taken up as a vaccine by the same DCs (Zhu et al., 2018). Vaccination with a mixture of DCs loaded with MSV / TRP-2 and DCs loaded with MSV TLR ligands did not generate strong anti-tumor immunity, suggesting that co-delivery of peptides and TLR ligands is critically important (Zhu et al., 2018). However, despite the progress made with MSV technology, the survival period of mice that can be extended by DC / MSV-based vaccination is limited (10 days), and it was further suggested that DC / MSV-based vaccination only delays tumor growth and cannot generate sufficient anti-tumor immunity to completely eliminate tumor cells.

[0011] Based on these studies, it was inferred that the current vaccine strategy was unable to generate sufficient immunity to completely eliminate cancer cells. Instead, immunosuppression in the tumor microenvironment inhibits the anti-tumor immunity induced by peptide vaccines. To understand why DCs loaded with CPP-conjugated therapeutic peptides are unable to induce anti-tumor immunity that eradicates cancer, it has been found that CPP-conjugated therapeutic peptides facilitate the intracellular delivery of antigenic peptides into DCs. Similarly, CPPs have been used to deliver various cargos, including proteins, DNA, siRNA, and mRNA, intracellularly (into target cells). However, it has been found that the CPP TAT-NY-ESO-1 peptide has difficulty generating a stable emulsion with the vaccine adjuvant Montanide ISA-51. The emulsion droplets of TAT-NY-ESO-1 and Montanide ISA-51 are unstable and diffuse in water within a short time, which can affect the efficacy of the vaccine. This unstable property of TAT-NY-ESO-1 and Montanide ISA-51 has motivated the inventors to further study solutions to this problem. One potential problem is that the positive charge (hydrophilicity) of the CPP (i.e., TAT) may be disrupting the emulsion of CPP-NY-ESO-1 and Montanide ISA-51.

[0012] Potent vaccines must contain innate immune signaling components. Recent studies by the inventors and co-researchers have found that co-delivery of antigenic 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 antigenic peptides and nucleic acid-based TLR ligands into the same DCs, thereby enhancing anti-tumor immunity. However, this approach does not solve the aforementioned basic problem, i.e., antigenic peptides and nucleic acid-based TLR3 and TLR9 ligands do not form complexes because the peptides are hydrophobic and the nucleic acids are negatively charged.

[0013] In view of the above points, there is a need for an improved delivery method for cancer therapeutic molecules comprising T cell epitopes, B cell epitopes, therapeutic nucleic acid molecules, and adjuvants. 2. Nanostructures Based on Self-Assembling Peptides

[0014] Self-assembly of molecules is the spontaneous formation of a regular structure that occurs under certain thermodynamic and kinetic conditions due to specific and local molecular interactions. Molecules are bound by hydrogen bonds, hydrophobic interactions, electrostatic interactions, and van der Waals forces and maintained in a stable low-energy state. Self-association occurs to form hierarchical structures at both the nano- and / or microscale, and these minimum energies are realized (Han et al., 2010).

[0015] Self-assembly inherently occurs spontaneously during protein folding, DNA double helix formation, and cell membrane formation (Korolkov et al., 2013). Self-assembled nanostructures made from natural biomolecular building blocks such as amino acids are biocompatible and are much preferred over their synthetic self-assembled monolayer (SAM) alternatives because of the ease of "bottom-up" fabrication (Yan et al., 2010; Tayebe Zohrabi et al., 2015). 3. Cell-Penetrating Peptides

[0016] Cell-penetrating peptides (CPPs) are generally described as short peptides of 8 to 30 amino acids that cross biological membranes, trigger the movement of various biomolecules into the cytoplasm across the cell membrane, and improve their intracellular trafficking, thereby facilitating their interaction with targets (see, e.g., U.S. Patent No. 9,598,465). CPPs are either protein-derived or chimeric sequences, and are usually amphipathic and have a positive net charge (Morris et al., 2008; Hansen et al., 2008; Heitz et al., 2009). Several CPPs have been identified from proteins, 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 factor (Lin et al., 1995; Rojas et al., 1998). The Tat peptide and membrane-translocating sequences (MTSs) 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] CPP can be subdivided into two major classes, one that requires a chemical bond to cargo and the other that is involved in the formation of stable non-covalent complexes. CPPs have been used to deliver a wide variety of cargo (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, the CPP transports the cargo into the cell mainly by its membrane translocation ability (Figure 1). In these applications, there is no therapeutic T cell epitope covalently linked to the CPP. Thus, CPPs are not amphiphilic and are different from CPP-T cell peptides, which are amphiphilic for self-assembling into nanoparticles with negatively charged molecules. Summary of the Invention Means for Solving the Problems

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

[0019] In certain embodiments, the inventors have demonstrated that multiple cationic CPPs, each covalently linked to a specific therapeutic peptide (e.g., an antigen peptide, preferably hydrophobic), can form dense, small-sized (50 - 100 nm) self-assembled nanoparticles and can be used to achieve efficient intracellular delivery of the therapeutic peptide. To facilitate nanoparticle formation and delivery across the cell membrane, other components such as negatively charged molecules (DNA, dsRNA, siRNA, or mRNA) may be incorporated into the nanoparticles (Figure 2). These nanoparticles comprise (i) a corona containing CPPs having a positively charged peptide covalently linked to a therapeutic peptide having a preferred hydrophobicity, and (ii) in addition to negatively charged molecules (DNA, dsRNA, siRNA, or mRNA), hydrophobic molecules (such as MPLA). Based on charge and hydrophobicity, the inventors designed and developed a novel technology of self-assembled CPP-T cell peptide nanoparticles comprising TLR ligands [CpG and MPLA (abbreviated as CM); CpG, MPLA, and poly(I:C) (abbreviated as CMI)], as schematically shown in Figures 2A and 2B. Amphiphobic or amphiphilic CPP-therapeutic peptides consisting of a CPP (such as TAT) having a positively charged peptide and covalently linked to a therapeutic peptide (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) by electrical interaction on the one hand, and with MPLA by the hydrophobicity inside the particles on the other hand.

Brief Description of the Drawings

[0020] This patent or application file contains at least one drawing created in color. Copies of this patent or patent application publication, including color drawing(s), will be issued by the office upon payment of the required fee for the copies.

[0021] The following drawings form a part of this application and are included to illustrate certain embodiments of the present invention. To facilitate an understanding of the principles of the present invention, reference is made to the embodiments or examples shown in the drawings and specific terms are used to describe them. Nevertheless, it should be understood that this is not intended to limit the scope of the present invention. Any changes and further modifications in the described embodiments, as well as any further applications of the principles of the present invention described herein, are contemplated as would normally occur to a person skilled in the art to which the present invention pertains.

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

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Mode for Carrying Out the Invention

[0048] Brief Description of the Sequences: SEQ ID NO: 1 is an exemplary therapeutic NY-ESO-1 specific peptide for use according to one aspect of the present disclosure.

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

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

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

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

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

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

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

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

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

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

[0060] SEQ ID NO: 13 is an exemplary amphiphilic model peptide specific cell permeable peptide sequence for use according to one aspect of the present disclosure.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0075] SEQ ID NO: 28 is an exemplary TAT binding peptide for use according to one aspect of the present disclosure. Description of Exemplary Embodiments

[0076] Exemplary embodiments of the present invention are described below. For the sake of clarity, not all features of actual implementations are described herein. It will of course be understood that in the development of any such actual embodiment, numerous implementation-specific decisions must be made in order to achieve the developer's specific goals, such as compliance with system-related constraints and business-related constraints (which vary from implementation to implementation). Further, such development efforts can be complex and time-consuming, but will be routine for those of ordinary skill in the art who have received the benefits of this disclosure.

[0077] Thus, in a first aspect, the present invention provides nanoparticles comprising a plurality of cationic CPPs each covalently linked to a hydrophobic therapeutic peptide, wherein the nanoparticles self-assemble under neutral conditions, e.g., at pH 7.0, and dissociate under acidic conditions, e.g., at pH 4.5 (FIGS. 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 to 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 containing 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 non-immunogenic peptide containing 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 non-covalently bound to the CPP, preferably 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 has a length of 20 to 24 bp. In some embodiments, poly(I:C) has a length of 0.2 to 1 kb.

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

[0082] The nanoparticles disclosed in this specification can be taken up by cells. In some embodiments, nanoparticles containing antigenic peptides or non-immunogenic peptides having T cell epitopes can be taken up by APCs (particularly DCs or macrophages) both in vitro and in vivo. In some embodiments, the TLR ligand activates 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 pharmaceutically acceptable carrier (preferably one having a pH of about 7.0).

[0084] In a third aspect, the present disclosure also provides a composition for generating the above-described nanoparticles, comprising a plurality of cationic CPPs each 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 of 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, self-assembling nanoparticles may be prepared and delivered to an animal where they are taken up by DCs or macrophages in vivo. Whether the uptake of the nanoparticles by DCs or macrophages is in vitro or in vivo, the self-assembling nanoparticles enter endosomes or lysosomes where the nanoparticles are disrupted at pH 4.5; the CPP-binding peptide and the TLR ligand are released. Endosome-localized TLR3, TLR7, TLR8, and TLR9 bind to the TLR ligand to trigger innate immune signaling, while the CPP-binding peptide binds to MHC class II molecules in the endosome for loading and presentation, or crosses the endosomal membrane to enter 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 to 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 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 8, 9, 10, or 11 amino acids presented by MHC class I molecules, or 9 to 25 amino acids presented by MHC class II molecules. The antigenic peptide or non-immunogenic peptide containing a T cell epitope is directed to a specific disease, such as a tumor, and an infectious disease.

[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 has a length of 15 to 24 bp. In some embodiments, poly(I:C) has a length of 0.2 to 1 kb. 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 an infectious disease. In a comprehensive and general sense, the method typically involves 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 an aggressive tumor, and such tumors can be structurally solid or non-solid depending on the particular disease. In certain embodiments, the cancerous tumor being treated is a primary tumor or a metastatic tumor (e.g., but not limited to, one or more melanomas or lung cancers).

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

[0091] Antigenic peptides or non-immunogenic peptides containing T cell epitopes in the nanoparticles are processed by APCs (particularly DCs or macrophages) and presented to T cells for an extended period of time by newly synthesized MHC class II molecules. TLR ligands contained within the nanoparticles stimulate DCs or immune cells to produce an innate immune response (e.g., release of type I interferon cytokines), present epitopes to T cells, co-stimulate the ability of DCs to co-stimulate T cells for activation, and enhance cytokine stimulation for the growth and expansion of T cells.

[0092] The nanoparticles described herein enhance the co-delivery of antigenic peptides or non-immunogenic peptides containing T cell epitopes and two or more TLR ligands into the same antigen-presenting cell (e.g., DC or macrophage), thereby enabling the generation of a strong and effective immune response. The resulting effects (e.g., anti-tumor effect or anti-pathogen effect) can be superior to those of several 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, while this detailed description and the specific examples shown represent preferred embodiments of the invention, various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description, which is presented for purposes of illustration only. Pharmaceutical preparation

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

[0095] In certain situations, the self-assembling nanoparticle composition in a properly formulated pharmaceutical vehicle of the present disclosure is desirably delivered to one or more cells, tissues, or organs within or surrounding the body of an animal by one or more standard delivery devices (including, but not limited to, delivery devices by subcutaneous, parenteral, intravenous, intramuscular, intrathecal, intratumoral, intraperitoneal, transdermal, topical, oral, or nasal inhalation, or by direct injection).

[0096] Also included as administration methods are 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 (which are hereby specifically incorporated by reference in their entireties). Solutions of the active compound as the free base or a pharmaceutically acceptable salt may be prepared in sterile water and may be appropriately mixed with one or more surfactants, such as hydroxypropylcellulose. Dispersion solutions may also be prepared in glycerol, liquid polyethylene glycol, oils, or mixtures thereof. Under normal conditions of storage and use, these preparations contain preservatives to prevent the growth of microorganisms.

[0097] For administration of aqueous injection solutions, although not limited thereto, the solution may be appropriately buffered if necessary, and the liquid diluent may be made isotonic first 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 pharmaceutically acceptable vehicles, including, for example, sterile aqueous media, buffers, diluents, etc. For example, a given dose of the active ingredient(s) may be dissolved in a specific volume of an isotonic solution (e.g., an isotonic NaCl-based solution) and then administered to the intended site of administration or further diluted with a vehicle suitable for intravenous injection (see, for example, "REMINGTON’S PHARMACEUTICAL SCIENCES", 15th edition, pp. 1035-1038 and 1570-1580). Depending on the condition of the control to be treated, the degree of treatment, and the site of administration, some variation in dosing will necessarily occur, but nevertheless, the person responsible for administration can determine the correct dosing regimen suitable for an individual subject using ordinary knowledge in the medical and pharmaceutical arts.

[0098] The sterile injectable composition may be prepared by admixing the required amount of the self-assembling nanoparticle composition of the present disclosure, optionally with some of the other ingredients listed above, in a suitable solvent and then filter sterilizing. Generally, the dispersion can be prepared by incorporating the selected sterilized active ingredient(s) into a sterile vehicle containing the base dispersion medium and the necessary ingredients of the other ingredients listed above. The self-assembling nanoparticle composition disclosed herein may also be formulated in neutral or salt form.

[0099] Pharmaceutically acceptable salts include acid addition salts (formed by the free amino groups of the protein), which are formed by inorganic acids (e.g., but not limited to, hydrochloric acid or phosphoric acid), or organic acids (e.g., but not limited to, acetic acid, oxalic acid, tartaric acid, mandelic acid, etc.). Salts formed by free carboxyl groups can also be obtained from inorganic bases (e.g., but not limited to, sodium, potassium, ammonium, calcium, or ferric hydroxide), and organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine, etc.). Once formulated, the solution is administered in a manner compatible with the dosage formulation and in an amount effective for the intended use. The 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 reagents, intranasal and inhalation formulations.

[0100] The amount, dosing regimen, formulation, and administration of the self-assembling nanoparticle compositions disclosed herein are within the purview of one of ordinary skill in the art having the benefit of this teaching. However, administration of a diagnostically effective (i.e., pharmaceutically effective) amount of one or more of the disclosed compositions may, in some cases, be achieved by a single administration, e.g., but not limited to, a single injection of a delivery agent in an amount sufficient to provide the desired benefit to a patient in need thereof. Alternatively, in some situations, multiple or continuous administrations of the self-assembling nanoparticle compositions of the present disclosure, over a relatively short or even relatively long period of time, may be desirable (as determined by a physician overseeing the administration of such a composition to a selected individual undergoing such a procedure(s), treatment, therapy, or diagnosis).

[0101] Typically, formulations of one or more self-assembling nanoparticle compositions described herein include at least an effective amount of a first active agent. Preferably, the formulation may include at least about 0.001% of each active ingredient, preferably at least about 0.01% of the active ingredient, although of course the percentage of the active ingredient(s) may vary and, for convenience, may be present in an amount of from about 0.01 to about 90 weight or volume percent, or from about 0.1 to about 80 weight or volume percent, or preferably from about 0.2 to about 60 weight or volume percent, based on the total formulation. Of course, the amount of the active compound(s) in each composition may be prepared in such a manner that an appropriate dosage is achieved in any given unit dose of the compound. Factors such as solubility, bioavailability, biological t 1 / 2 , route of administration, shelf life of the product, etc., as well as other pharmacological considerations, are contemplated and so various dosages and treatment regimens may be desirable.

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

[0103] Pharmaceutical formulations containing one or more self-assembling nanoparticle compositions disclosed herein may further comprise one or more excipients, buffers, or diluents that are specifically formulated for contact with mammalian cells (and particularly human cells) and / or for administration to a mammalian subject (e.g., a human patient). The composition may further comprise, optionally, one or more diagnostic or prognostic agents, and / or may be formulated with an 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 the body of a mammalian patient (and particularly a human patient).

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

[0105] The specific amounts of the self-assembling nanoparticle compositions employed, and the specific administration times, or dosing regimens, for the compositions employing the formulations of the present disclosure are within the purview of one of ordinary skill in the art having the benefit of this teaching. However, administration of the formulations of the present disclosure can be accomplished by administration of one or more doses of the formulation for an effective time to provide the desired benefit to the patient being so treated. Such dosing regimens can be determined by a physician overseeing the administration of the compound, depending on the particular condition or patient, the degree or duration of the treatment being performed, and the like.

[0106] The pharmaceutical formulations comprising one or more of the self-assembling nanoparticle compositions disclosed herein are not limited solely to use in humans, or even primates or mammals. In certain embodiments, the methods and compositions disclosed herein can be used with avians, amphibians, reptiles, or other animal species. However, in preferred embodiments, the compositions of the present disclosure are formulated for administration to mammals (particularly humans) in various regimens for diagnosing, ameliorating, and / or treating one or more diseases in the body of a patient, and particularly for treating one or more types of tumors or cancer cells, or for treating one or more infections. As described above, such compositions are not limited solely to use in humans, and can also be formulated for veterinary administration, including administration to selected livestock, exotic or domesticated animals, companion animals (including pets, etc.), non-human primates, and biological specimens from animals or other specimens captured by other means. Compositions for the preparation of pharmaceuticals

[0107] Another important aspect of the present invention relates to a method for using the self-assembling nanoparticle compositions (and formulations containing the same) of the present disclosure for 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). The use of the self-assembling nanoparticle compositions of the present disclosure is particularly intended for use in the diagnosis and / or prognosis of cancer, the detection and / or prediction of cancer metastasis, or the monitoring of its extent, and / or the treatment of one or more abnormal conditions (e.g., the treatment of one or more cancer cell types in vivo, ex vivo, and / or in situ).

[0108] Such use generally involves administering to a mammal in need thereof, in an amount sufficient and for a sufficient time to diagnose, treat, alleviate, or ameliorate tumor formation, or one or more symptoms of cancer growth and / or metastasis in an affected mammal, at least one self-assembling nanoparticle composition of the present disclosure comprising at least a first active agent. Pharmaceutical formulations comprising one or more self-assembling nanoparticle compositions of the present disclosure also form part of the present disclosure and further form these compositions comprising at least a first pharmaceutically acceptable excipient for use in the treatment and / or amelioration of one or more symptoms of cancer in an affected mammal. Self-assembling 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) for forming nanoparticles. The resulting nanoparticles have (i) a core comprising a 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, and the hydrophobic TLR ligand may be monophosphoryl lipid A (MPLA), or R848.

[0111] Without wishing to be bound by any theory, self-assembly is thought to occur due to the hydrophobicity of the therapeutic peptide (and, optionally, the hydrophobic molecule), the hydrophilicity of the CPP, and the electrostatic binding by the positively and negatively charged molecules in an aqueous solution at about pH 7.0.

[0112] The amounts of the components used to form the nanoparticles of the present invention can be determined by those skilled in the art. If more negatively charged molecules are used, more CPPs are involved in nanoparticle formation. Furthermore, due to the difference in zeta potential, the size and shape of the nanoparticles, as well as the in vivo distribution, change as the amounts of some components change.

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

[0114] As described above, the nanoparticles of the present invention self-organize under neutral conditions (e.g., pH 7.0) and are disrupted under acidic conditions (e.g., pH 4.5). Thus, these nanoparticles are delivered as dense and small-sized particles to dendritic cells or macrophages and then disrupted inside endosomes where the pH becomes 4.5, releasing the CPP together with the therapeutic peptide (preferably an antigen peptide or a non-immunogenic peptide containing a T cell epitope) and other molecules (preferably TLR ligands) into the cytoplasm. Thereafter, the antigen peptide or non-immunogenic peptide containing a T cell epitope binds to MHC class I or II molecules, and its epitope is presented to T cells, while the TLR ligand binds to TLRs and triggers the TLR-mediated signaling pathways (NF-κB and type I interferon), producing inflammatory cytokines and inducing strong natural and acquired immune responses. Chemotherapeutic methods and uses

[0115] An important aspect of the present disclosure relates to a method for using the self-assembling nanoparticle formulations of the present disclosure for treating or ameliorating the symptoms of one or more forms of cancer, including, for example, tumors or metastatic cancers (including, but not limited to, melanoma metastases to the mammalian lung). Such methods generally involve administering to a mammalian subject (and, in particular, to a human in need thereof), in an amount sufficient and for a time sufficient to treat cancer (or, alternatively, to ameliorate one or more symptoms of the cancer), one or more self-assembling nanoparticle compositions of the present disclosure comprising at least a first anti-cancer therapeutic agent.

[0116] In certain embodiments, the self-assembling nanoparticle compositions described herein may be provided to an animal in one mode of treatment (either as a single administration or, alternatively, as multiple administrations 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 over a period of months or longer, or incorporate the treatment in combination with one or more additional modes of therapy. 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-assembling 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, provides for use in the manufacture of a medicament for treating and / or ameliorating one or more symptoms of a mammalian infection or cancer (e.g., a human infection, a cancerous tumor, and including linkers).

[0118] The present invention also provides for the use of one or more of the self-assembling 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 infections and / or cell hyperproliferation (i.e., cancer), etc.). Therapeutic kit

[0119] Also preferred embodiments of the present disclosure are therapeutic kits comprising one or more of the self-assembling nanoparticle compositions of the present disclosure and instructions for using the kit in a particular mode of treatment. These kits may further optionally include 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 optionally include one or more delivery devices (such as syringes, injectors, etc.) adapted to deliver the self-assembling nanoparticle composition(s) to an animal. Such kits typically include at least one vial, test tube, flask, bottle, syringe, or other container, which may contain the self-assembling nanoparticle composition(s), preferably in appropriately aliquoted amounts. If a second medicament is also provided, the kit may also include a second separate container, which may contain this second composition. Alternatively, a plurality of self-assembling nanoparticles as disclosed herein may be prepared as a single mixture (such as a suspension or solution) and packaged in a single container (such as a vial, flask, syringe, catheter, cannula, bottle, or other suitable single container).

[0121] The kit of the present invention also typically includes a holding mechanism adapted to tightly enclose and hold vial(s) or other container(s) for commercial sale, for example, an injection-molded or blow-molded plastic container that can hold the desired vial(s) or other container(s) internally to reduce or prevent breakage, exposure to sunlight, or other undesirable factors, or to enable rapid use of the composition(s) included in the kit. Cell-penetrating peptide

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

[0100] Since CPPs can translocate across cell membranes, they have been used as drug delivery vehicles over a long period of time (Gupta et al., 2005). CPPs (short peptides less than 30 amino acids in cationicity), and CPPs based on polyarginine (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), the membrane translocation of CPPs is based on hydrogen bond interactions between the guanidium groups of arginine residues and the carboxyl, phosphoryl, or sulfuryl groups of carbohydrates and phospholipids on the cell surface. The pathway of CPP translocation across the membrane was initially explained by a receptor / endocytosis-independent mechanism, but now new CPP internalization mechanisms 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 factor.

[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. YARAARRAARR (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 (amphiphilic 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] An extensive list of known CPPs can be found online at the publicly available website CPPsite 2.0, which is an updated version of the Cell - Penetrating Peptide Database (CPPsite).

[0127] In the present disclosure, the cationic CPP is covalently linked to the T cell epitope peptide and is present in the corona portion of the nanoparticle. The positively charged or neutral corona attaches the nanoparticle to the negatively charged cell surface and is then taken up by cells such as DCs or macrophages with improved uptake efficiency.

[0128] The CPP also helps an antigen peptide containing a T cell epitope or a weakly immunogenic peptide (the T cell epitope is bound to the peptide) to directly bind to MHC class II molecules in the endosome for antigen presentation on the cell surface, or helps an antigen peptide or a non-immunogenic peptide containing a T cell epitope to escape from the endosome and then enter the ER and Golgi, where it binds to newly synthesized MHC class I molecules for presentation on the cell surface for T cell activation.

[0129] The cationic CPP is required in the present invention as described above. However, non-cationic CPPs may be modified by adding or attaching some amino acids such as Lys, Arg, and His to the backbone chain, as is well known to those skilled in the art. For example, polyLys or Arg peptides are synthesized as cationic CPPs. Antigen peptide

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

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

[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 prostate acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. 4 Other target molecules belong to a group of transformation-related molecules such as the oncogene HER-2 / Neu / ErbB-2. Yet another group of target antigens are oncofetal 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, for example, NY-ESO-1, CT83, the MAGE gene family, and neoantigens. 4 。

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

[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] Furthermore, as described by Renkvist et al. (2001), there are numerous antigens known in the art. A variety of T cell determinant epitopes encoded by tumor antigens and recognized by T cells (either cytotoxic CD8 + or helper CD4 + are listed in PCT International Patent Application Publication No. WO02 / 064057, which is specifically incorporated herein by reference in its entirety.

[0137] Analogs or artificially modified epitopes are not listed, but those skilled in the art recognize methods for their acquisition or generation 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 confirmed in the Ludwig Institute for Cancer Research database, which can be readily found by those skilled in the art 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. As is well known to those skilled in the art, antigen peptides that are not hydrophobic can 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. T cell epitope

[0139] The immunogenic peptide forming the core portion of the nanoparticle of the present invention contains a T cell epitope.

[0140] Since T cell epitopes do not need to be presented on the surface of a carrier to elicit an immune response, they may 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 are known from the literature and may also be predicted by algorithms based on the existing protein sequences of specific pathogens or cancer antigens, or newly designed.

[0142] There is a wealth of known T cell epitopes available from scientific literature. These T cell epitopes may be selected from specific pathogens, from cancer-specific peptide sequences, or may be 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 by reference in its entirety). There are generally available databases, such as the "MHCBN VERSION 4.0", which is a database of MHC, or the "Protein Data Bank", which is a database of PDB, or other databases, that contain thousands of different T cell epitopes.

[0143] Helper T cell (HTL) epitopes can be made highly immunogenic by incorporating them into peptide sequences that are not originally immunogenic or attaching them to non-peptidic antigens. The Pan-DR-binding peptide HTL epitope PADRE has been widely used in vaccine design for malaria, Alzheimer's disease, and many other vaccines.

[0144] According to the definition of the MHCBN database (above), a T cell epitope is a peptide with a binding affinity (IC 50 value) of less than 50,000 nM for the corresponding MHC molecule. Such peptides are considered MHC binders. According to this definition, by August 2006, in version 4.0 of the MHCBN database, the following data were available: 20,717 MHC binders and 4,022 MHC non-binders.

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

[0146] Described herein, preferred HTL epitopes for design have a binding affinity (IC 50A peptide sequence that binds with a value). These are considered weak binders. Preferably, these epitopes bind to MHC II molecules with an IC stronger than 50 nM as measured by biophysical methods or predicted by NetMHCIIpan. 50 They bind with a value). These are considered strong binders.

[0147] T cell epitopes can be incorporated at several positions within a non-immunogenic peptide. To achieve this, specific sequences with T cell epitopes must follow the rules of MHC binding. The rules for this binding to MHC molecules are incorporated into MHC binding prediction programs that use advanced algorithms to predict MHC-binding peptides.

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

[0149] Generally, in the present invention, a non-immunogenic peptide containing a T cell epitope needs to be hydrophobic so that it can be incorporated into nanoparticles and delivered to endosomes. A non-immunogenic peptide containing a non-hydrophobic T cell epitope may 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 are homologs of the Drosophila Toll protein, which has 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 highly conserved structural motifs that 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 confirmed in humans.

[0152] TLRs are expressed in innate immune cells such as dendritic cells (DCs) and macrophages, as well as in 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 within endosomes and include TLR3, TLR7, TLR8, TLR9, TLR11, TLR12, and TLR13.

[0153] Stimulation of TLRs by the corresponding PAMP or DAMP initiates a signaling cascade that activates transcription factors such as AP-1, NF-κB, and interferon regulatory factor (IRF). Signaling by TLRs elicits various cellular responses, including the production of interferons (IFNs), pro-inflammatory cytokines, and effector cytokines that induce an adaptive immune response.

[0154] TLR signaling consists of at least two distinct pathways, namely the MyD88-dependent pathway that causes the production of inflammatory cytokines, as well as the TRIF-dependent pathway associated with the stimulation of IFN-β and the maturation of dendritic cells. TLR ligand

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

[0156] Pathogen-associated molecules that meet this requirement are crucial for pathogen function, are difficult to change by mutation, and are said to be evolutionarily conserved. Features that are somewhat conserved in pathogens include lipopolysaccharides (LPS), lipoproteins, lipopeptides, and lipoarabinomannan on the cell surface of bacteria; proteins such as flagellin derived from bacterial flagella; double-stranded RNA of viruses; or unmethylated CpG islands in bacterial and viral DNA; and CpG islands found in the promoters of eukaryotic DNA; as well as certain other RNA and DNA molecules. CpG-A and CpG-B

[0157] Table 1 lists some well-known TLRs and their common ligands. Table 1 Well-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 in which TLRs are variously distributed. Therefore, in order to trigger a strong T cell response, the ligands in Table 1, and other ligands described herein or elsewhere, may be shaped into nanoparticles. 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. Hydrophobic TLR ligands may be located in the core part of the nanoparticle together with hydrophobic therapeutic peptides, while negatively charged TLR ligands may be present in the corona part of the nanoparticle together with cationic CPPs. The electrical interaction between the positively charged CPP and the negatively charged TLR ligand is thought to result in a more stable and dense nanostructure. In one embodiment, two negatively charged TLR ligands are included in the corona part of the nanoparticle. In another embodiment, one hydrophobic TLR ligand is included in the core part of the nanoparticle and two negatively charged TLR ligands are included in the corona part of the nanoparticle. The presence of many types of TLR ligands in the nanoparticle clearly improves the T cell response. TLR ligands may also be modified to have desired properties, as is well known to those skilled in the art. Exemplary definitions

[0160] According to the present invention, polynucleotides, nucleic acid segments, nucleic acid sequences, etc. include, but are not limited to, DNA (including, but not limited to, genomic DNA or extra-genomic 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 prepared or synthesized in other ways in whole or in part by human hands.

[0161] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The following references provide those of ordinary skill in the art with general definitions of many of the terms used in this 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 (eds.), 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. (eds.), Springer-Verlag (1991); and The HarperCollins Dictionary of Biology, W.G. Hale and J.P. Margham (eds.), HarperCollins (1991).

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

[0163] In accordance with longstanding patent law convention, the words “a” and “an,” as used throughout this specification and in the claims, mean “one or more.”

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

[0165] "Biocompatible" refers to a material that, when exposed to living cells, maintains the appropriate cellular activity of those cells without causing undesirable effects in the cells, such as changes in the cell life cycle, changes in the cell growth rate, or cytotoxic effects.

[0166] As used herein, the term "biologically functionally equivalent" is well understood in the art and is further defined in more detail 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 that are identical or functionally equivalent to one or more nucleotide sequences provided herein are expected to be useful, in particular, in the practice of the methods and compositions defined in this application.

[0167] As used herein, "biomimetic" means an analogue of a synthetic material that is similar to a substance that naturally occurs in the human body and that 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 a composition of one or more that resists fluctuations in pH when an acid or alkali is added to a solution or composition containing the buffer, or an aqueous solution thereof. 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 exhibiting buffering activity is referred to as a buffer or buffer solution. A buffer generally does not have an infinite ability to maintain the pH of a solution or composition, but rather typically can maintain the pH within a certain range, for example, a pH of about 5 to 7.

[0169] As used herein, the term "carrier" is intended to include any solvent(s), dispersion medium(s), coating(s), diluent(s), buffer(s), isotonic agent(s), solution(s), suspension(s), colloid(s), inert(s), etc., or combinations thereof, that are pharmaceutically acceptable for administration to the relevant animal. The use of one or more delivery vehicles for chemical compounds, particularly chemotherapeutic agents, is well known to those of ordinary skill in the pharmaceutical arts. Any conventional medium or agent is expected to be used in diagnostic, prophylactic, and therapeutic compositions, except when it is incompatible with the active ingredient. One or more supplementary active ingredient(s) may also be incorporated into one or more of the disclosed chemotherapeutic compositions or administered with one or more of the disclosed chemotherapeutic compositions.

[0170] As used herein, the term "DNA segment" refers to a DNA molecule isolated from the total genomic DNA of a particular species without contamination. Thus, a DNA segment obtained from a biological sample using one of the compositions disclosed herein refers to one or more DNA segments 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, as well as smaller fragments of such DNA segments, and 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 in another way.

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

[0173] As used herein, a "heterologous" sequence is defined in relation to a predetermined reference sequence, for example, a polynucleotide or polypeptide sequence. For example, with respect to a structural gene sequence, a heterologous promoter is defined as a promoter that does not naturally occur adjacent to the referenced structural gene and 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 a polynucleotide, means sequences that, although arising from different sources, have the same basic nucleotide sequence. Typically, homologous nucleic acid sequences are derived from organisms having closely related genes, or genomic sequences that are substantially similar in one or more respects. In contrast, "similar" polynucleotides share the same function as polynucleotides from different species or organisms, but may have significantly different primary nucleotide sequences (nucleotide sequences encoding one or more proteins or polypeptides that perform similar functions or have similar biological activities). Similar polynucleotides often originate 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 the exact same primary sequence as a second nucleic acid or amino acid sequence, the term "identity" can be used in place 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 evaluate whether a given sequence is identical or homologous to another selected sequence.

[0176] The term "identical" or percent "identity", in the context of two or more nucleic acid or polypeptide sequences, refers to sequences or subsequences that are the same, or to two or more sequences or subsequences that, when compared and aligned to maximize correspondence using one of the following sequence comparison algorithms (or other algorithms available to those of skill in the art), or by visual inspection, have a specified percentage of identical amino acid residues or nucleotides.

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

[0178] The phrases "isolated" or "biologically pure" refer to a material that substantially or essentially lacks the components that are normally associated with that material when found in its natural state.

[0179] As used herein, the term "kit" may be used to represent a portable, self - contained variety of enclosures that contain at least one set of reagents, components, or pharmaceutically formulated compositions for performing one or more assay methods of the present invention. Optionally, such kits may 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 functionally connecting 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, etc.

[0181] As used herein, the term "naturally occurring," when applied to an object, refers to the object being found in nature. For example, a polypeptide or polynucleotide sequence that is present in a living organism (including a virus), isolatable from a natural source, and not intentionally modified by human hand in a laboratory is naturally occurring. As used herein, a laboratory strain of rodents that could have been selectively bred according to classical genetics is considered a naturally occurring animal.

[0182] As used herein, the term "nucleic acid" includes one or more types of polynucleotides, including polydeoxyribonucleotides (including 2-deoxy-D-ribose), polynucleotides (including 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 non-base moieties). As used herein, the term "nucleic acid" also includes polymers of ribonucleosides or deoxyribonucleosides that are typically covalently linked by phosphodiester bonds between subunits, but in some cases by phosphorothioate, methylphosphonate, etc. "Nucleic acid" includes single-stranded and double-stranded DNA, as well as single-stranded 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 transiently expressed small RNA (stRNA), etc., as well as any combination thereof.

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

[0184] As used herein, the term "patient" (also interchangeably referred to as "host" or "subject") refers to any host that can receive one or more pharmaceutical compositions disclosed herein. Preferably, said 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, without limitation, any mammalian host. Preferably, the term refers to any mammalian host, the latter including, but not limited to, humans and non-human primates, cattle, dogs, caprine, cavines, crows, epines, horses, cats, hircine, lapin, leporine, wolves, murine, sheep, pigs, frogs, racines, foxes, etc. (including domestic animals, biological specimens, exotic animals, as well as companion animals, pets, and any animal under veterinary care). A patient can be of any age capable of mounting a response by generating an immune response to vaccination with the present vaccine. In certain embodiments, the mammalian patient is preferably a human.

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

[0186] As used herein, "pharmaceutically acceptable salts" preferably refer to salts that retain the desirable biological activity of the compound and do not impart any undesirable toxic effects. Such salts include, for example, but are not limited to, acid addition salts formed with inorganic acids (such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, etc.); and 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, alginic acid, naphthoic acid, polyglutamic acid, naphthalenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid); salts with polyvalent metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, etc.; 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 within a bacterial host cell (e.g., E. coli) and a selectable marker for detecting the bacterial host cell containing the plasmid. The plasmids and vectors of the present invention may contain one or more gene elements described herein arranged such that the inserted coding sequences can be transcribed and translated within a suitable 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 from 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 includes repeating structural units. A polymer can be configured in multiple forms and compositions or combinations of compositions.

[0189] As used herein, the term "polypeptide" is intended to encompass both the singular "polypeptide" and the plural "polypeptides" and includes any chain(s) 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 "continuous 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 modifications (e.g., including, 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). For polynucleotide and polypeptide structures, there are conventional nomenclatures in the art.

[0190] For example, to describe amino acids, the one-letter and three-letter notations are widely used: alanine (A; Ala), arginine (R; Arg), asparagine (N; Asn), aspartic acid (D; Asp), cysteine (C; Cys), glutamine (Q; Gln), 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 preferably exist as the "L" isomer. However, any L-amino acid residue may be substituted with a residue of the "D" isomer 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 administering a compound alone or in a pharmaceutical composition before the clinical symptoms of a disease state develop such that any symptom, aspect, or feature of the disease state is prevented. Such prevention and inhibition need not be absolute in order to be considered medically useful.

[0192] "Protein" is used interchangeably herein with "peptide" and "polypeptide" and includes peptides and polypeptides synthesized, recombinantly produced, or produced in vitro, as well as peptides and polypeptides expressed in vivo after administration of a nucleic acid sequence to a host animal or human subject. The term "polypeptide" is preferably intended to refer to any length of amino acid chain and includes polypeptides of short peptides of about 2 to about 20 amino acid residues, oligopeptides of about 10 to about 100 amino acid residues, and longer polypeptides of about 100 amino acid residues or more. Further, the term is also intended to include enzymes, i.e., functional biomolecules containing at least one amino acid polymer. The polypeptides and proteins of the present invention also include polypeptides and proteins that are post-translationally modified or have been modified, and also include any sugar or other derivative(s) or conjugate(s) 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 removed from substantially all other compounds or entities, i.e., 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 naturally found with it, such as cellular materials such as cellular proteins and / or nucleic acids.

[0194] The term "recombinant" refers to a material (e.g., polynucleotide or polypeptide) that has been artificially or synthetically (non-naturally) modified by human intervention. The modification may be performed on a material in its natural environment or state, or on a material removed from its natural environment or state. Specifically, for example, a promoter sequence is "recombinant" when it is produced by the expression of a nucleic acid segment that has been engineered by human hands. For example, a "recombinant nucleic acid" is a nucleic acid that is produced by recombining nucleic acids during, for example, 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 the expression of a recombinant nucleic acid; and a "recombinant virus", e.g., a recombinant AAV virus, is produced by the expression of a recombinant nucleic acid.

[0195] As used herein, the term "regulatory element" refers to the region(s) 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 does not include total cellular RNA of a particular species. Thus, an RNA segment may refer to one or more RNA segments that are isolated or purified from other RNAs (of natural or synthetic origin). The term "RNA segment" includes RNA segments and even smaller fragments of such segments.

[0197] The term "an array essentially represented by SEQ ID NO: X" means that the array substantially corresponds to the portion of SEQ ID NO: X and contains a relatively small number of nucleotides (amino acids in the case of a polypeptide sequence) that are not identical to the nucleotides (amino acids) of SEQ ID NO: X, or that the nucleotides (amino acids) are biologically functionally equivalent to those of SEQ ID NO: X. The term "biologically functionally equivalent" is well understood in the art and is further defined in more detail herein. Accordingly, sequences having about 85% to about 90%; more preferably about 91% to about 95%; or even more preferably about 96% to about 99% of 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 present invention.

[0198] Suitable standard hybridization conditions for nucleic acids for use in the present invention include, for example, hybridization at 42 °C for 16 hours in 50% formamide, 5× Denhardt's solution, 5× SSC, 25 mM sodium phosphate, 0.1% SDS, and 100 μg / mL denatured salmon sperm DNA, followed by sequential washing at 60 °C for 1 hour with 0.1× SSC, 0.1% SDS solution to remove the desired amount of background signal. Lower stringency hybridization conditions for the present invention include, for example, hybridization at 42 °C for 16 hours in 35% formamide, 5× Denhardt's solution, 5× SSC, 25 mM sodium phosphate, 0.1% SDS, and 100 μg / mL denatured salmon sperm DNA or E. coli DNA, followed by sequential washing at 55 °C with 0.8× SSC, 0.1% SDS. It will be understood by those skilled in the art that such hybridization conditions can 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) to which treatment with a composition according to the invention can be provided. Mammalian species that can benefit from the treatment methods of the present disclosure include, but are not limited to, apes; chimpanzees; orangutans; humans; monkeys; domesticated animals such as dogs and cats; livestock 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 target sequence (e.g., an oligonucleotide sequence) is substantially complementary to all or part of a selected sequence, and thus specifically binds to a portion of the mRNA encoding that selected sequence. Thus, typically, the sequence is highly complementary to the mRNA "target" sequence and has about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 or fewer base mismatches over the entire complementary portion of the sequence. In many instances, the sequence is completely complementary, i.e., there are zero mismatches along the complementary stretch, to the sequence to which the oligonucleotide specifically binds, and thus it may be desirable in some cases. Thus, highly complementary sequences typically bind very specifically to the target sequence region of the mRNA and are thus very efficient in reducing and / or even inhibiting translation of the target mRNA sequence into its polypeptide product.

[0202] Substantially complementary nucleic acid sequences have a complementarity (or "percent identity") to the corresponding nucleic acid target sequence to which the nucleic acid specifically binds that is greater than about 80 percent, more preferably 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 nucleic acid sequences that are even more substantially complementary for use in the practice of the present invention, and in such instances, the nucleic acid sequence has a complementarity to the corresponding target sequence to which the nucleic acid specifically binds that is greater than about 90 percent. In certain embodiments, the complementarity to the corresponding target sequence to which the nucleic acid specifically binds may be greater than about 95 percent, and even up to (including that value) about 96%, about 97%, about 98%, about 99% complementarity to all or a portion of the target sequence to which the designed nucleic acid specifically binds, and even up to about 100% percent identity complementarity.

[0203] The percent similarity or percent complementarity of any of the nucleic acid sequences of the present disclosure can be determined by comparing the sequence information, for example, using the GAP computer program, version 6.0, available from the 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 of the two sequences. Preferred initial parameters for the GAP program include the following: (1) for nucleotides, a unitary comparison matrix (including a value of 1 for a match and 0 for a mismatch), 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 of" or "essentially free of" in relation to the amount of a component preferably refer to a composition in which the content of a certain compound is less than about 10% by weight, preferably less than about 5% by weight, more preferably less than about 1% by weight. In preferred embodiments, these terms refer to less than about 0.5% by weight, less than about 0.1% by weight, or less than about 0.01% by weight.

[0205] As used herein, the term "structural gene" is generally intended to describe a polynucleotide (e.g., a gene) that is expressed to produce a coded 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) to which treatment with a composition according to the present invention can be provided. Mammalian species that can benefit from the treatment methods of the present disclosure include, but are not limited to, humans, non-human primates (apes; chimpanzees; monkeys, and orangutans, etc.), domesticated animals (including dogs and cats), as well as livestock such as horses, cows, pigs, sheep, and goats, or other mammalian species (including, but not limited to, mice, rats, guinea pigs, rabbits, hamsters).

[0207] As used herein, the terms "substantially corresponding", "substantially homologous", or "substantial identity" represent features of nucleic acid or amino acid sequences, where a selected nucleic acid sequence or a selected amino acid sequence has at least about 70 or about 75 percent sequence identity as 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, and even 85 percent sequence identity, more preferably at least about 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 percent sequence identity. Even more preferably, sequences with higher homology 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 the sequence is compared.

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

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

[0210] The term "therapeutically meaningful period" means the period for which one or more active agents are necessary for being therapeutically effective. The term "therapeutically effective" refers to a reduction in the severity and / or frequency of one or more symptoms, the removal of one or more symptoms and / or the underlying factors, the prevention of the occurrence of symptoms and / or the underlying factors, and the improvement or alleviation of damage.

[0211] A "therapeutic agent" can be any physiological or pharmacologically active substance that can bring about a desired biological effect at a target site in a subject. Therapeutic agents can be chemotherapeutic agents, immunosuppressive agents, cytokines, cytotoxic agents, nuclease-degrading compounds, radioisotopes, receptors, and prodrug-activating enzymes, which may be natural, may be produced by synthetic or recombinant methods, or may be 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 can also be used as therapeutic agents. Examples of such cytokines are lymphokines, monokines, and conventional polypeptide hormones. Cancer chemotherapeutic agents can be preferred therapeutic agents. For a more detailed description of anticancer agents and other therapeutic agents, those skilled in the art are referred to any number of treatises, including but not limited to the Physician’s Desk Reference, as well as 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) identified as a site for sequence-specific interaction of one or more transcription factors, often taking 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] "Transcription regulatory element" refers to a polynucleotide sequence that activates transcription, either alone or in combination with one or more other nucleic acid sequences. Transcription regulatory elements can 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 that is operably linked to at least a first cis-acting promoter sequence and, optionally, to one or more other cis-acting nucleic acid sequences necessary for efficient transcription of a structural gene sequence, including at least a first structural gene, and at least a first distal regulatory element that may be required for appropriate tissue-specific developmental transcription of the structural gene sequence operably disposed under the control of the promoter and / or enhancer element, as well as any additional cis sequences necessary 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, plasmid, or recombinant DNA or RNA molecule) into a host cell or protoplast, where the exogenous polynucleotide is either integrated into at least a first chromosome or can replicate autonomously within the transformed host cell. Transfection, electroporation, and the uptake of "naked" nucleic acids all represent examples of techniques used to transform host cells 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 kind of medical or surgical management. Treating may include, but is not limited to, administering a composition comprising a therapeutic agent to a subject. "Treating" is 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, and includes any administration or application of a compound or composition of the invention to a subject. In certain embodiments, the compositions of the invention may also be administered prophylactically, prior to the occurrence of any symptoms or signs of a condition, when prophylaxis is warranted. Typically, in such cases, the subject is diagnosed as being "at risk" of developing such a disease or disorder as a result of a family history, medical record, or the results of any one or more diagnostic or prognostic tests that suggest a tendency to develop such a disease or disorder later.

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

[0219] In certain embodiments, one or more nucleic acid segments of the invention are advantageously utilized in combination with a suitable detectable marker (i.e., a "label"), such as in cases where a polynucleotide probe labeled is utilized to determine 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 and include, but are not limited to, fluorescent, radioactive, enzymatic, or other ligands (e.g., avidin / biotin), which can be detected in a suitable assay. In certain embodiments, also, one or more fluorescent labels or enzyme tags such as urease, alkaline phosphatase, or peroxidase may be utilized in place of radioactive or other environmentally less desirable reagents. In the case of enzyme tags, colorimetric, chromogenic, or fluorogenic indicator substrates can be utilized to provide a method of detecting a sample visible to the human eye, or can be utilized in analytical methods such as scintigraphy, fluorescence quantification, spectrophotometry, etc. to identify specific hybridization with a sample 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 characteristic or parameter (e.g., maximum of the emission and / or excitation spectrum), and further label a second oligonucleotide probe with a second label having a second detection characteristic or parameter different from the first label (i.e., discreet or distinguishable). The use of multiplexed assays is well known to those of skill in the art of molecular genetics, particularly in the context of gene amplification / detection protocols. Biological functional equivalence

[0220] It is possible to obtain a functional system that modifies and alters the structure of nucleic acids, vectors containing nucleic acids, and the mRNAs, polypeptides, or therapeutic agents encoded thereby, and still contains one or more therapeutic agents with desirable properties. As described above, it is often desirable to introduce one or more mutations into a specific polynucleotide sequence. In certain situations, the resulting encoded polypeptide sequence is altered by this mutation, or in other cases, the sequence of this polypeptide remains unchanged by one or more mutations in the encoding polynucleotide.

[0221] When it is desirable to create equivalent or even improved second-generation molecules by changing the amino acid sequence of a polypeptide, this can be achieved by changing one or more codons of the coding DNA sequence according to Table 2.

[0222] For example, a particular amino acid can substitute for other amino acids in a protein structure without significantly impairing the interactive binding ability to a structure such as, for example, the antigen-binding region of an antibody or the binding site on a substrate molecule. Since the biological functional activity of a protein is defined by its interactive ability and properties, it is possible to obtain a protein with similar properties by making certain amino acid sequence substitutions in the protein sequence (and, of course, the underlying DNA coding sequence). Thus, the inventors intend to be able to make various changes to the peptide sequence of the compositions of the present disclosure, or the corresponding DNA sequence encoding the peptide, without significantly impairing its biological utility or activity.

[0223] Table 2 [Table 2]

[0224] When making such changes, the hydrophobicity-hydrophilicity index of the amino acids may be considered. The importance of the hydrophobicity-hydrophilicity amino acid index in conferring interactive biological functions on proteins is generally recognized in the art (Kyte and Doolittle, 1982, which is hereby incorporated by reference). It is accepted that the relative hydrophobicity-hydrophilicity characteristics of amino acids contribute to the secondary structure of the protein, which in turn defines the interaction of that protein with other molecules (e.g., enzymes, substrates, receptors, DNA, antibodies, antigens, etc.). Each amino acid has been assigned a hydrophobicity-hydrophilicity index based on its hydrophobic 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); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0225] It is known in the art that a protein can be obtained in which a particular amino acid is replaced by another amino acid having a similar hydrophobicity-hydrophilicity index or score and still has a similar biological activity, i.e., a protein with still equivalent biological function can be obtained. When making such changes, substitutions of amino acids with a hydrophobicity-hydrophilicity index within ±2 are preferred, substitutions within ±1 are particularly preferred, and substitutions within ±0.5 are especially preferred. It is also understood in the art that substitutions of similar amino acids can be effectively made based on hydrophilicity. U.S. Patent No. 4,554,101 (which is specifically incorporated herein by reference in its entirety) 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 that 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 substituting one amino acid with another having a similar hydrophilicity value can still result in a biologically equivalent, particularly immunologically equivalent, protein. In such cases, substitutions of amino acids with hydrophilicity values within ±2 are preferred, particularly substitutions within ±1, and even more particularly substitutions within ±0.5 are especially preferred.

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

[0228] The section headings used throughout are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents (including, but not limited to, patents, patent applications, articles, books, and treatises) cited in this application are hereby expressly incorporated by reference in their entirety by express reference. If one or more of the incorporated documents or similar materials define a term in a manner that conflicts with the definition of that term in this application, this application controls.

Example

[0229] The following examples are included to illustrate exemplary embodiments of the present invention. Those skilled in the art should understand that the techniques disclosed in these examples are those that have been found to function well in the practice of the present invention and, therefore, may be considered to constitute a preferred mode for carrying out the present invention. However, those skilled in the art should understand that, in view of the present disclosure, many changes may 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] In addition to NY-ESO-1 peptide and Montanide ISA-51, many clinical trials have been reported using CpG or poly(I:C), but the clinical responses and immune responses are generally weak or moderate. One reasonable reason is that the 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 has been demonstrated that the DC / TAT-TRP-2 vaccine can generate strong protective (but not therapeutic) immunity (Wang et al., 2002). In a phase I clinical trial, the TAT-ESO-1 peptide was mixed with Montanide ISA-51 for the vaccine, and this vaccine only generated a weak T cell response. During the course of this trial, the inventors found two potential problems: 1) TAT-ESO-1 was unable to form a stable complex with Montanide ISA-51, probably due to the positive charge at the N-terminus; 2) TAT-ESO-1 formed a precipitate when mixed with CpG. Based on charge and hydrophobicity, the inventors designed and developed a novel technology of self-assembling CPP-T cell peptide nanoparticles (PEP-NANO) using TLR ligands [CpG, MPLA, and poly(I:C) (abbreviated as CMI)], as schematically shown in FIGS. 2A and 2B. Amphiphobic or amphiphilic CPP therapeutic peptides consisting of CPPs such as TAT, which have positively charged peptides and are covalently linked to therapeutic peptides (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) by electrical interaction on the one hand, and with MPLA by hydrophobicity inside the particles on the other hand. 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 Concentration and combination of TLR ligands

Table 3

[0233] Self-assembly of TAT-peptide nanoparticles was triggered by dropwise addition of the TAT-peptide within 1 minute while sonication was performed in an ice-cold water bath. TAT-TRP2 exhibits hydrophilicity / positive charge and hydrophobicity and promotes electrostatic interaction with CpG ODN. The hydrophobic C-terminus of the TRP2 peptide forms a hydrophobic core with MPLA by hydrophobic interaction. Electrostatic and hydrophobic interactions drive the self-assembly of TAT-TRP2 with double or triple TLR agonists, forming spherical TAT-TRP2-CM nanoparticles as shown by AFM analysis for both the height and DMT modulus distribution across a representative cross-section (red line). In contrast, the non-TAT-modified TRP2 peptide could not form a complex with CpG-MPLA and formed long fibers.

[0234] To formulate stable and highly effective peptide vaccine nanoparticles, the inventors optimized the formulation using various different ratios of TAT-TRP2 peptide:TLR ligand as shown in Table 3 and analyzed the size of each nanoparticle (80 - 150 nm) of different combinations of TAT-TRP2 and TLR ligands (Figure 4). The gray bars indicate unstable / polydisperse complexes with a 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 various nitrogen(+) / phosphate(-) (N / P) ratios (N = nitrogen derived from amino acid residues; P = CpG ODN phosphate groups). The zeta potential of the TAT-TRP2-CM nanoparticles changed according to the difference in the N / P ratio (Figure 5). For subsequent studies, condition #2 was applied to TAT-TRP2-CM and condition #18 was applied to 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 the CPP peptide nanoparticles are pH-dependent

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

[0237] To further characterize the self - organization and nanoparticle size at different pH values from pH 4 to 7, the inventors found that the self - organization 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 the size increased at pH 6.0 and decreased at pH 5.0. The nanoparticles of TAT - TRP2 - CM were completely disrupted and separated (Figure 9A). Based on these results, the inventors inferred that TAT - TRP2 - CM nanoparticles were taken up by APCs through phagocytosis into endosomes / lysosomes, where the nanoparticles were disrupted at pH 4 - 5. Due to the acidification within the endosome / lysosome compartment, the positive charge of TAT - TRP2 increased and the CpG ODN was neutralized. Thus, the TAT - TRP2 peptide was released into the cytoplasm and presented by MHC class I or II molecules in the ER, while the TLR ligand bound to TLRs, triggered the innate immune response, produced cytokines, thereby enhancing antigen presentation and T - cell activation (Figure 9B). In contrast, TAT - TRP2 alone entered APCs by cell permeability and was presented to T cells by APCs, without an innate immune response and cytokine production. This pH - dependent property was further shown to apply to TAT - TRP2 - CM, TAT - TRP2 - CMI, TAT - ESO - l - CM, and TAT - ESO - l - CMI (Figure 9C). Example 5 Innate immune response and cytokine production triggered by CPP - peptide nanoparticles with different combinations of TLR ligands

[0238] To identify the optimal combination of TLR ligands that stimulate the innate immune response, the inventors newly isolated bone marrow-derived DCs and then treated them with different TLR ligands (alone only), double combinations, or triple combinations (Figures 9A, 9B, 9C, and 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 the cell supernatant. Double combinations of poly(I:C) / CpG, CpG / MPLA, and the triple combination of CpG / poly(I:C) / MPLA were found to be superior to other groups in triggering innate cytokine production. The triple combination of CpG / poly(I:C) / MPLA is the strongest activator for inducing cytokine production (Figure 10). Example 6 The formation and size of CPP peptide nanoparticles are pH-dependent

[0239] In addition to NY-ESO-1 peptide and Montanide ISA-51, many clinical trials have been reported using CpG or poly(I:C), but the clinical responses and immune responses are generally weak or moderate. One of the plausible reasons is that the 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 the DC / TAT-TRP-2 vaccine could generate strong protective (but not therapeutic) immunity (Wang et al., 2002). In a phase I clinical trial, the TAT-ESO-1 peptide was mixed with Montanide ISA-51 for the vaccine, and this vaccine only generated a weak T cell response. Overall, mouse and human clinical trials have shown that the current vaccine approach does not result in a strong immune response and clinical response. The main problem is that the cancer antigen peptide / protein and TLR ligands are not co-delivered to the same APC. In many cases, only one type of TLR ligand is used instead of two or three ligands being co-delivered. In the following examples, the inventors demonstrate that the SAPEP-NANO technology, which utilizes amphiphilic CPP therapeutic peptides such as TAT-TRP2 or TAT-ESO-1 to form nanoparticles with negatively charged CpG and / or poly(I:C) by electrical interaction on the one hand and with MPLA by hydrophobicity on the other hand, can generate strong anti-tumor immunity in a mouse model. Example 7 Generation of Robust Anti-Tumor Immunity by DCs Loaded with TAT-TRP2 and TLR Ligand Nanoparticles

[0240] To enhance anti-tumor immunity, the inventors hypothesized that the TAT-TRP-2 peptide forms a complex with TLR ligands such as CpG and MPLA based on physical properties (positive / negative charge, hydrophilicity, and hydrophobicity) and induces therapeutic immunity. To test this prediction, the inventors used the B16 mouse model and the tyrosinase-related protein-2 (TRP-2) peptide as an experimental system. The TAT-TRP-2 (YGRKKRRQRRRSYVDFFVWL) (SEQ ID NO: 17) peptide formed a tight complex with CpG / MPLA (TAT-TRP2-CM), whereas the TRP2 peptide was unable to form a complex with CpG / MPLA (TRP2-CM) (Figures 3A-1, 3A-2, 3B-1, 3B-2, 3C-1, and 3C-2). DCs loaded with TAT-TRP2-CM or TRP2-CM were prepared and intravenously injected into B16 tumor-bearing mice. After 16 days, lung metastases were 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 was unable to suppress the number of lung metastases compared to the DC / β-gal-CM control group (Figure 11).

[0241] Multistage vehicle (MSV) nanotechnology has recently been shown to be able to load peptides, CpG, and MPLA into silicon particles and induce a strong immune response against B16 tumor cells (Zhu et al., 2018). To compare the ability of DC / MSV vaccine and DC / PEP-NANO vaccine to induce anti-tumor immunity and survival, the inventors 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). Clearly, the DC / TAT-TRP2 / CpG / MPLA group (#3) and the DC / MSV-TAT-TRP2 / CpG / MPLA group (#5) induced stronger therapeutic immunity and were able to suppress B16 lung metastasis (Figure 12B) compared to DC / TRP-2 / CpG / MPLA (#2) and DC / MSV / TRP-2 / CpG / MPLA (#4), independent of MSV, suggesting that the TAT sequence, rather than MSV, is critically required to generate the strongest immune response. More importantly, the inventors showed that mice immunized with DC / TAT-TRP-2 / CpG / MPLA survived much longer than those in the DC / MSV-TAT-TRP-2 / CpG / MPLA group (all mice died within 35 days after B16 tumor injection) (Figure 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 inoculation. These results suggested that the TAT-TRP-2 / CpG / MPLA vaccine was the best among the various different vaccine groups tested. Example 8 Generation of Robust Anti-Tumor 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 can induce potent anti-tumor immunity against RM1 / A2-ESO-1 tumor cells, experiments were conducted by vaccinating with DC / control peptide, DC / TAT-ESO-CM, or DC / TAT-ESO-CMI. Tumor growth was monitored every two days. DC / TAT-ESO-CM vaccination was observed to significantly suppress RM1 / HLA-A2-NY-ESO-1 tumor growth compared to the control group (Figures 13A and 13B). Importantly, DC / TAT-ESO-CMI showed a much stronger immunity than the DC / TAT-ESO-CM vaccine (Figures 13A and 13B). Further analysis of the immune cell response confirmed that the antigen-specific response in DC / TAT-ESO-CMI was superior to that in DC / TAT-ESO-CM (Figures 14A and 14B). These results suggested that both DC / TAT-ESO-CM and DC / TAT-ESO-CMI vaccines generate potent anti-tumor immunity.

[0243] To further demonstrate whether DC / TAT-ESO-CM can induce therapeutic anti-tumor immunity in other tumor models, mammary carcinoma E0771 / A2-ESO tumor cells were used as the 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 strong therapeutic anti-tumor immunity

[0244] Most studies for vaccines use DCs loaded with antigen peptides, MSV particles, or combinations of CPP peptides and TLR nanoparticles, but such processes are complex and very labor-intensive, especially for clinical trials. Thus, the inventors hypothesized whether CPP peptides / TLR nanoparticles could be used directly for vaccination to generate strong anti-tumor immunity. To verify 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 to a much higher extent than DC / TAT-ESO-CMI (Figures 16B and 16C). In contrast, as expected, rapid tumor growth was observed in the control group. 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 cancers (Figures 17A-17D, 18A, and 18B), which is consistent with previous reports (Fukuyama et al., 2006; Paret et al., 2015). Thus, CT83 is expected to function as an immune target for cancer vaccines and immunotherapy.

[0246] To verify 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 CMI generated strong T cell responses against the CT83-A2 peptide (Figures 19A, 19B, 19C, 19D, and 20A-20D). HLA-DR13 and HLA-DP4 restricted T cells were generated after in vitro peptide stimulation.

[0247] To confirm whether TAT-CT83-CMI could elicit potent antitumor immunity, for each mouse, a TAT-CT83 peptide vaccine was prepared by mixing 100 μg of a TAT-CT83 peptide mixture (containing 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)) under sonication. The experimental design using HLA-A2 transgenic mice is shown in Figure 20A. It was shown that the TAT-CT83-CMI vaccine could strongly induce potent antitumor immunity against mouse breast cancer E0771 / A2 / CT83 cells (Figures 20B and 20C). Furthermore, such antitumor immunity could be further enhanced by anti-PD-1 blockade therapy (Figures 20B and 20C). Importantly, T cells induced by the vaccine infiltrated into the tumor tissue compared to mice without the vaccine (Figure 20D).

[0248] Table 4 TAT-conjugated CT83 peptide

Table 4

[0249] Similarly, experiments were conducted in HLA-A2 Tg mice using E0771 / A2-ESO breast cancer cells. A single vaccination with DC / TAT-ESO-CMI completely suppressed tumor growth 10 days after tumor injection (1×10 6 cells / mouse) compared to the control treatment group (Figures 21A, 21B, and 21C). Furthermore, vaccination with TAT-ESO-CMI without DC was shown to elicit potent antitumor immunity and suppress tumor cell growth in the treatment model (Figure 21D). 2. Combination therapy with TAT-TRP2-CMI or TAT-ESO-CMI vaccines including anti-PD-1 blockade

[0250] To verify whether the SAPNANO vaccine can be combined with immune checkpoint therapy, when anti-PD-1 therapy was combined with TAT-TRP-2-CMI SAPNANO vaccination, it was shown that anti-tumor immunity was further enhanced and the survival period of mice could be extended compared with TAT-TRP-2-CMI SAPNANO alone (Figure 22A, Figure 22B, and Figure 22C). In particular, when anti-PD-1 was combined with TAT-TRP2-CMI, the survival period of mice was significantly extended (Figure 22C).

[0251] To further verify 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 (Figure 23A and Figure 23B). It was shown that the tumor growth was significantly inhibited by the SAPNANO vaccine alone (Figure 22A and Figure 22B). The anti-tumor immunity induced by the SAPNANO vaccine could be further enhanced by anti-PD-1 blockade therapy (Figure 23A and Figure 23B). 3. Combination therapy of TAT-ESO-CMI vaccine with TCR-T cell immunotherapy

[0252] To verify whether the inventors' new SAPNANO vaccine can boost A2-ESO TCR-T cell-mediated immunity against breast cancer, experiments were conducted using E0771 / A2-ESO tumor cells. After adoptively transferring A2-ESO TCR-T cells and then inoculating with TAT-ESO-CMI vaccine, the inhibition of E0771 / A2-ESO tumor growth was better than either alone (Figure 24A). Notably, the TAT-ESO-CMI vaccine induces stronger anti-tumor immunity than adoptive transfer of TCR-T cells (Figure 24A). Consistent with this, the TAT-ESO-CMI vaccine (25.9%) increased tumor-infiltrating A2-ESO TCR-T cells compared with A2-ESO TCR-T cells alone (5.5%) (Figure 24B). These results suggest that A2-ESO TCR-T cells can be increased in vivo by the TAT-ESO-CMI vaccine.

[0253] To further verify this combination therapy in humanized mice, human PBMCs were injected into NSG mice 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. 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. However, by combining the TAT - ESO - CMI vaccine with ESO - specific TCR - recombinant T cell therapy, a stronger antitumor effect than either alone could be achieved in the MDA - MB - 231 - A2 - ESO breast cancer model compared to the TCR - T cell alone group (Figures 25A, 25B, 25C, 25D, and 25E). References

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[0328] The examples and embodiments described herein are for illustrative purposes only, and various modifications or changes contemplated thereby are suggested to those skilled in the art, and it is to be understood that they are included within the spirit and scope of this application and the scope of the appended claims. All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein. The description of ranges of values herein is merely a shorthand way of referring individually to each separate value falling within the range, and each separate value is incorporated herein as if it were individually recited herein.

[0329] Any description in this specification of any aspect or embodiment of the invention using terms such as "comprising", "having", "including", or "containing" in relation to one or more elements is, unless otherwise stated or clearly contradicted by the context, intended to support that one or more particular elements "consist of", "consist essentially of", or "substantially comprise" similar aspects or embodiments of the invention. (For example, a composition described herein as comprising a particular element should be understood, unless otherwise stated or clearly contradicted by the context, to also describe a composition consisting of that element.)

[0330] All of the compositions and methods described and claimed herein can be made or executed without undue experimentation, with reference to this disclosure. Although the compositions and methods of the invention have been described in preferred aspects, it will be apparent to those skilled in the art that changes may be made to the compositions and methods, as well as to the steps or series of steps of the methods described herein, without departing from the concept, spirit, and scope of the invention. More specifically, it is apparent that certain chemically and / or physiologically related agents may be substituted for the agents described herein, and still achieve the same or similar results.

[0331] All such similar substitutions and modifications apparent to those skilled in the art are considered to be within the scope of 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 composed of a plurality of cationic cell-penetrating peptides that are self-organizing at neutral pH and covalently linked to a hydrophobic therapeutic peptide ligand; (b) A pharmaceutically acceptable buffer, diluent, carrier, or vehicle Comprising a composition. (Item 2) The composition according to item 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 according to item 1 or item 2, further comprising an amphiphilic 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 according to any of the preceding items, which is adapted or configured to increase IFN-I expression when introduced into a suitable mammalian cell; preferably adapted or configured to increase the expression of IFN-α4 or IFN-β. (Item 5) The composition according to any of the preceding items, contained in an isolated population of mammalian cells such as tumor cells or dendritic cells. (Item 6) The composition according to any of the preceding items, comprising one or more cationic cell-penetrating peptides disclosed in any one of SEQ ID NOs: 3 to 8 or SEQ ID NOs: 11 to 16. (Item 7) (c) A chemotherapeutic agent, immunomodulatory agent, neuroactive agent, anti-inflammatory agent, anti-lipidemic agent, hormone, receptor agonist, receptor antagonist, anti-infective agent, antibody, antigen-binding fragment of an antibody, ribozyme, cofactor, steroid, or any combination thereof, further comprising the composition according to any of the preceding items. (Item 8) The composition according to 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, isabepirone, eribulin, lapatinib, carmustine, nitrogen mustard, sulfamustine, platinum tetranitrate, vincristine, etoposide, camptothecin, and any combination thereof. (Item 9) The composition according to any of the preceding items, further comprising an adjuvant. (Item 10) The composition according to any of the preceding items, further comprising an antigen, an antigen polypeptide, or an antigen peptide fragment thereof. (Item 11) The composition according to any of the preceding items, which is formulated with 1) a population of liposomes, nanoparticles, or microparticles; or 2) mixed with 1 or more surfactants, niosomes, ethosomes, transferosomes, phospholipids, or sphingosomes. (Item 12) The composition according to any of the preceding items, which is mixed with 1 or more pharmaceutically acceptable carriers, buffers, diluents, vehicles, or excipients. (Item 13) The composition according to any of the preceding items, which is formulated for systemic administration to a mammal, preferably for intravenous administration to a human. (Item 14) The composition according to any of the preceding items, which is 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. The composition according to any of the preceding items. (Item 15) A composition according to 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) A composition according to any of the preceding items for use in the treatment, prevention or amelioration of one or more symptoms of human cancer or infection. (Item 17) An isolated population of mammalian cells comprising a composition according to any of the preceding items. (Item 18) An isolated population of mammalian cells according to item 17, characterized in that it is human dendritic cells. (Item 19) 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) The use according to item 19, wherein the mammalian subject is a human, non-human primate, companion animal, exotic animal or livestock. (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 preventing, diagnosing, treating or ameliorating one or more symptoms of a disease, dysfunction, abnormal condition or trauma in a mammal. (Item 22) A method of treating or ameliorating one or more symptoms of cancer or infection in an animal in need thereof, comprising administering to the animal an effective amount of a composition according to any one of items 1 to 16 for a time sufficient to treat or ameliorate the one or more symptoms of the cancer or the infection in the animal. (Item 23) The method according to item 22, wherein the cancer has been diagnosed or identified as a treatment-resistant, metastatic, recurrent or treatment-resistant cancer. (Item 24) The method according to item 22 or 23, wherein the animal is a human. (Item 25) The method according to any one of items 22 to 24, wherein the composition is systemically administered to the animal in a single dose, or over a period of 1 day or more days, over a period of 1 week or more weeks, or over a period of 1 month or more months or a longer period in a plurality of successive administrations. (Item 26) The method according to any one of items 22 to 25, wherein the composition further comprises a second distinct chemotherapeutic agent or a population of second distinct self-assembling nanoparticles comprising a second distinct therapeutic agent. (Item 27) A method of administering a diagnostic agent, a therapeutic agent, or a prophylactic agent to one or more cells, tissues, organs, or the whole body of a mammalian subject in need thereof, comprising administering to the subject an effective amount of the composition according to any one of items 1 to 16. (Item 28) The method according to item 27, wherein the one or more cells are human dendritic cells. (Item 29) The method according to item 27, wherein the one or more tissues are neoplastic. (Item 30) 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, comprising administering to the patient in need thereof for a period effective to provide the therapeutic composition to at least one cell, at least one tissue, or at least one organ of the patient 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.

Claims

[Claim 1] The invention described in the specification.

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