Self-assembling peptide nanoparticles and their uses

Abstract

Disclosed are self-assembling nanoparticle compositions comprising a plurality of cationic cell-penetrating peptides, each of which is covalently linked to a hydrophobic therapeutic molecule (e.g., an antigenic peptide, mRNA, siRNA, DNA, etc.) and optionally non-covalently bound to at least one TLR (Toll-like receptor) ligand. Also disclosed are methods for using the nanoparticle compositions to treat, prevent, and / or ameliorate one or more symptoms of a mammalian disease, including, but not limited to, cancer, infection, inflammation, and related diseases and abnormal conditions.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 791,795, filed January 12, 2019, the contents of which are specifically incorporated herein by express reference in their entirety.

[0003] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0004] not applicable.

[0005] Names of parties to the joint research agreement

[0006] not applicable. Technical Field

[0007] The present disclosure relates to nanoparticles comprising: a plurality of cationic cell penetrating peptides (CPPs), each of which is covalently linked to a hydrophobic therapeutic peptide, e.g., an antigenic peptide; and optionally at least one or more TLR (Toll-like receptor) ligands, which are non-covalently bound to the CPP-linked therapeutic peptide. The amphiphilic nature of the CPP-linked therapeutic peptide enables self-assembly with negatively charged nucleic acids (e.g., CpG, poly(I:C), mRNA, siRNA, and DNA) and hydrophobic MPLA to form nanoparticles under neutral conditions (pH = 7.0), but is destroyed under acidic conditions (pH < 5). The resulting self-assembled nanoparticles containing CPP-linked therapeutic peptides and TLR ligands or mRNA allow for co-delivery to antigen-presenting cells (APCs) for efficient presentation to activate T cells, thereby generating potent immunity to cancer and other diseases. Therefore, the present disclosure also provides methods for treating and / or preventing cancer (including various tumors or infectious diseases) by employing nanoparticles assembled with CPP-T cell peptides / TLR ligands. Background Art

[0008] 1. Cancer immunotherapy

[0009] Cancer is the leading cause of death in the United States and worldwide, posing a major public health problem. Cancer immunotherapy has been a promising approach for treating cancer (Di Lorenzo et al., 2011; Lesterhuis et al., 2011; Rosenberg, 2011; Wang and Wang, 2017). Several immunotherapy-based checkpoint blockade drugs, such as cytotoxic T lymphocyte-associated protein 4 (CTLA-4) monoclonal antibodies (Abs), ipilimumab (Yervoy), programmed cell death (PD)-1 Abs, and pembrolizumab (Keytruda), have been approved by the U.S. Food and Drug Administration (FDA) for the treatment of many types of cancer (Bagcchi, 2014; Hodi, 2010; Kantoff et al., 2010; Bender, 2017). Furthermore, cell-based immunotherapy using T cells engineered with T cell receptors (TCRs) or chimeric antigen receptors (CARs) has shown promising clinical responses in blood cancers such as leukemias and lymphomas.

[0010] Despite these rapid advances, most cancer patients generally do not respond to checkpoint blockade therapy. For example, approximately 20% of lung cancer patients respond to immune checkpoint therapy. Only 13%-18% of breast cancer and prostate cancer patients respond to immune checkpoint therapy (Nanda et al., 2016; Kwon et al., 2014). CAR-T cell immunotherapy technology is very effective in blood cancers (Sadelain et al., 2017; Johnson and June, 2017), but it is not very effective in solid cancers, which may be due to immunosuppression in the tumor microenvironment. Recent studies have shown that the clinical effectiveness of immune checkpoint blockade therapy depends on the presence of tumor-reactive T cells in tumor tissue and is associated with tumor-infiltrating T cells, PD-L1 expression, and mutation load (Sharma et al., 2017). Cancer patients whose tumor tissue lacks tumor-infiltrating antigen-specific T cells generally fail to respond to immune checkpoint therapy. To overcome these problems, cancer vaccines can increase tumor-specific T cells to control tumors. An alternative strategy is to adoptively transfer tumor-specific T cells that are derived from cancer patients or engineered to express tumor antigen-specific TCRs or CARs on T cells.

[0011] Immunotherapy using cancer vaccines offers the potential for high tumor-specific cytotoxicity and is therefore a very attractive approach to cancer treatment. In fact, the first therapeutic cancer vaccines (Sipuleu-cel-T; Previ Dendreon was approved by the FDA in 2010 for the treatment of metastatic prostate cancer (Kantov et al., 2010). However, cancer vaccines have generally achieved only limited clinical success. Even for the FDA-approved siprexa-T Vaccines, there is no obvious clinical response, but the survival of patients is extended to 4.1 months compared with the control group. Vaccination with antigenic peptides or dendritic cells (DCs) pulsed with antigenic peptides can produce anti-tumor immunity, but it has failed to produce sufficient immune responses and has not been able to obtain significant clinical benefits in several types of cancer tested (Melero et al., 2014; Rosenberg et al., 2004). DC / peptide or protein vaccines alone may not be strong enough to produce a potent and long-lasting anti-tumor response (Rosenberg et al., 2004).

[0012] Previous studies have shown that intracellular delivery of cancer antigen peptides such as tyrosinase-related protein 2 (TRP-2) to DCs via covalent cell-penetrating peptides (CPPs) enhances antigen-specific T cell responses and anti-tumor immunity against cancer, primarily due to prolonged antigen presentation time from DCs to T cells (Wang and Wang, 2002; Wang et al., 2002). Based on these preclinical studies, clinical studies using the TAT-NY-ESO-1 peptide were initiated, and this type of peptide vaccine was found to be safe and to induce antigen-specific T cell responses in 6 of 9 prostate cancer patients evaluated, which correlated with a two-fold increase in PSA in vaccinated patients (Sonpavde et al., 2014). However, the overall immune response was too weak and short-lived to induce cancer regression. Therefore, new strategies are urgently needed to develop more potent vaccines against cancer and other diseases.

[0013] Toll-like receptors (TLRs) have recently emerged as a crucial component of the innate immune system, detecting microbial infections and activating DC maturation programs to induce adaptive immune responses (Iwasaki and Medzhitov, 2004; Akira and Takeda, 2016). Triggering innate immune receptors in DCs (such as TLRs, Nod-like receptors (NLRs), and RIG-like receptors (RLRs)) with their corresponding ligands activates nuclear factor-κB (NFκB), type I interferons (IFNs), and inflammatory responses. These signaling pathways produce proinflammatory cytokines and induce robust innate and adaptive immune responses. Administering antigens together with TLR ligands can increase the antigen's immunogenicity and enhance the ability of DCs to elicit T cell responses (Blander and Medzhitov, 2006). Loading DCs with antigenic peptides and TLR ligands can be effective in generating strong T cell responses using peptides and TLR ligands (Palucka and Banchereau, 2013).

[0014] Nanotechnology, such as multistage vectors (MSVs), can load more antigens into nanoliposomes or nanoparticles and generate stronger antitumor immunity against breast cancer compared to traditional DC vaccines (Xia et al., 2015). In melanoma, it was found that the cancer antigen peptide TRP-2 must be co-loaded with TLR ligands (CpG and MPLA) into MSVs and then taken up by the same DCs as the vaccine (Zhu et al., 2018). Vaccination with a mixture of MSV / TRP-2-loaded DCs and DCs loaded with MSVTLR ligands did not produce potent antitumor immunity, indicating that co-delivery of peptides and TLR ligands is crucial (Zhu et al., 2018). However, despite advances in MSV technology, DC / MSV-based vaccination can only extend mouse survival for a limited period (10 days), further suggesting that DC / MSV-based vaccination only delays tumor growth but does not generate sufficient antitumor immunity to completely eliminate tumor cells.

[0015] Based on these studies, it is inferred that current vaccine strategies fail to generate sufficient immunity to completely eliminate cancer cells. Alternatively, immunosuppression in the tumor microenvironment inhibits the anti-tumor immunity induced by peptide vaccines. To understand why DCs loaded with CPP-linked therapeutic peptides fail to trigger anti-tumor immunity to eradicate cancer, it was found that CPP-linked therapeutic peptides promote intracellular delivery of antigenic peptides into DCs. Similarly, CPPs have been used to deliver various cargoes (including proteins, DNA, siRNA, and mRNA) into cells, into target cells. However, it was found that the CPP TAT-NY-ESO-1 peptide was difficult to produce a stable emulsion with the vaccine adjuvant Montanide ISA-51. Emulsion drops of TAT-NY-ESO-1 and Montanide ISA-51 are unstable in water and diffuse within a short period of time, which may affect vaccine efficacy. This unstable nature of TAT-NY-ESO-1 and Montanide ISA-51 prompted the inventors to further investigate how to overcome this problem. One potential problem is that the (hydrophilic) positive charge of the CPP (ie, TAT) may disrupt the emulsion of CPP-NY-ESO-1 and Montanide ISA-51.

[0016] A potent vaccine must contain innate immune signaling components. Recent studies by the inventors and their collaborators have shown that co-delivery of antigenic peptides and TLR ligands to the same DC is essential for generating a potent and effective immune response (Zhu et al., 2018). Although the MSV-based approach improves the co-delivery of antigenic peptides and nucleic acid-based TLR ligands to the same DC, it enhances anti-tumor immunity. However, this approach does not solve the fundamental problem, that is, due to the hydrophobicity of the peptide and the negatively charged nucleic acid, the antigenic peptide will not form a complex with the nucleic acid-based TLR3 and TLR9 ligands.

[0017] In view of the foregoing, there is a need for improved delivery methods for cancer therapeutic molecules comprising T cell epitopes, B cell epitopes, therapeutic nucleic acid molecules, and adjuvants.

[0018] 2. Peptide-based self-assembled nanostructures

[0019] Molecular self-assembly is the spontaneous formation of ordered structures, and it occurs under thermodynamic and kinetic conditions due to specific and local molecular interactions. Hydrogen bonding, hydrophobic interactions, electrostatic interactions, and van der Waals forces combine to maintain molecules in a stable, low-energy state. Self-association to form hierarchical structures occurs at both the nanoscale and / or microscale to achieve these energy minima (Han et al., 2010).

[0020] Self-assembly occurs spontaneously in nature during protein folding, DNA double helix formation, and cell membrane formation (Korolkov et al., 2013). Due to their biocompatibility and the ease of "bottom-up" fabrication (Yan et al., 2010), self-assembled nanostructures made from natural biomolecular building blocks such as amino acids are highly preferred as synthetic self-assembled monolayer (SAM) alternatives (Tayebe Zohrabi et al., 2015).

[0021] 3. Cell-penetrating peptides

[0022] Cell penetrating peptides (CPPs) are generally described as short peptides of 8-30 amino acids that can penetrate biological membranes to trigger the movement of various biomolecules across the cell membrane into the cytoplasm and improve their intracellular routes, thereby promoting interaction with targets (see, e.g., U.S. Patent No. 9,598,465). CPPs are derived from proteins or from chimeric sequences, are generally amphipathic and have a net positive 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). Tat peptides and membrane translocation sequences (MTS) have been used to transduce 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).

[0023] CPPs can be subdivided into two main categories, the first requiring chemical attachment to a cargo, and the second involving the formation of stable non-covalent complexes. CPPs have been used to deliver a wide range of cargoes (plasmid DNA, oligonucleotides, siRNA, PNA, proteins, peptides, liposomes, nanoparticles) into various cell types and in vivo models (Morris et al., 2008; Beggars and Sagan, 2013; Huang et al., 2015; Marcus et al., 2016; Gungor et al., 2014). In these cases, CPPs primarily deliver cargoes into cells via their membrane translocation ability ( Figure 1In these applications, there are no therapeutic T cell epitopes covalently linked to the CPP. Therefore, CPP does not have amphipathic properties and is different from CPP-T cell peptides, which have amphipathic properties to self-assemble into nanoparticles with negatively charged molecules. Summary of the Invention

[0024] The present invention overcomes these and other limitations inherent in the prior art by providing a novel vaccine with a targeted delivery system. Disclosed are self-assembling nanoparticles comprising a population of cationic cell-penetrating peptides (CPPs) linked to one or more hydrophobic therapeutic peptide ligands, including TLRs (toll-like receptors) and antigenic peptides. The amphiphilic nature of the resulting nanoparticles (i.e., having both a hydrophilic and a hydrophobic portion) further facilitates the inclusion of one or more therapeutic mRNAs, siRNAs, and / or DNA molecules. The resulting particles self-assemble at neutral pH and can be delivered to antigen-presenting cells (APCs) such as dendritic cells for presentation to T cells, thereby activating the immune system, or the particles can be delivered directly as a vaccine.

[0025] In a specific embodiment, the inventors have demonstrated that cationic CPPs each covalently linked to a certain therapeutic peptide (e.g., an antigenic peptide, which is preferably hydrophobic) can form compact and small-sized (50-100 nm) self-assembled nanoparticles and can be used to achieve effective intracellular delivery of therapeutic peptides. Other components such as negatively charged molecules (DNA, dsRNA, siRNA or mRNA) can be included in the nanoparticles to promote nanoparticle formation and nanoparticle delivery across the cell membrane (Figure 2). The nanoparticles include: (i) a crown including a CPP with a positively charged peptide, the CPP covalently linked to a therapeutic peptide with preferably hydrophobic properties, and (ii) a negatively charged molecule (DNA, dsRNA, siRNA or mRNA) plus a hydrophobic molecule such as MPLA. Based on the charge and hydrophobic properties, a new technology for self-assembling CPP-T cell peptide nanoparticles with TLR ligands [CpG and MPLA, abbreviated as CM; CpG and MPLA and poly (I: C), abbreviated as CMI] was designed and developed, such as Figure 2A and Figure 2B As schematically shown in FIG. , an amphiphilic or amphiphilic CPP-therapeutic peptide forms nanoparticles with negatively charged CpG and / or poly(I:C) through electrical interactions, and with MPLA through hydrophobicity within the particle. The CPP-therapeutic peptide is composed of a CPP such as TAT ​​with a positively charged peptide covalently linked to a therapeutic peptide (typically hydrophobic) such as NY-ESO-1 [SLLMWITQCFLPV (SEQ ID NO: 1)] and TRP-2 [SYVDFFVWL (SEQ ID NO: 2)]. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with one or more color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0027] The following drawings form part of this specification and are included to illustrate certain aspects of the present invention. To facilitate an understanding of the principles of the present invention, reference will now be made to the embodiments or examples illustrated in the drawings, and specific language will be used to describe them. However, 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 and any further applications of the principles of the present invention as described herein are contemplated as would normally occur to one of ordinary skill in the art to which the present invention relates.

[0028] The present invention may be better understood by reference to the following description taken in conjunction with the accompanying drawings, in which like reference numerals identify like elements, and in which:

[0029] Figure 1 demonstrated that CPPs function as carriers to deliver cargo into cells;

[0030] Figure 2A and Figure 2B It was shown that cationic CPP-T cell epitope peptides possess amphiphilic properties to form nanoparticles and be delivered into endosomes. Figure 2A Cationic CPP (positively charged) is covalently linked to a T cell epitope peptide (hydrophobic) to create amphipathic properties. CpG and poly(I:C) are negatively charged, while MPLA is hydrophobic. The CPP-T cell peptide forms nanoparticles with CpG and / or poly(I:C) through electrical interactions, while hydrophobic interactions with MPLA form nanoparticles within the particles. Figure 2B CPP-peptide / TLR ligand nanoparticles are taken up by DCs or macrophages and delivered to endosomes, where they are destroyed at pH 4.0. The CPP-peptide is processed by MHC class I or II molecules and presented to T cells, while the TLR ligand binds to TLR3, TLR4, and TLR9 to trigger innate immune responses and cytokine production, thereby improving the efficiency and quality of T cell responses.

[0031] Figure 3A-1 、 Figure 3A-2 、 Figure 3B-1 、 Figure 3B-2 、 Figure 3C-1 and Figure 3C-2Figure 2 shows self-assembled nanoparticles of TAT-TRP2 with CpG and MPLA (CM). TRP2 alone cannot form particles with CpG and MPLA. AFM analysis indicates the size of the nanoparticle cross-section.

[0032] Figure 4 The DLS measurements of the size distribution of the self-assembled nanoparticles of TAT-TRP2 and TLR ligands at different ratios are shown. The combination of TAT-TRP2 and TLR ligands (different ratios) is listed in Table 3. The grey bar indicates an unstable / polydisperse complex with a large PDI (PDI>0.5);

[0033] Figure 5 The zeta potential of the TAT-TRP2-CM complex composed of TAT-TRP2, CpG, and MPLA at various nitrogen (+) to phosphate (-) (N / P) ratios is shown;

[0034] Figure 6A and Figure 6B Characterization of the TAT-TRP2-CM complex is shown. DLS measurement of the size of the TAT-TRP2-CM complex in H2O ( Figure 6A ). TAT-TRP2 (or TRP2 in H2O TAT ), CpG, MPLA and the zeta potential of the TAT-TRP2-CM complex ( Figure 6B ). PDI, polydispersity index;

[0035] Figure 7A 、 Figure 7B 、 Figure 7C 、 Figure 7D 、 Figure 7E and Figure 7F Nanoparticles of TAT-TRP2 and TAT-ESO-1 with CpG and MPLA (TAT-TRP2-CM, TAT-ESO-1-CM) and nanoparticles of TAT-TRP2 and TAT-ESO-1 with CpG, MPLA and poly(I:C) (TAT-TRP2-CMI and TAT-ESO-1-CMI) are shown. Schematic representation of TAT-TRP2 and TAT-ESO-1 nanoparticles ( Figure 7A TAT-TRP2 and TAT-ESO-1 interact with CM ( Figure 7B ) or CMI( Figure 7C and Figure 7D ) of nanoparticle size. Figure 7C SEQ ID NO: 30 is disclosed. Figure 7E and Figure 7F: Zeta potential of TAT-TRP2-CM, TAT-TRP2-CMI, TAT-ESO-1-CM and TAT-ESO-1-CMI;

[0036] Figure 8A 、 Figure 8B and Figure 8C The characterization of the TAT-TRP2-CM complex at different pH values ​​is shown. ( Figure 8A and Figure 8B ) at pH 7.0( Figure 8A ) and pH 4.0( Figure 8C ) DLS measurement of the size of the TAT-TRP2-CM complex in potassium phosphate buffer. ( Figure 8C ) in pH 7.0 and pH 4.0 potassium phosphate buffers. TAT ), zeta potential of CpG, MPLA, and TAT-TRP2-CM complex. PDI, polydispersity index;

[0037] Figure 9A 、 Figure 9B 、 Figure 9C and Figure 9D The assembly of TAT-TRP2 / TLR and TAT-ESO-1 / TLR nanoparticles was shown to be pH dependent. Figure 9A Shown are DLS measurements of the size changes of the TAT-TRP2-CM complex in pH 4-7 potassium phosphate buffer. Figure 9B Schematic illustration of the process of TAT-TRP2 or TAT-TRP2-CM complex uptake and pH-dependent complex disassembly in DCs. Figure 9C and Figure 9D The results show that TAT-TRP2-CM ( Figure 9C ), TAT-ESO-1-CM, TAT-TRP2-CMI( Figure 9D ) and DLS measurements of size changes of the TAT-ESO-1-CMI complex;

[0038] Figure 10 shows a combination of TLR ligands for stimulating innate immune responses and cytokine production. Bone marrow-derived DCs were isolated and then treated with different TLR ligands (single, double, or triple combinations). The production of cytokines (TNF-α, IL-6, IFN-α, and IFN-β) in the cell supernatant was determined by ELISA. Poly(I:C) / CpG, CpG / MPLA double combinations, and CpG / poly(I:C) / MPLA triple combinations were more effective than other groups in triggering innate immune cytokine production.

[0039] Figure 11The lung metastasis model of B16 tumors in C57BL / 6 mice vaccinated with DC / TAT-TRP2-CM and DC / TRP2-CM vaccines is shown. B16 tumor cells (0.2×10 6 cells / mouse) were injected (intravenously) into C57BL / C mice and inoculated with DC / TAT-TRP2-CM, DC / TRP2-CM, or DC / β-gal-CM (5×10 6 The mice were sacrificed on day 18. The number of lung metastases was counted.

[0040] Figure 12A 、 Figure 12B and Figure 12C Shown are lung metastasis and survival rates of B16-bearing C57BL / 6 mice after various vaccinations. Figure 12A The tumor model and vaccine scheme are shown. Figure 12B On day 0, B16 tumor cells (0.2×10 6 cells / mouse) were injected (intravenously) into C57BL / C mice, and on day 5, the mice were injected with 5 different vaccine groups (5×10 6 All mice were sacrificed on day 18. The number of lung metastases was counted. Figure 12C B16 injection and vaccine are shown with Figure 12B Same as in . Survival of B16-bearing mice vaccinated with different groups of vaccines was monitored for 55 days. Error bars represent standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001;

[0041] Figure 13A and Figure 13B The results show that DC / TAT-ESO-CM and TAT-ESO-CMI vaccines produce strong anti-tumor immunity. HLA-A2 Tg mice were injected with RM1 / A2-ESO-1 tumor cells on day 0. Tumor-bearing mice were treated with the vaccine (DC / control, DC / TAT-ESO-CM, or DC / TAT-ESO-CMI). Tumor growth was monitored every two days. Figure 13A Tumor sizes on day 15 are shown. Figure 13B Tumor growth curves are shown. P values ​​between different groups are indicated;

[0042] Figure 14A and Figure 14B T cell responses in mice vaccinated with DC / control, DC / TAT-ESO-CM, or DC / TAT-ESO-CMI vaccines are shown ( Figure 14A ). Figure 14B CD8 +IFN-γ% in cT cells;

[0043] Figure 15 The results show significant inhibition of breast cancer growth following DC / TAT-ESO-CM vaccination. HLA-A2Tg mice were injected with E0771 / A2-ESO-1 tumor cells on day 0. Tumor-bearing mice were treated with either DC / control or DC / TAT-ESO-CM. Tumor growth was monitored every two days. Vaccination with the DC / TAT-ESO-CM vaccine significantly inhibited breast cancer growth.

[0044] Figure 16A 、 Figure 16B and Figure 16C It was shown that direct immunization with TAT-ESO-CMI generated potent therapeutic anti-tumor immunity compared to DC / TAT-ESO-CMI vaccination. Figure 16A In this study, HLA-A2 Tg mice were injected with RM1 / A2-ESO tumor cells on day 0 and then injected with TAT-ESO-CMI three times on days 10, 13, and 18 or vaccinated with DC / TAT-ESO-CMI vaccine on day 10. Tumor growth was monitored every two days. Figure 16B In the figure, the tumor size of each group is shown. Figure 16C In Figure 2, tumor growth of the three groups after vaccination was recorded. P values ​​of significance between the groups are shown.

[0045] Figure 17A 、 Figure 17B 、 Figure 17C and Figure 17D Shown is CT83 expression in breast cancer samples and cell lines. Figure 17A Shown is RF-PCR analysis of CT83 expressing breast cancer cell lines. Figure 17B Shown is the expression of CT83 in breast cancer samples analyzed using RT-PCR. NY-ESO-1 was used as a positive control. Figure 17C Western blot analysis of breast cancer cell lines using anti-CT83 antibody. Figure 17D Antibody staining of normal and breast cancer tissues for CT83 expression is shown in FIG.

[0046] Figure 18A and Figure 18B Shown is CT83 expression in lung cancer samples and cell lines. Figure 18A Shown are RF-PCR analyses of CT83 expressing lung cancer cell lines and cancer samples. Figure 18B Western blot analysis of CT83 expression in lung cancer cell lines using anti-CT83 antibodies is shown. MDA-468 was used as a positive control;

[0047] Figure 19A 、 Figure 19B 、 Figure 19C and Figure 19D Shown is the generation of CD83-specific T cells using self-assembled TAT-CT83 peptide nanoparticles with CMI. Figure 19A Shown is intracellular staining of IFN-γ released from splenocytes of mice immunized with TAT-CT83-CMI. Figure 19B IFN-γ release assay by ELISA is shown. Figure 19C Shown are T cell responses to CT83 peptide after one culture period. Figure 19D shows the establishment of CT83-A2 restricted peptide T cell clones;

[0048] Figure 20A 、 Figure 20B 、 Figure 20C and Figure 20D The results showed that TAT-CT83-CMI vaccine produced potent anti-tumor immunity against EO771-A2-CT83 breast cancer. Figure 20A ) Establishment of E0771-A2-CT83 breast cancer model in HLA-A2 transgenic mice and vaccination protocol. Figure 20B ) Images of EO771-A2-CT83 breast tumors after 2 immunizations with the indicated vaccine formulations alone or together with anti-PD1 blockade therapy (10 mg / kg BW, intraperitoneally). ( Figure 20C ) Tumors from mice vaccinated with the indicated formulations with or without anti-PD1 antibody were isolated on day 16 and their weights were measured. ( Figure 20D ) CD3 at tumor sites with or without TAT-CT83-CMI vaccination + immunohistochemical staining for T cell infiltration;

[0049] Figure 21A 、 Figure 21B 、 Figure 21C and Figure 21D It was shown that vaccination with TAT-ESO-CMI produces potent therapeutic immunity against breast cancer. Figure 21A ) Schematic representation of vaccine experimental design. ( Figure 21B ) shows the tumor growth of HLA-A2 Tg mice in each group. ( Figure 21C ) Images of mice and tumor size. Figure 21D ) TAT-ESO-CMI vaccination without DCs significantly inhibited tumor growth. *P < 0.05, **P < 0.01;

[0050] Figure 22A 、 Figure 22B and Figure 22CTAT-TRP2-CMI vaccine alone or in combination with anti-PD-1 therapy is shown. Figure 22A and Figure 22B ) Lung images and number of lung metastases in mice that received TAT-TRP-2 / CMI vaccination alone or in combination with anti-PD-1 treatment. ( Figure 22C ) Mouse survival after TAT-TRP-2 / CMI vaccination alone or in combination with anti-PD-1 therapy;

[0051] Figure 23A and Figure 23B TAT-ESO-CMI vaccine alone or in combination with anti-PD-1 therapy is shown. Figure 23A ) Tumor images of RM1 / A2-ESO tumor cells in HLA-A2-Tg mice that received TAT-ESO-CMI vaccination alone or in combination with anti-PD-1 treatment compared to the control group. ( Figure 23B ) Tumor growth curves after TAT-ESO-CMI vaccination alone or in combination with anti-PD-1 therapy compared with the control group;

[0052] Figure 24A and Figure 24B It was shown that TCR-T cell transfer followed by SAPNANO vaccine expanded tumor-infiltrating T cells and significantly inhibited tumor growth. Figure 24A ) Tumor growth in different treatment groups. *P value < 0.05. ( Figure 24B ) after administration of A2-ESO TCR-T cells alone or in combination with the TAT-ESO-CMI vaccine, the percentage of tumor-infiltrating A2-ESO TCR-T cells increased. A2-ESO TCR-T cells were detected using an anti-TCR human νβ13 antibody that was assayed on anti-CD3 positive T cells. Endogenous T cells induced by the TAT-ESO-CMI vaccine were not detected using the anti-TCR human νβ13 antibody; and

[0053] Figure 25A 、 Figure 25B 、 Figure 25C 、 Figure 25D and Figure 25E The combination of NY-ESO TCR-T therapy and TAT-ESO-CMI vaccination produced a strong anti-tumor response in a humanized NSG mouse model. Figure 25A ) Schematic diagram of animal experiments. 3 to 4 weeks before tumor incubation, 1×10 7Human PBMCs were used to humanize 3- to 4-week-old NSG mice to reconstitute the human immune system. HLA-A2 and NY-ESO-positive human breast cancer cells (MDA-MB-231-A2-ESO) were subcutaneously injected into the fat pads of humanized NSG mice (1 million per mouse). Tumor-bearing mice were treated with NY-ESO TCR-T cells on day 5. Three doses of human IL-2 (50,000 IU) and four doses of TAT-ESO-CMI vaccine were administered via intravenous injection. Mice were sacrificed on day 30. Figure 25B ) Human lymphocytes were detected by FACS in NSG mice 3 weeks after humanization. ( Figure 25C ) Tumor growth was monitored after treatment. Data are expressed as mean ± SEM. PBS control group (N = 4), other groups (N = 5), two-way ANOVA test was used for statistical analysis. *p < 0.05, **p < 0.01. ( Figure 25D and Figure 25E Tumors were imaged and weighed after isolation from mice. (Mean ± SEM, T-test was used for statistical analysis. *p < 0.05, **p < 0.01, ***p < 0.001)

[0054] Brief description of the sequence:

[0055] SEQ ID NO: 1 is an exemplary therapeutic NY-ESO-1 specific peptide for use according to one aspect of the present disclosure;

[0056] SEQ ID NO: 2 is an exemplary therapeutic TRP-2 specific peptide for use according to one aspect of the present disclosure;

[0057] SEQ ID NO: 3 is an exemplary HIV Tat 47-57 specific cell penetrating peptide sequence used according to one aspect of the present disclosure;

[0058] SEQ ID NO: 4 is an exemplary TAT-PTD-4 specific cell penetrating peptide sequence used according to one aspect of the present disclosure;

[0059] SEQ ID NO: 5 is an exemplary TAT-PTD-5 specific cell penetrating peptide sequence used according to one aspect of the present disclosure;

[0060] SEQ ID NO: 6 is an exemplary DPV3-specific cell-penetrating peptide sequence used according to one aspect of the present disclosure;

[0061] SEQ ID NO: 7 is an exemplary DPV6-specific cell-penetrating peptide sequence used according to one aspect of the present disclosure;

[0062] SEQ ID NO: 8 is an exemplary DPV7-specific cell-penetrating peptide sequence for use according to one aspect of the present disclosure;

[0063] SEQ ID NO: 9 is an exemplary nine-residue poly-arginine cell-penetrating peptide sequence for use in accordance with one aspect of the present disclosure;

[0064] SEQ ID NO: 10 is an exemplary nine-residue polylysine cell-penetrating peptide sequence for use in accordance with one aspect of the present disclosure;

[0065] SEQ ID NO: 11 is an exemplary FHV capsid-specific cell-penetrating peptide sequence used according to one aspect of the present disclosure;

[0066] SEQ ID NO: 12 is an exemplary signal peptide II-specific cell-penetrating peptide sequence used according to one aspect of the present disclosure;

[0067] SEQ ID NO: 13 is an exemplary amphiphilic model peptide-specific cell-penetrating peptide sequence for use according to one aspect of the present disclosure;

[0068] SEQ ID NO: 14 is an exemplary HSV VP22-specific cell-penetrating peptide sequence for use according to one aspect of the present disclosure;

[0069] SEQ ID NO: 15 is an exemplary peptide carrier-specific cell-penetrating peptide sequence for use according to one aspect of the present disclosure;

[0070] SEQ ID NO: 16 is an exemplary CL22-specific cell-penetrating peptide sequence for use according to one aspect of the present disclosure;

[0071] SEQ ID NO: 17 is an exemplary TRP-2 specific cell penetrating peptide sequence for use according to one aspect of the present disclosure;

[0072] SEQ ID NO: 18 is an exemplary TAT-linked peptide for use according to one aspect of the present disclosure;

[0073] SEQ ID NO: 19 is an exemplary TAT-linked peptide for use according to one aspect of the present disclosure;

[0074] SEQ ID NO: 20 is an exemplary TAT-linked peptide for use according to one aspect of the present disclosure;

[0075] SEQ ID NO: 21 is an exemplary TAT-linked peptide for use according to one aspect of the present disclosure;

[0076] SEQ ID NO: 22 is an exemplary TAT-linked peptide for use according to one aspect of the present disclosure;

[0077] SEQ ID NO: 23 is an exemplary TAT-linked peptide for use according to one aspect of the present disclosure; and

[0078] SEQ ID NO: 24 is an exemplary TAT-linked peptide for use in accordance with one aspect of the present disclosure.

[0079] SEQ ID NO: 25 is an exemplary TAT-linked peptide for use according to one aspect of the present disclosure;

[0080] SEQ ID NO: 26 is an exemplary TAT-linked peptide for use according to one aspect of the present disclosure;

[0081] SEQ ID NO: 27 is an exemplary TAT-linked peptide for use according to one aspect of the present disclosure; and

[0082] SEQ ID NO: 28 is an exemplary TAT-linked peptide for use according to one aspect of the present disclosure. DETAILED DESCRIPTION

[0083] Illustrative embodiments of the present invention are described below. For the sake of clarity, not all features of an actual implementation are described in this specification. It should of course be understood that in the development of any such actual implementation, many implementation-specific decisions must be made to achieve the developer's specific goals, such as complying with system-related and business-related constraints, which will vary from implementation to implementation. Furthermore, it should be understood that such development efforts may be complex and time-consuming, but will be routine work for those of ordinary skill in the art having the benefit of this disclosure.

[0084] Thus, in a first aspect, the present invention provides nanoparticles comprising a plurality of cationic CPPs each covalently linked to a hydrophobic therapeutic peptide, said nanoparticles self-assembling under neutral conditions (e.g., at pH 7.0) and dissociating under acidic conditions (e.g., at pH 4.5). Figure 2A and Figure 2B ).

[0085] 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, transporter, amphipathic model peptide, HSV VP22 and CL22. In some embodiments, the cationic CPP consists of 8-30 amino acids. In one embodiment, the cationic CPP is Tat.

[0086] 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 9-25 amino acids. Antigenic peptides or non-immunogenic peptides containing T cell epitopes are involved in specific diseases such as tumors and infectious diseases.

[0087] The nanoparticles of the present invention may further include 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, a Poly(I:C), or a combination thereof. In some embodiments, the CpG oligodeoxynucleotide is 20-24 bp in length. In some embodiments, the Poly(I:C) is 0.2 kb to 1 kb in length.

[0088] The nanoparticles disclosed herein may also carry at least one hydrophobic molecule, preferably a hydrophobic TLR ligand, non-covalently bound to a therapeutic peptide. In some embodiments, the hydrophobic molecule is monophosphoryl lipid A (MPLA), R848, or a combination thereof.

[0089] Nanoparticles disclosed herein can be taken up by cells. In some embodiments, nanoparticles containing antigenic peptides or non-immunogenic peptides with T cell epitopes can be taken up by APCs, particularly DCs or macrophages, in vitro and in vivo. In some embodiments, TLR ligands activate one or more TLR signaling pathways.

[0090] In another aspect, the present disclosure provides a pharmaceutical composition comprising the nanoparticles of the present invention and a pharmaceutically acceptable carrier, preferably at about pH 7.0.

[0091] In a third aspect, the present disclosure also provides a composition for producing the above-mentioned nanoparticles, the composition comprising a cationic CPP each covalently linked to a hydrophobic therapeutic peptide and optionally at least one negatively charged molecule and / or at least one hydrophobic molecule.

[0092] Before the nanoparticles are administered, the components in the composition can be mixed in a medium of about pH 7.0. The nanoparticles self-assemble in the medium, are taken up by APC in vitro by endocytosis, and are then used for vaccines. Alternatively, self-assembling nanoparticles can be prepared and delivered to animals, where they are taken up by DCs or macrophages in the animal body. Regardless of whether DCs or macrophages take up nanoparticles in vitro or in vivo, the self-assembling nanoparticles will enter endosomes or lysosomes, where they will be destroyed at pH 4.5; the peptides and TLR ligands connected by CPPs are released. When the TLR3, TLR7, TLR8, and TLR9 localized in the endosomal region bind to the TLR ligands to trigger innate immune signaling, the peptides connected by CPPs will bind to the MHC class II molecules in the endosomes for loading and presentation, or pass through the endosomal membranes into the cytoplasm, ER, and Golgi apparatus for antigen processing and presentation to T cells by MHC class I molecules.

[0093] 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, transporter, amphipathic model peptide, HSV VP22 and CL22. In some embodiments, the cationic CPP consists of 8-30 amino acids. In one embodiment, the cationic CPP is Tat. In some embodiments, the therapeutic peptide is an antigenic peptide or a non-immunogenic peptide 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 8, 9, 10 or 11 amino acids presented by MHC class I molecules or 9-25 amino acids presented by MHC class II molecules. Antigenic peptides or non-immunogenic peptides containing T cell epitopes are related to specific diseases such as tumors and infectious diseases.

[0094] 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 length of the CpG oligodeoxynucleotide is 15bp to 24bp. In some embodiments, the length of the Poly (I: C) is 0.2kb to 1kb. In some embodiments, the hydrophobic molecule is a hydrophobic TLR ligand non-covalently. In some embodiments, the hydrophobic molecule is monophosphoryl lipid A (MPLA), R848 or a combination thereof.

[0095] In a fourth aspect, the present disclosure provides a method for treating, preventing and / or ameliorating at least one symptom of a cancer or infectious disease. In a general and general sense, the method generally comprises providing a therapeutically effective amount of a pharmaceutical formulation comprising the nanoparticles disclosed herein to a subject in need thereof.

[0096] In some embodiments, cancer can be a dangerous tumor, and such tumors can be solid or non-solid in composition, depending on the particular disease. In certain embodiments, the cancerous tumor to be treated is a primary tumor or a metastatic tumor, such as, but not limited to, one or more melanomas or lung cancers.

[0097] In other embodiments, treatment of disease is envisaged, in particular, for example, treatment of one or more viral, fungal and / or bacterial infections.

[0098] Antigenic peptides or non-immunogenic peptides containing T cell epitopes in nanoparticles are processed by APCs, particularly DCs or macrophages, and presented to T cells for a long time via newly synthesized MHC class II molecules. TLR ligands contained in the nanoparticles stimulate DCs or immune cells to produce innate immune responses, such as the release of type I interferon cytokines, which enhance the ability of DCs to present epitopes to T cells and co-stimulate T cell activation, as well as the stimulation of T cell growth and expansion by cytokines.

[0099] The nanoparticles disclosed herein can enhance the co-delivery of antigenic peptides or non-immunogenic peptides containing T cell epitopes with two or more TLR ligands to the same antigen-presenting cells, such as DCs or macrophages, thereby generating a potent and effective immune response. The resulting anti-tumor or anti-pathogen effects can be superior to those of other delivery platforms, such as MSV.

[0100] Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. However, it should be understood that although the detailed description and specific examples indicate preferred embodiments of the present invention, they are given by way of illustration only, as various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art based on this detailed description.

[0101] Drug formulations

[0102] In certain embodiments, the present disclosure relates to self-assembling nanoparticle compositions prepared in the form of pharmaceutically acceptable formulations for administration to one or more cells or tissues of an animal, alone or in combination with one or more other diagnostic, preventive and / or therapeutic modalities. The formulation of pharmaceutically acceptable excipients and carrier solutions is well known to those of ordinary skill in the art, as is the development of suitable dosages and treatment regimens for using the self-assembling nanoparticle compositions described herein in a variety of therapeutic, preventive, diagnostic and prognostic protocols.

[0103] In certain cases, it is desirable to deliver a suitably formulated pharmaceutical vehicle containing the disclosed self-assembling nanoparticle compositions to one or more cells, tissues, or organs in or around the body of an animal by one or more standard delivery devices, including but not limited to subcutaneously, parenterally, intravenously, intramuscularly, intrathecally, intratumorally, intraperitoneally, transdermally, topically, by oral or nasal inhalation, or by direct injection.

[0104] The method of administration can also include those described in U.S. Patent Nos. 5,543,158, 5,641,515 and 5,399,363, each of which is incorporated herein by reference in its entirety. Solutions of the active compound in the form of a free alkali or a pharmaceutically acceptable salt can be prepared in sterile water and can be suitably mixed with one or more surfactants such as hydroxypropyl cellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, oil or a mixture thereof. Under normal storage and use conditions, these preparations contain preservatives to prevent microbial growth.

[0105] For the use of injectable aqueous solution, be not limited to that, if necessary, described solution can be compatibly buffered, and liquid diluent is first isotonic with enough saline or glucose.These specific aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous (subcutaneous), percutaneous, subcutaneous (subdermal) and / or intraperitoneal administration.In this respect, compositions of the present invention can be deployed in one or more pharmaceutically acceptable vehicles, and described vehicle comprises for example sterile aqueous medium, buffer, diluent etc.For example, the active component of given dose can be dissolved in the isotonic solution (for example, isotonic NaCl base solution) of specific volume, and then inject in the application site of suggestion, or further dilute (referring to for example, " Lei Mingdun pharmaceutical science (REMINGTON ' S PHARMACEUTICAL SCIENCES) " the 15th edition, 1035-1038 page and 1570-1580 page) in the vehicle being applicable to intravenous infusion. While some variation in dosage will inevitably occur depending on the condition of the subject being treated, the extent of the treatment, and the site of administration, the person responsible for administration will be able to determine the proper dosage regimen for an individual subject using ordinary knowledge in the medical and pharmaceutical arts.

[0106] Sterile injectable compositions can be prepared by incorporating the disclosed self-assembling nanoparticle compositions in the desired amount into an appropriate solvent along with several other ingredients enumerated above, as needed, followed by filtration sterilization. Typically, dispersions can be prepared by incorporating the selected sterilized active ingredient into a sterile vehicle containing a base dispersion medium and the desired other ingredients from those enumerated above. The self-assembling nanoparticle compositions disclosed herein can also be formulated in neutral or salt form.

[0107] Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of proteins), and pharmaceutically acceptable salts are formed with inorganic acids such as, but not limited to, hydrochloric acid or phosphoric acid, or organic acids such as, but not limited to, acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. The salts formed with free carboxyl groups can also be derived from inorganic bases such as, but not limited to, sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc. After formulation, the solution will be applied in a manner compatible with the dosage formulation and in an amount effective for the intended application. The formulations of the compounds of the present invention can be applied in various dosage forms, such as injectable solutions, topical preparations, oral formulations, including sustained-release capsules, hydrogels, colloids, viscous gels, transdermal agents, intranasal formulations, and inhalation formulations.

[0108] The amounts, dosage regimens, formulations, and administration of the self-assembling nanoparticle compositions disclosed herein will be within the capabilities of those of ordinary skill having the benefit of the present teachings. However, it is likely that administration of a diagnostically effective (i.e., pharmaceutically effective) amount of one or more of the disclosed compositions can be achieved through a single administration, such as without limitation to a single injection of a sufficient amount of the delivered agent to provide the desired benefit to a patient in need thereof. Alternatively, in some cases, it may be desirable to provide multiple or continuous administrations of the disclosed self-assembling nanoparticle compositions over a relatively short or even relatively long period of time, as may be determined by a medical practitioner overseeing the administration of such compositions to an individual selected to undergo such a procedure, treatment, therapy, or diagnosis.

[0109] Typically, the formulation of one or more of the self-assembling nanoparticle compositions described herein will contain at least an effective amount of the first active agent. Preferably, the formulation may contain at least about 0.001% of each active ingredient, preferably at least about 0.01% of the active ingredient, but the percentage of the active ingredient can of course vary and can conveniently be present in an amount of about 0.01 to about 90% by weight or volume, or about 0.1 to about 80% by weight or volume, or more preferably about 0.2 to about 60% by weight or volume of the total formulation. Naturally, the amount of active ingredient in each composition can be prepared in such a way that a suitable dosage will be obtained in any given unit dose of the compound. One of ordinary skill in the art of preparing such pharmaceutical formulations will envision properties such as solubility, bioavailability, bioavailability, etc. 1 / 2 , route of administration, product shelf life, and other pharmacological considerations, and as such, various dosages and treatment regimens may be desired.

[0110] While it is contemplated that systemic administration is effective in many embodiments of the present invention, it is also contemplated that the formulations disclosed herein are suitable for direct injection into one or more organs, tissues, or cell types in the body. For example, the disclosed nanoparticles can be administered directly to a specific discreet location in the body, or directly to a tumor, tumor stem cell, cancerous tissue, and / or cancer stem cell using suitable means known to those of ordinary skill in the relevant medical oncology fields.

[0111] Pharmaceutical formulations comprising one or more of the self-assembling nanoparticle compositions disclosed herein may further comprise one or more excipients, buffers, or diluents specifically formulated for contact with mammalian cells, and in particular human cells, and / or for administration to mammalian subjects such as human patients. The compositions may further optionally comprise one or more diagnostic or prognostic agents, and / or may be formulated with additional microsphere populations, microparticle populations, nanosphere populations, or nanoparticle populations, or may be formulated to contain one or more additional therapeutic and / or diagnostic agents for administration to one or more cells, tissues, organs, or bodies of mammalian patients (and in particular human patients).

[0112] The formulation of pharmaceutically acceptable excipients and carrier solutions is well known to those skilled in the art, as is the development of suitable dosage, diagnostic and / or therapeutic regimens using the specific self-assembling nanoparticle compositions described herein in a variety of ways (including, for example, but not limited to, oral, parenteral, intravenous, intranasal, intratumoral, and intramuscular administration routes).

[0113] The specific amount of the self-assembling nanoparticle composition employed and the specific time of administration or dosage regimen of the composition using the disclosed formulation will be within the ability of one of ordinary skill in the art having the benefit of the present teachings. However, it is likely that administration of the disclosed formulations can be achieved by administering one or more doses of the formulation over a period of time effective to provide the desired benefit to the patient receiving such treatment. Such dosage regimens can be determined by the medical practitioner supervising the administration of the compound, depending on the specific condition or patient, the extent or duration of the therapy being administered, etc.

[0114] The pharmaceutical formulations comprising one or more self-assembling nanoparticle compositions as disclosed herein are not in any way limited to being used only for humans, or even primates or mammals. In certain embodiments, birds, amphibians, reptiles or other animal species can be used to adopt the methods and compositions disclosed herein. However, in a preferred embodiment, the compositions of the present disclosure are preferably formulated for use in various schemes for diagnosing, improving and / or treating one or more diseases in a patient's body and in particular for treating one or more types of tumors or cancer cells or for treating one or more infections to mammals and in particular to humans. As described above, such compositions are not limited only to humans, but can also be formulated for use in veterinary applications including but not limited to selected livestock, exotic species or domestic animals, companion animals (including pets, etc.), non-human primates, and animal specimens or other captive specimens.

[0115] Composition for preparing medicine

[0116] Another important aspect of the present invention relates to methods for using the disclosed self-assembling nanoparticle compositions (and formulations comprising the same) in the preparation of a medicament for preventing, diagnosing, treating, and / or ameliorating one or more symptoms of one or more diseases, disorders, abnormal conditions, or conditions in animals (including, for example, vertebrate mammals). It is particularly contemplated to use the disclosed self-assembling nanoparticle compositions for the diagnosis and / or prognosis of cancer, the detection and / or prediction of cancer metastasis, or for monitoring its extent and / or for treating one or more abnormal conditions, such as in vivo, ex vivo, and / or in situ treatment of one or more cancer cell types.

[0117] Such use generally involves administering to a mammal in need thereof one or more of the disclosed self-assembling nanoparticle compositions comprising at least a first active agent in an amount sufficient and for a time sufficient to diagnose, treat, alleviate, or ameliorate one or more symptoms of tumor formation or cancer growth and / or metastasis in the infected mammal. Pharmaceutical formulations comprising one or more of the disclosed self-assembling nanoparticle compositions also form part of the present disclosure, and particularly those compositions further comprising at least a first pharmaceutically acceptable excipient for the treatment and / or amelioration of one or more symptoms of cancer in an infected mammal.

[0118] Self-assembled nanoparticles

[0119] The present disclosure describes the use of cationic cell penetrating peptides (each covalently linked to a hydrophobic therapeutic peptide, and optionally at least one negatively charged molecule and / or at least one hydrophobic molecule) to form nanoparticles. The resulting nanoparticles have: (i) a core comprising the hydrophobic therapeutic peptide and optionally the hydrophobic molecule, and (ii) a corona comprising the CPP and optionally the negatively charged molecule.

[0120] The negatively charged molecule may be a TLR ligand, such as CpG oligodeoxynucleotide, Poly(I:C), DNA and RNA (mRNA or siRNA). The hydrophobic molecule may preferably be a hydrophobic TLR ligand, which may be monophosphoryl lipid A (MPLA) or R848.

[0121] While not wishing to be bound by any theory, it is believed that due to the hydrophobicity of the therapeutic peptide (and optional hydrophobic molecules) and the hydrophilicity of the CPP, self-assembly occurs in aqueous solution at approximately pH 7.0 through electronic bonding of positively and negatively charged molecules.

[0122] The amounts of components used to form the nanoparticles of the present invention can be determined by one skilled in the art. In the case of more negatively charged molecules, more CPPs will participate in nanoparticle formation. Furthermore, due to different zeta potentials, varying the amounts of some components can alter the size and shape of the nanoparticles, as well as their in vivo biodistribution.

[0123] In one embodiment, a cationic CPP (positively charged)-antigenic peptide or a weakly immunogenic peptide containing a T cell epitope (usually hydrophobic) has been designed and synthesized and mixed with a CpG oligonucleotide (negatively charged) and monophosphoryl lipid A (MPLA, hydrophobic) in phosphate buffered saline (PBS). Although both CPP-antigenic peptide (10 mM) and CpG (10 mM) are soluble in PBS, once mixed (1:1), precipitation or aggregates are observed. These aggregates are round nanoparticles with a diameter of 100 nM. Self-assembly is believed to occur through electrical interactions of the positively charged CPP with negatively charged molecules and hydrophobic interactions between the peptide itself and monophosphoryl lipid A (MPLA). Further studies using different ratios (positive: negative charge or molar concentration) found that different ratios of CPP-therapeutic peptide, CpG and MPLA can produce nanoparticles of 100 nM size at pH 7, but the zeta potential (surface charge) and assembly efficiency of each component are different.

[0124] As described above, the nanoparticles of the present invention self-assemble under neutral conditions (e.g., at pH 7.0) and are destroyed under acidic conditions (e.g., at pH 4.5). Therefore, the nanoparticles are delivered to dendritic cells or macrophages in the form of compact and small-sized particles, and are then destroyed in the endosomes where the pH becomes 4.5, thereby releasing the CPP and therapeutic peptides, preferably antigenic peptides or non-immunogenic peptides containing T cell epitopes, and other molecules, preferably TLR ligands, into the cytoplasm. Thereafter, the antigenic peptides or non-immunogenic peptides containing T cell epitopes are bound to MHC class I or class II molecules and the epitopes are presented to T cells, while the TLR ligands are bound to TLRs to trigger TLR-mediated signaling pathways (NF-κB and type I interferons), thereby producing proinflammatory cytokines and inducing strong innate and adaptive immune responses.

[0125] Chemotherapy and uses

[0126] Important aspects of the present disclosure relate to methods for treating or ameliorating symptoms of one or more forms of cancer using the disclosed self-assembling nanoparticle formulations, including, for example, tumors or metastatic cancers, such as, but not limited to, melanoma metastases to the lungs of mammals. Such methods generally involve administering to a mammal (and particularly to a human in need thereof) one or more of the disclosed self-assembling nanoparticle compositions comprising at least a first anti-cancer therapeutic agent in an amount sufficient and for a time sufficient to treat (or alternatively ameliorate) (one or more symptoms of) cancer in the affected mammal.

[0127] In certain embodiments, the self-assembling nanoparticle compositions described herein may be provided to animals as a single treatment (as a single administration, or alternatively, multiple administrations over a period of time ranging from several hours (hrs) to several days (or even several weeks or months), as needed to treat a particular disease, condition, dysfunction, or abnormal condition. Alternatively, in some embodiments, it may be desirable to continue treatment or combine it with one or more additional therapy modalities for a period of several months or longer. In other embodiments, it may be desirable to provide therapy in combination with one or more conventional treatment regimens.

[0128] The present disclosure also provides the use of one or more of the disclosed self-assembling nanoparticle compositions in the manufacture of a medicament for treating and / or ameliorating one or more symptoms of infection or cancer, and in particular for the manufacture of a medicament for treating and / or ameliorating one or more symptoms of mammalian infection or cancer, including, for example, human infection, cancerous tumors, etc.

[0129] The present invention also provides the use of one or more of the disclosed self-assembling nanoparticle compositions in the manufacture of a medicament for treating a disease or condition in a mammal, and in particular for treating one or more human diseases such as infection and / or cell hyperproliferation (i.e., cancer).

[0130] Therapeutic kits

[0131] Therapeutic kits comprising one or more of the disclosed self-assembling nanoparticle compositions and instructions for using the kit in a specific therapeutic manner also represent preferred aspects of the present disclosure. These kits may further optionally comprise one or more additional therapeutic compounds, one or more diagnostic agents, or any combination thereof.

[0132] The kit of the present invention can be packaged for commercial distribution and can further optionally include one or more delivery devices (e.g., syringes, injections, etc.) suitable for delivering the self-assembling nanoparticle composition to an animal. Such kits typically contain at least one vial, test tube, flask, bottle, syringe or other container in which the self-assembling nanoparticle composition can be placed and preferably appropriately aliquoted. In the case where a second drug is also provided, the kit may also contain a second different container in which the second composition can be placed. Alternatively, a variety of self-assembling nanoparticles as disclosed herein can be prepared into a single mixture such as a suspension or solution, and the plurality of self-assembling nanoparticles can be packaged in a single container, such as a vial, flask, syringe, catheter, cannula, bottle or other suitable single container.

[0133] The kits of the present invention may also typically include a holding mechanism suitable for containing or maintaining vials or other containers in a sealed space for commercial sale, for example, injection- or blow-molded plastic containers, in which the desired vials or other containers may be retained to minimize or prevent breakage, exposure to sunlight or other undesirable factors, or to allow immediate use of the composition contained in the kit.

[0134] Cell-penetrating peptides

[0135] Cell-penetrating amphiphilic peptides such as HIV-Tat-based peptides and chimeric cell-penetrating peptides have also been applied to deliver therapeutic cargoes to their targets (Magzoub et al., 2004).

[0136] CPPs have long been used as drug delivery vehicles because they can translocate cell membranes (Gupta et al., 2005). Short cationic peptide CPPs of less than 30 amino acids and polyarginine-based CPPs with a length of 8-10 arginine residues have shown the most efficient membrane penetration (Fuchs et al., 2006).

[0137] According to the classical mechanism (Fuchs et al., 2006), membrane penetration of CPPs is based on hydrogen bonding interactions between the guanidinium groups of arginine residues and the carboxyl, phosphoryl, or sulfonyl groups of carbohydrates and phospholipids on the cell surface. Initially, the pathways by which CPPs translocate across membranes were defined by mechanisms independent of receptors and endocytosis, but now, novel mechanisms of CPP internalization have been demonstrated.

[0138] 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.

[0139] Some examples of cell penetrating peptides include, but are not limited to:

[0140] 1.YGRKKRRQRRR(HIV Tat 47-57)(SEQ ID NO:3);

[0141] 2YARAAARQARA(TAT-PTD-4)(SEQ ID NO:4);

[0142] 3.YARAARRAARR(TAT-PTD-5)(SEQ ID NO:5);

[0143] 4.RKKRRRESRKKRRRES(DPV3)(SEQ ID NO:6);

[0144] 5.GRPRESGKKRKRKRLKP(DPV6)(SEQ ID NO:7);

[0145] 6.GKRKKKGKLGKKRDP(DPV7)(SEQ ID NO:8);

[0146] 7. RRRRRRRRR (polyarginine, R9) (SEQ ID NO: 9);

[0147] 8. KKKKKKKKK (polylysine, K9) (SEQ ID NO: 10);

[0148] 8. RRRRNRTRRNRRRVR (FHV shell) (SEQ ID NO: 11);

[0149] 9. GALFLGWLGAAGSTMGAWSQPKKKRKV (signal peptide II) (SEQ ID NO: 12);

[0150] 10. KLALKLALKALKAALKLA (amphiphilic model peptide) (SEQ ID NO: 13);

[0151] 11.DAATATRGRSAASRPTERPRAPARSASRPRRPVE (HSV VP22) (SEQ ID NO: 14);

[0152] 12. KETWWETWWTEWSQPKKKRKV (peptide carrier) (SEQ ID NO: 15); and

[0153] 13. KKKKKKGGFLGFWRGENGRKTRSAYERMCNILKGK (CL22) (SEQ ID NO: 16).

[0154] A complete list of known CPPs can be found online at the publicly available CPPsite 2.0 website, which is an updated version of the Cell Penetrating Peptide Database (CPPsite).

[0155] In the present disclosure, cationic CPPs are covalently linked to T cell epitope peptides and present at the corona portion of the nanoparticles. The positively charged or nearly neutral corona enables the nanoparticles to attach to negatively charged cell surfaces and then be taken up by cells such as DCs or macrophages with improved uptake efficiency.

[0156] CPPs also help antigenic peptides or weakly immunogenic peptides containing T cell epitopes linked thereto to bind directly to MHC class II molecules in endosomes for antigen presentation on the cell surface, or escape from endosomes and then enter the ER and Golgi apparatus, where antigenic peptides or non-immunogenic peptides containing T cell epitopes bind to newly synthesized MHC class I molecules for presentation on the cell surface to activate T cells.

[0157] As described above, the present invention requires a cationic CPP. However, as is well known to those skilled in the art, non-cationic CPPs can be modified by adding or attaching amino acids such as Lys, Arg, and His to the backbone. For example, poly-Lys peptides or poly-Arg peptides are synthesized as cationic CPPs.

[0158] Antigenic peptide

[0159] In the context of the present disclosure, the term "antigenic peptide" refers to a peptide antigen that is shared by a specific tumor or pathogen and that binds to an MHC molecule.

[0160] The tumor antigens disclosed herein are preferably derived from cancer, including but not limited to primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, leukemia, uterine cancer, cervical cancer, bladder cancer, kidney cancer and adenocarcinoma, such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, etc. In one embodiment, the tumor antigens disclosed herein include one or more cancer antigen epitopes that are immunologically recognized by tumor infiltrating lymphocytes (TILs) derived from mammalian cancerous tumors.

[0161] Malignant tumors express many 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 GP 100 in melanoma, and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. 4Other target molecules belong to the group of transformation-related molecules, such as the oncogenes HER-2 / Neu / ErbB-2. Yet another group of target antigens are carcinoembryonic antigens, such as carcinoembryonic antigen (CEA).

[0162] Examples of tumor antigens such as cancer testis antigens and mutation-derived neoantigens include, but are not limited to, NY-ESO-1, CT83, MAGE gene family, and neoantigens 4 .

[0163] Similarly, oncogene product peptide antigens common to specific tumor types have been identified. These polypeptides will be used as agents in the polypeptide complexes of the present invention, which can generally be used to stimulate T cell responses to effectively react with tumors carrying such antigens, including but not limited to HER-2 / neu associated with human breast cancer and gynecological cancer, and carcinoembryonic antigen (CEA) associated with pancreatic cancer.

[0164] Tumor antigens and their cancer antigen epitopes can be purified and isolated from natural sources, such as from primary clinical isolates, cell lines, etc. Cancer peptides and their antigenic epitopes can also be obtained by chemical synthesis or by recombinant DNA technology 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).

[0165] In addition, there are many antigens known in the art, as described in Renkvist et al. (2001). PCT International Patent Application Publication No. WO 02 / 064057 lists antigens encoded by tumors and targeted by T cells (cytotoxic CD8 + or helper CD4 + ) and the PCT International Patent Application Publication is expressly incorporated herein by reference in its entirety.

[0166] Although analogs or artificially modified epitopes are not listed, the skilled person knows how to obtain or generate analogs or artificially modified epitopes by standard means in the art. Other antigens identified by antibodies and detected by Serex technology are identified in the database of the Ludwig Institute for Cancer Research [see Sahin et al., (1997) and Chen et al., (2000)], which can be easily found on the World Wide Web.

[0167] Antigenic peptides of the present invention need to be hydrophobic so that they are encapsulated in nanoparticles and delivered to endosomes. As is well known to those skilled in the art, non-hydrophobic antigenic peptides can be modified to increase the antigenicity of the peptide by adding or attaching one or more amino acids such as Gly, Ala, Val, Leu, Ile, Pro, Phe, Met and Trp to the backbone.

[0168] T cell epitopes

[0169] The immunogenic peptides forming the core portion of the nanoparticles of the present invention include T cell epitopes.

[0170] Since T cell epitopes do not need to be displayed on the surface of the carrier to elicit immunity, T cell epitopes can be incorporated into the core of the nanoparticle.

[0171] T cell epitopes can be selected from different sources. For example, T cell epitopes can be determined experimentally. Such epitopes are known in the literature and can also be predicted by algorithms based on existing protein sequences of specific pathogens or cancer antigens, or such epitopes can be designed from scratch.

[0172] A large number of known T cell epitopes can be obtained in the scientific literature. These T cell epitopes can be selected from specific pathogens, cancer-specific peptide sequences, or the T cell epitopes can be peptides designed from scratch with specific characteristics, for example, PADRE peptides (see, for example, U.S. Patent No. 5,736,142, which is expressly incorporated herein by reference in its entirety), which bind to many different MHC II molecules, making them so-called promiscuous T cell epitopes. There are commonly available databases containing thousands of different T cell epitopes, such as the MHC database "MHCBN version 4.0" or the PDB database "Protein Data Bank" etc.

[0173] It is well known and documented that incorporating helper T cell (HTL) epitopes into otherwise non-immunogenic peptide sequences or attaching them to non-peptide antigens can make these HTL epitopes more immunogenic. The Pan-DR binding peptide HTL epitope PADRE has been widely used in vaccine design for malaria, Alzheimer's, and many other vaccines.

[0174] According to the definition of the MHCBN database (supra), a T cell epitope is a binding affinity (IC 50 Peptides with a β-catenin (β-catenin) value less than 50,000 nM are considered MHC binders. Based on this definition, as of August 2006, the following data were available in the MHCBN database version 4.0: 20,717 MHC binders and 4,022 MHC non-binders.

[0175] Suitable T cell epitopes can also be obtained by using prediction algorithms. These prediction algorithms can scan existing protein sequences from pathogens or cancer antigens to find putative T cell epitopes, or they can predict whether a de novo designed peptide will bind to a specific MHC molecule. Many such prediction algorithms are generally available on the internet. Examples are SVRMHCdb (Wan et al., 2006), SYFPEITHI, MHCPred, motif scanners, or NetMHCIIpan for MHC II binding molecules and NetMHCpan for MHC I binding epitopes.

[0176] The HTL epitopes as described herein and preferably used for design are peptide sequences that bind to the HTL with a binding affinity (IC) higher than 500 nM as measured by biophysical methods or predicted by NetMHCIIpan. 50 These are considered weak binders. Preferably, these epitopes bind to any MHC II molecule with an IC higher than 50 nM as measured by biophysical methods or predicted by NetMHCIIpan. 50 Values ​​bind to MHC II molecules. These are considered strong binders.

[0177] T cell epitopes can be incorporated into several places within non-immunogenic peptides. To achieve this, the specific sequence with the T cell epitope must comply with the MHC binding rules. The rules for binding to MHC molecules are incorporated into MHC binding prediction programs, which use complex algorithms to predict MHC binding peptides.

[0178] There are many different HLA molecules, each of which has amino acid restrictions in its sequence to which it will bind optimally. Binding motifs are summarized in Table 3 of U.S. Patent No. 8,546,337, which is expressly incorporated herein by reference in its entirety. In the table, the motif indicates positions with an x ​​that can have any amino acid, and in square brackets indicates (a list of) amino acids that can only be located at specific positions in the binding motif.

[0179] Generally, in the present invention, the non-immunogenic peptide containing a T cell epitope needs to be hydrophobic so that it can be encapsulated in the nanoparticle and delivered to the endosome. As is well known to those skilled in the art, the non-hydrophobic non-immunogenic peptide containing a T cell epitope can be modified by adding or attaching amino acids such as Gly, Ala, Val, Leu, Ile, Pro, Phe, Met and Trp to the backbone.

[0180] T OLL TLR-like receptors and TLR signaling

[0181] 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 infection. TLRs recognize highly conserved structural motifs called pathogen-associated microbial patterns (PAMPs), which are expressed only by microbial pathogens, or danger-associated molecular patterns (DAMPs), which are endogenous molecules released from necrotic or dying cells.

[0182] TLRs include TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, and TLR13, although the latter two are not found in humans.

[0183] TLRs are expressed in innate immune cells such as dendritic cells (DCs) and macrophages, as well as non-immune cells such as fibroblasts and epithelial cells. TLRs are mainly classified into two subfamilies based on their localization, cell surface TLRs and intracellular TLRs. Cell surface TLRs include TLR1, TLR2, TLR4, TLR5, TLR6, and TLR10, while intracellular TLRs are located in endosomes and include TLR3, TLR7, TLR8, TLR9, TLR11, TLR12, and TLR13.

[0184] Stimulation of TLRs by corresponding PAMPs or DAMPs initiates a signaling cascade, leading to the activation of transcription factors such as AP-1, NF-κB, and interferon regulatory factors (IRFs). TLR signaling triggers a variety of cellular responses, including the production of interferons (IFNs), proinflammatory cytokines, and effector cytokines that direct adaptive immune responses.

[0185] TLR signaling consists of at least two distinct pathways: a MyD88-dependent pathway that results in the production of inflammatory cytokines, and a TRIF-dependent pathway that is associated with stimulation of IFN-β and maturation of dendritic cells.

[0186] TLR ligands

[0187] TLRs (and other innate immune receptors) are not easily altered during evolution due to their specificity; these receptors recognize molecules that are constantly associated with threats (e.g., pathogens or cellular stress) and are highly specific to those threats.

[0188] Pathogen-associated molecules that meet this requirement are considered essential for pathogen function and difficult to change through mutation; they are said to be evolutionarily conserved. Some conserved features among pathogens include bacterial cell surface lipopolysaccharides (LPS), lipoproteins, lipopeptides, and lipoarabinomannan; proteins such as flagellin from bacterial flagella; double-stranded RNA from viruses; or unmethylated CpG islands in bacterial and viral DNA; and CpG islands found in promoters of eukaryotic DNA; as well as certain other RNA and DNA molecules.

[0189] C P GA and C P GB

[0190] Table 1 lists some well-known TLRs and their common ligands:

[0191] Table 1

[0192] Commonly known TLRs and their ligands

[0193]

[0194]

[0195] The nanoparticles of the present invention will be taken up by antigen presenting cells (APCs) in which TLRs are distributed in different ways, especially dendritic cells (DCs) or macrophages. Therefore, in order to trigger a robust T cell response, the ligands in Table 1 and other ligands described herein or elsewhere can be formed into nanoparticles. For different diseases to be treated or prevented, TLRs can be selected for nanoparticle formation based on the main type of antigen presenting cells involved in the immune response.

[0196] One or more, preferably two or more TLR ligands can be contained in a nanoparticle. Hydrophobic TLR ligands can be located in the core portion of the nanoparticle together with the hydrophobic therapeutic peptide, while negatively charged TLR ligands can be present in the crown portion of the nanoparticle together with the cationic CPP. The electrical interaction of the positively charged CPP with the negatively charged TLR ligand is believed to provide a more stable and compact nanostructure. In one embodiment, two negatively charged TLR ligands are contained in the crown portion of the nanoparticle. In another embodiment, the core portion of the nanoparticle contains one hydrophobic TLR ligand, and the crown portion of the nanoparticle contains two negatively charged TLR ligands. The presence of multiple types of TLR ligands in the nanoparticle significantly improves T cell responses. As is well known to those skilled in the art, TLR ligands can also be modified to have desired properties.

[0197] Exemplary Definition

[0198] 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 extragenomic DNA), genes, peptide nucleic acids (PNA), RNA (including but not limited to rRNA, mRNA and tRNA), nucleosides and suitable nucleic acid segments obtained from natural sources, chemically synthesized, modified, or otherwise prepared or synthesized in whole or in part by humans.

[0199] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the invention pertains. The following references provide general definitions of many of the terms used in the present invention for those skilled in the art: Dictionary of Biochemistry and Molecular Biology, (2nd ed.) J. Stenesh (ed.), Wiley-Interscience (1989); Dictionary of Microbiology and Molecular Biology (3rd ed.), P. Singleton and D. Sainsbury (eds.), Wiley-Interscience (2007); Chambers Dictionary of Science and Technology (2nd ed.), P. Walker (ed.), Chambers (2007); Glossary of Genetics (2nd ed.). Genetics) (5th ed.), R. Rieger et al. (eds.), Springer-Verlag (1991); and The HarperCollins Dictionary of Biology, W. G. Hale and J. P. Margham (eds.), HarperCollins (1991).

[0200] Although any methods and compositions similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and compositions are described herein. For purposes of the present invention, for clarity and ease of reference, the following terms are defined as follows:

[0201]

[0026] Following long-standing patent law convention, the terms "a" and "an" are used throughout this application and claims to mean "one or more."

[0202] As used herein, the terms "about" and "approximately" are interchangeable and should generally be understood to refer to a numerical range surrounding a given number, as well as all numbers within the recited numerical range (e.g., "about 5 to 15" means "about 5 to about 15" unless otherwise specified). Furthermore, all numerical ranges herein should be understood to include every integer within the stated range.

[0203] "Biocompatible" refers to a material that, when exposed to living cells, will support proper cellular activity of the cells without causing adverse effects within the cells, such as changes in cell life cycle, changes in cell proliferation rate, or cytotoxic effects.

[0204] The term "biologically functional equivalent" is well known in the art and is further defined in detail herein. Thus, nucleotide sequences having about 85% to about 90% identity or functional equivalence to one or more of the nucleotide sequences provided herein; or more preferably, about 91% to about 95%; or even more preferably, about 96% to about 99% identity or functional equivalence are specifically contemplated as being useful in the practice of the methods and compositions described herein.

[0205] As used herein, "bionic" shall mean the similarity of a synthetic material to a substance naturally occurring in the human body and not being rejected by the human body (eg, not causing an adverse reaction in the human body).

[0206] As used herein, the term "buffer" includes one or more compositions or aqueous solutions thereof that resist fluctuations in pH when an acid or base is added to the solution or composition comprising the buffer. This resistance to pH changes is due to the buffering properties of such solutions and may be a function of one or more specific compounds contained in the composition. Therefore, solutions or other compositions that exhibit buffering activity are referred to as buffers or buffered solutions. Buffers generally do not have the ability to maintain the pH of a solution or composition indefinitely; rather, buffers are generally capable of maintaining the pH within a certain range, such as a pH of about 5 to 7.

[0207] As used herein, the term "carrier" is intended to include any solvent, dispersion medium, coating agent, diluent, buffer, isotonic agent, solution, suspension, colloid, inert agent, etc. or a combination thereof that is pharmaceutically acceptable for administration to the relevant animal. The use of one or more delivery vehicles for chemical compounds in general and for chemotherapeutic agents in particular is well known to those of ordinary skill in the pharmaceutical field. Unless any conventional medium or agent is incompatible with the active ingredient, it is envisioned that it will be used in diagnostic, prophylactic and therapeutic compositions. One or more supplementary active ingredients can also be incorporated into one or more chemotherapeutic compositions in the disclosed chemotherapeutic compositions, or administered in combination with one or more chemotherapeutic compositions in the disclosed chemotherapeutic compositions.

[0208] As used herein, the term "DNA segment" refers to a DNA molecule isolated from the total genomic DNA of a particular species. Thus, a DNA segment obtained from a biological sample using one of the compositions disclosed herein refers to one or more DNA segments that have been isolated or purified from the total genomic DNA of the particular species from which the one or more DNA segments were obtained. The term "DNA segment" encompasses DNA segments and smaller fragments of such segments, as well as recombinant vectors, including, for example, plasmids, cosmids, phages, viruses, and the like.

[0209] As used herein, the term "effective amount" refers to an amount capable of treating or ameliorating a disease or condition or otherwise producing the desired therapeutic effect.

[0210] As used herein, the terms "for example" or "eg," are used merely as examples and are not intended to be limiting, and should not be construed to refer only to those items explicitly enumerated in the specification.

[0211] As used herein, a "heterologous" sequence is defined as a sequence related to a predetermined reference sequence, such as 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 near the reference structural gene but has been localized through laboratory manipulation. Similarly, a heterologous gene or nucleic acid segment is defined as a gene or segment that does not naturally occur near a reference promoter and / or enhancer element.

[0212] As used herein, when referring to polynucleotides, "homologous" means sequences with the same basic nucleotide sequence, although from different sources. Typically, homologous nucleic acid sequences are derived from closely related genes or organisms with one or more substantially similar genomic sequences. In contrast, "similar" polynucleotides are polynucleotides that have the same function as polynucleotides from different species or organisms, but may have significantly different primary nucleotide sequences encoding one or more proteins or polypeptides that achieve similar functions or have similar biological activities. Similar polynucleotides can typically be derived from two or more (e.g., genetically or phylogenetically) not closely related organisms.

[0213] As used herein, the term "homology" refers to the degree of complementarity between two or more polynucleotide or polypeptide sequences. When a first nucleic acid or amino acid sequence has an identical primary sequence to a second nucleic acid or amino acid sequence, the term "identity" may be substituted for the term "homology." Sequence homology and sequence identity can be determined by analyzing two or more sequences using algorithms and computer programs known in the art. Such methods can be used to assess whether a given sequence is identical or homologous to another selected sequence.

[0214] The terms "identical" or "percent identity" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms (or other algorithms available to one of ordinary skill) or by visual inspection.

[0215] As used herein, the phrase "in need of treatment" refers to a judgment made by a health care professional, such as a physician or veterinarian, that a patient requires (or will benefit in one or more ways from) treatment. Such a judgment can be made based on a variety of factors within the health care professional's expertise and can include the recognition that the patient is ill due to a disease state that can be treated by one or more compounds or pharmaceutical compositions as described herein.

[0216] The phrases "isolated" or "biologically pure" refer to material that is substantially or essentially free from components that normally accompany the material as it is found in its native state.

[0217] As used herein, the term "kit" may be used to describe a portable, self-contained housing comprising at least one set of reagents, components, or pharmaceutical compositions for performing one or more assays of the invention. Optionally, such kits may include one or more sets of instructions for using the accompanying reagents, for example, in laboratory or clinical applications.

[0218] "Linking" or "joining" refers to any method known in the art for functionally linking one or more proteins, peptides, nucleic acids or polynucleotides, including but not limited to recombinant fusion, covalent bonding, disulfide bonding, ionic bonding, hydrogen bonding, electrostatic bonding, etc.

[0219] As used herein, the term "naturally occurring" when applied to an object refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence present in an organism (including a virus) that can be isolated from a source in nature and has not been intentionally modified by humans in a laboratory is naturally occurring. As used herein, a strain of laboratory rodents that may have been selectively bred according to classical genetics is considered a naturally occurring animal.

[0220] As used herein, the term "nucleic acid" includes one or more types of the following: polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), and any other type of polynucleotides, which are N-glycosides of purine or pyrimidine bases or modified purine or pyrimidine bases (including abasic sites). As used herein, the term "nucleic acid" also includes polymers of ribonucleosides or deoxyribonucleosides, typically through phosphodiester bonds between subunits, but in some cases through covalent bonds such as 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 (snORA), small nuclear RNA (snRNA), and small sequential RNA (stRNA), etc., and any combination thereof.

[0221] As used herein, the terms "operably linked" and "operably connected" refer to the association of nucleic acid sequences linked in such a manner that the coding regions are contiguous and in the correct reading frame. Such sequences are typically contiguous or substantially contiguous. However, because enhancers typically function when separated from the promoter by several kilobases and intronic sequences can be of variable length, some polynucleotide elements may be operably linked but not contiguous.

[0222] 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, the subject is a vertebrate, which is intended to represent any animal species (and preferably, a mammalian species, such as humans). In certain embodiments, "patient" refers to any animal host, including but not limited to any mammalian host. Preferably, the term refers to any mammalian host, including but not limited to humans and non-human primates, cattle, dogs, goats (caprine), cavine, crows (corvine), epine, horses, cats, goats (hircine), rabbits (lapine), hares, wolves, mice, sheep, pigs, frogs, racine, foxes, etc., including livestock, animal specimens, exotic species, and companion animals, pets, and any animals cared for by veterinarians. The patient can be at any age at which the patient is able to respond to the vaccination of this vaccine by generating an immune response. In a specific embodiment, the mammalian patient is preferably a human.

[0223] The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that preferably do not produce an allergic or similar untoward reaction when administered to a mammal, and particularly when administered to a human.

[0224] As used herein, "pharmaceutically acceptable salts" refers to salts that preferably retain the desired biological activity of the parent compound and do not impart any undesirable toxicological effects. Examples of such salts include, but are not limited to, acid addition salts formed with inorganic acids (e.g., 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 (embonic 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.

[0225] As used herein, the term "plasmid" or "vector" refers to a genetic construct consisting of genetic material (i.e., nucleic acid). Typically, a plasmid or vector contains an origin of replication that functions in a bacterial host cell, for example, Escherichia coli, and a selectable marker for detecting bacterial host cells containing the plasmid. The plasmids and vectors of the present invention may comprise one or more genetic elements as described herein, arranged so that the inserted coding sequence can be transcribed and translated in a suitable expression cell. Additionally, the plasmid or vector may comprise one or more nucleic acid segments, genes, promoters, enhancers, activators, multiple cloning regions, or any combination thereof, including segments obtained or derived from one or more natural and / or artificial sources.

[0226] As used herein, "polymer" means a chemical compound or mixture of compounds formed by polymerization and comprising repeating structural units. Polymers can be constructed in a variety of forms and compositions or combinations of compositions.

[0227] As used herein, the term "polypeptide" is intended to encompass singular "polypeptide" as well as plural "polypeptides", and comprises any one or more chains of two or more amino acids. Thus, as used herein, terms including but not limited to "peptide", "dipeptide", "tripeptide", "protein", "enzyme", "amino acid chain" and "continuous amino acid sequence" are all encompassed within the definition of "polypeptide", and the term "polypeptide" can be used in place of any of these terms, or interchangeably with any of these terms. The term further encompasses polypeptides that have undergone one or more post-translational modifications, including, for example, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization, proteolytic cleavage, post-translational processing, or modifications comprising one or more non-naturally occurring amino acids. Conventional nomenclature for polynucleotide and polypeptide structures exists in the art.

[0228] For example, one-letter and three-letter abbreviations are widely used to describe amino acids: alanine (A; Ala), arginine (R; Arg), asparagine (N; Asn), aspartic acid (D; Asp), cysteine ​​(C; Cys), glutamine (Q; Gin), glutamic acid (E; Glu), glycine (G; Gly), histidine (H; His), isoleucine (I; Ile), leucine (L; Leu), methionine (M; Met), phenylalanine (F; Phe), proline (P; Pro), serine (S; Ser), threonine (T; Thr), tryptophan (W; Trp), tyrosine (Y; Tyr), valine (V; Val), and lysine (K; Lys). The amino acid residues described herein are preferably in the "L" isomeric form. However, residues in the "D" isomeric form may replace any L-amino acid residue, provided that the desired properties of the polypeptide are retained.

[0229] As used herein, the terms "prevent," "preventing," "prevention," "suppress," "suppressing," and "suppression" as used herein refer to the administration of a compound, alone or as contained in a pharmaceutical composition, prior to the onset of clinical symptoms of a disease state to prevent any symptom, aspect, or characteristic of a disease state. Such prevention and suppression are not necessarily to be considered absolute to be medically useful.

[0230] "Protein" is used interchangeably with "peptide" and "polypeptide" herein, and encompasses peptides and polypeptides that are synthetic, recombinant, or produced in vitro, as well as peptides and polypeptides that are expressed in vivo following administration of the nucleic acid sequence to a host animal or human subject. The term "polypeptide" is preferably intended to refer to any amino acid chain length, including short peptides of about 2 to about 20 amino acid residues in length, oligopeptides of about 10 to about 100 amino acid residues in length, and longer polypeptides, including those of about 100 amino acid residues or more in length. In addition, the term is also intended to encompass enzymes, i.e., functional biomolecules comprising at least one amino acid polymer. The polypeptides and proteins of the present invention also encompass polypeptides and proteins that are or have been post-translationally modified, and encompass any sugar or other derivative or conjugate added to the backbone amino acid chain.

[0231] As used herein, "purified" means separated from a number of other compounds or entities. A compound or entity can be partially purified, substantially purified, or pure. A compound or entity is considered pure when it is removed from substantially all other compounds or entities, i.e., preferably at least about 90%, more preferably at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than 99% pure. A partially or substantially purified compound or entity can be removed from at least 50%, at least 60%, at least 70%, or at least 80% of the material in which it naturally occurs, such as cellular material, such as cellular proteins and / or nucleic acids.

[0232] The term "recombinant" indicates that a material (e.g., a polynucleotide or polypeptide) has been artificially or synthetically (i.e., non-naturally) altered by human intervention. The alteration can be performed on the material within its natural environment or native state, or it can be performed on material removed from its natural environment or native state. Specifically, for example, a promoter sequence is "recombinant" when it is produced by the expression of an artificially engineered nucleic acid segment. For example, a "recombinant nucleic acid" is made by recombining a nucleic acid, such as during cloning, DNA shuffling, or other procedures, or by chemical or other mutagenesis; a "recombinant polypeptide" or "recombinant protein" is a polypeptide or protein produced by expression of a recombinant nucleic acid; and a "recombinant virus," for example, a recombinant AAV virus, is produced by expression of a recombinant nucleic acid.

[0233] As used herein, the term "regulatory element" refers to one or more regions of a nucleic acid sequence that regulates transcription. Exemplary regulatory elements include, but are not limited to, enhancers, post-transcriptional elements, transcriptional control sequences, and the like.

[0234] The term "RNA segment" refers to an RNA molecule isolated from the total cellular RNA of a particular species. Thus, an RNA segment can refer to one or more RNA segments (of natural or synthetic origin) that have been separated from other RNA or purified from other RNA. Included within the term "RNA segment" are RNA segments and smaller fragments of such segments.

[0235] The term "sequence substantially as shown in SEQ ID NO: X" means that the sequence corresponds substantially to a portion of SEQ ID NO: X and has relatively few nucleotides (or amino acids in the case of polypeptide sequences) that are different from the nucleotides (or amino acids) of SEQ ID NO: X or that are not biologically functional equivalents of said nucleotides (or amino acids). The term "biologically functional equivalents" is well known in the art and is defined in further detail herein. Thus, nucleotide sequences having about 85% to about 90% identity or functional equivalence to one or more of the nucleotide sequences provided herein; or more preferably, about 91% to about 95%; or even more preferably, about 96% to about 99% identity or functional equivalence are specifically contemplated as being useful in the practice of the present invention.

[0236] Suitable standard hybridization conditions for nucleic acids of 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 continuous washing at 60°C with 0.1 × SSC, 0.1% SDS solution for 1 hour 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 continuous washing at 55°C with 0.8 × SSC, 0.1% SDS. One of ordinary skill in the art will recognize that such hybridization conditions can be easily adjusted to obtain the desired stringency level for a particular application.

[0237] As used herein, the term "structural gene" is intended to generally describe a polynucleotide, such as a gene, that is expressed to produce an encoded peptide, polypeptide, protein, ribozyme, catalytic RNA molecule, or antisense molecule.

[0238] As used herein, the term "subject" describes an organism, including mammals, such as primates, to whom treatment with the compositions according to the present invention can be provided. Mammalian species that can benefit from the disclosed treatment methods include, but are not limited to, apes; chimpanzees; orangutans; humans; monkeys; domestic animals, such as dogs and cats; livestock, such as horses, cattle, pigs, sheep, goats, and chickens; and other animals, such as mice, rats, guinea pigs, and hamsters.

[0239] In some embodiments, the present invention provides the method for the present invention to provide the method for the present invention of the present invention.When being used to define amino acid or nucleotide sequence, term " substantially complementary " means specific subject sequence, for example oligonucleotide sequence, with all or part of the sequence of selection substantially complementary, and therefore will be specifically bonded to the part of the mRNA that the sequence of selection is encoded.Like this, sequence will be usually with mRNA " target " sequence height complementarity, and will have the base mispairing that is no more than about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9 or about 10 in the complementary part of whole sequence.In many cases, may expect sequence accurate match, that is, with the sequence complete complementarity of oligonucleotide specific binding, and therefore have zero mispairing along complementary section.Like this, highly complementary sequence will be very specifically bonded to the target sequence region of mRNA usually, and therefore will be very effective aspect the translation of reducing and / or even suppressing target mRNA sequence to polypeptide product.

[0240] A substantially complementary nucleic acid sequence will have greater than about 80% complementarity (or "% exact match") with the corresponding nucleic acid target sequence to which the nucleic acid specifically binds, and more preferably will have greater than about 85% complementarity with the corresponding target sequence to which the nucleic acid specifically binds. In certain aspects, as described above, it is desirable to have even more substantially complementary nucleic acid sequences for use in the practice of the present invention, and in such cases, the nucleic acid sequence will have greater than about 90% complementarity with the corresponding target sequence to which the nucleic acid specifically binds, and in certain embodiments, may have greater than about 95% complementarity with the corresponding target sequence to which the nucleic acid specifically binds, and even up to and including about 96%, about 97%, about 98%, about 99%, and even about 100% exact match complementarity with all or a portion of the target sequence to which the designed nucleic acid specifically binds.

[0241] The percent similarity or percent complementarity of any disclosed nucleic acid sequence can be determined, for example, by comparing sequence information using the GAP computer program version 6.0, available from the University of Wisconsin Genetics Computer Group (UWGCG). The GAP program uses the alignment method of Needleman and Wunsch (1970). Briefly, the GAP program defines similarity as the number of similar aligned symbols (i.e., nucleotides or amino acids) divided by the total number of symbols in the shorter of the two sequences. Preferred default parameters for the GAP program include: (1) a unary comparison matrix for nucleotides (containing a value of 1 for identity and 0 for non-identity), and a 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 end gaps.

[0242] As used herein, the terms "substantially free" or "essentially free" in relation to the amount of a component preferably refer to a composition containing less than about 10%, preferably less than about 5%, and more preferably less than about 1% by weight of the compound. In preferred embodiments, these terms refer to less than about 0.5%, less than about 0.1%, or less than about 0.01% by weight.

[0243] As used herein, the term "structural gene" is intended to generally describe a polynucleotide, such as a gene, that is expressed to produce an encoded peptide, polypeptide, protein, ribozyme, catalytic RNA molecule, or antisense molecule.

[0244] As used herein, the term "subject" describes an organism, including mammals, such as primates, to whom treatment with the compositions according to the present invention can be provided. Mammalian species that can benefit from the disclosed treatment methods include, but are not limited to, humans, non-human primates, such as apes; chimpanzees; monkeys and orangutans, domestic animals, including dogs and cats, and livestock, such as horses, cattle, pigs, sheep, and goats, or other mammalian species, including, but not limited to, mice, rats, guinea pigs, rabbits, hamsters, and the like.

[0245] As used herein, the terms "substantially correspond to," "substantially homologous," or "substantially identical" refer to the property of a nucleic acid or amino acid sequence in which the selected nucleic acid sequence or the selected amino acid sequence has at least about 70% or about 75% sequence identity compared to a selected reference nucleic acid or amino acid sequence. More typically, the selected sequence and the reference sequence will have at least about 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, or even 85% sequence identity, and more preferably, at least about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95% sequence identity. Still more preferably, highly homologous sequences typically share greater than at least about 96%, 97%, 98%, or 99% sequence identity between the selected sequence and the reference sequence to which it is compared.

[0246] As used herein, "synthetic" shall mean that the material is not of human or animal origin.

[0247] A "targeting moiety" is any factor that can facilitate targeting of a particle to a specific site. For example, the targeting moiety can be a chemical targeting moiety, a physical targeting moiety, a geometric targeting moiety, or a combination thereof. A chemical targeting moiety can be a chemical group or molecule on the surface of the particle; a physical targeting moiety can be a specific physical property of the particle, such as surface or hydrophobicity; and a geometric targeting moiety comprises the size and shape of the particle. Further, the chemical targeting moiety can be a dendrimer, an antibody, an aptamer, which can be a thioaptamer, a ligand, an antibody, or a biomolecule that binds to a specific receptor at the target site. The physical targeting moiety can be a surface charge. Charge can be introduced during particle manufacture by using chemical treatments such as specific washes. For example, immersing a porous silica or silicon oxide surface in water can result in a negative charge on the surface.

[0248] The surface charge can also be provided by additional layers or chemical chains such as polymer chains on the particle surface. For example, polyethylene glycol chains can be the source of negative charge on the surface. Polyethylene glycol chains can be coated or covalently coupled to the surface using methods known to those of ordinary skill in the art.

[0249] The term "therapeutic treatment period" means the period of time during which one or more active agents are necessary for the treatment to be effective. The term "therapeutically effective" means reducing the severity and / or frequency of one or more symptoms, eliminating one or more symptoms and / or underlying causes, preventing the occurrence of symptoms and / or their underlying causes, and ameliorating or repairing damage.

[0250] " therapeutic agent " can be any physiological or pharmacologically active substance that can produce the biological effect that expects at the target site in the subject.Therapeutic agent can be chemotherapeutic agent, immunosuppressant, cytokine, cytotoxic agent, nucleolytic compound, radioisotope, receptor and prodrug activating enzyme, and the therapeutic agent can be naturally occurring, produced by synthesis or recombinant method or its combination. Medicines affected by classical multidrug resistance, such as vinca alkaloids (for example, vinblastine (vinblastine) and vincristine (vincristine)), anthracyclines (for example, doxorubicin (doxorubicin) and daunorubicin (daunorubicin)), RNA transcription inhibitors (for example, actinomycin-D (actinomycin-D)) and microtubule stabilizing drugs (for example, paclitaxel (paclitaxel)), can have the specific use as therapeutic agent. Cytokine can also be used as therapeutic agent. The example of this type of cytokine is lymphokine, monokine and traditional polypeptide hormone. Cancer chemotherapy agent can be preferred therapeutic agent. For a more detailed description of anticancer and other therapeutic agents, one skilled in the art is referred to any number of manuals, including but not limited to the Physician's Desk Reference and Hardman and Limbird (2001).

[0251] As used herein, "transcription factor recognition site" and "transcription factor binding site" refer to polynucleotide sequences or sequence motifs that have been identified as sites of sequence-specific interaction of one or more transcription factors, typically in the form of direct protein-DNA binding. Typically, transcription factor binding sites can be identified by DNA footprinting, gel mobility shift assays, etc., and / or can be predicted based on known consensus sequence motifs or by other methods known to those of ordinary skill in the art.

[0252] "Transcriptional regulatory element" refers to a polynucleotide sequence that activates transcription alone or in combination with one or more other nucleic acid sequences. A transcriptional regulatory element may, for example, include one or more promoters, one or more response elements, one or more negative regulatory elements and / or one or more enhancers.

[0253] A "transcription unit" refers to a polynucleotide sequence comprising: at least a first structural gene, said gene operably linked to at least a first cis-acting promoter sequence and optionally operably linked to one or more other cis-acting nucleic acid sequences necessary for efficient transcription of the structural gene sequence; and at least a first distal regulatory element, which may be required for proper tissue-specific and developmental transcription of the structural gene sequence, said structural gene sequence being operably located under the control of promoter and / or enhancer elements; and any additional cis-sequences necessary for efficient transcription and translation (e.g., polyadenylation sites, mRNA stability control sequences, etc.).

[0254] As used herein, the term "transformation" is intended to generally describe the process of introducing an exogenous polynucleotide sequence (e.g., a viral vector, a plasmid, or a recombinant DNA or RNA molecule) into a host cell or protoplast, wherein the exogenous polynucleotide is incorporated into at least the first chromosome or is capable of autonomous replication within the transformed host cell. Transfection, electroporation, and "naked" nucleic acid uptake all represent examples of techniques for transforming a host cell with one or more polynucleotides.

[0255] As used herein, the term "transformed cell" is intended to refer to a host cell whose nucleic acid complement has been altered by the introduction of one or more exogenous polynucleotides into the cell.

[0256] As used herein, "treating" or "treatment of" refers to providing any type of medical or surgical management to a subject. Treatment may include, but is not limited to, administering to a subject a composition comprising a therapeutic agent. "Treatment" comprises administering or applying to a subject a compound or composition of the present invention for purposes such as 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 manifestations of a disease, disorder, or condition. In certain aspects, the compositions of the present invention may also be administered prophylactically before the onset of any symptoms or manifestations of the condition, where such prevention is necessary. Typically, in such cases, the subject will be one who has been diagnosed as "at risk" for such a disease or condition due to family history, medical records, or completion of one or more diagnostic or prognostic tests that indicate a tendency to subsequently develop such a disease or condition.

[0257] As used herein, the term "vector" refers to a nucleic acid molecule (usually comprising DNA) that can replicate in a host cell and / or can be operably linked to another nucleic acid segment to bring about replication of the linked segment. Plasmids, cosmids, or viruses are exemplary vectors.

[0258] In certain embodiments, it may be advantageous to employ one or more nucleic acid segments of the present invention in combination with an appropriate detectable marker (i.e., a "label"), such as in the case of employing a labeled polynucleotide probe to determine the presence of a given target sequence in a hybridization assay. A variety of suitable indicator compounds and compositions for labeling oligonucleotide probes are known in the art, including but not limited to fluorescent ligands, radioactive ligands, enzyme ligands, or other ligands that can be detected in a suitable assay, such as avidin / biotin, etc. In particular embodiments, one or more fluorescent labels or enzyme tags such as urease, alkaline phosphatase, or peroxidase may also be employed rather than radioactive or other environmentally less desirable reagents. In the case of enzyme tags, known colorimetric, chromogenic, or fluorescent indicator substrates may be used to provide methods for detecting samples visible to the human eye, or to identify specific hybridization to a sample containing one or more complementary or substantially complementary nucleic acid sequences by analytical methods such as scintigraphy, fluorimetry, spectrophotometry, etc. In the case of so-called "multiplexed" assays, in which two or more labeled probes are detected simultaneously or sequentially, it may be desirable to label a first oligonucleotide probe with a first label having a first detection property or parameter (e.g., emission and / or excitation spectral maximum), and to label a second oligonucleotide probe with a second label having a second detection property or parameter that is different (i.e., discrete or distinguishable) from the first label. The use of multiplexed assays is well known to those of ordinary skill in the art of molecular genetics, particularly in the context of gene amplification / detection protocols.

[0259] biologically functional equivalents

[0260] The structure of the nucleic acid or the vector comprising the nucleic acid, as well as the mRNA, polypeptide or therapeutic agent encoded thereby, can be modified and altered and still obtain a functional system containing one or more therapeutic agents with desired properties. As described above, it is generally desirable to introduce one or more mutations into a particular polynucleotide sequence. In some cases, the resulting encoded polypeptide sequence is altered by such mutations, or in other cases, the sequence of the polypeptide is not altered by one or more mutations in the encoding polynucleotide.

[0261] When it is desired to alter the amino acid sequence of a polypeptide to produce an equivalent or even improved second generation molecule, the amino acid change can be accomplished by altering one or more of the codons encoding the DNA sequence according to Table 2.

[0262] For example, certain amino acids can be substituted for other amino acids in a protein structure without significantly losing the ability to interact with structures such as the antigen-binding region of an antibody or a binding site on a substrate molecule. Since the ability and properties of a protein to interact define its biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and of course, in its underlying DNA coding sequence, and still obtain a protein with similar properties. Therefore, the inventors contemplate that various changes can be made to the peptide sequence of the disclosed composition or the corresponding DNA sequence encoding the peptide without significantly losing the biological utility or activity of the peptide sequence.

[0263] Table 2

[0264]

[0265] When making such changes, the hydropathic index of amino acids can be considered. The importance of the hydropathic amino acid index in imparting interactive biological functions to proteins is generally understood in the art (Kyte and Doolittle, 1982, incorporated herein by reference). It is accepted that the relative hydrophilicity of amino acids contributes to the secondary structure of the resulting protein, which in turn defines the interaction of the protein with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc. A hydropathic index has been assigned to each amino acid based on the hydrophilicity and charge characteristics of the amino acid (Kyte and Doolittle, 1982). These values ​​are: 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).

[0266] It is known in the art that certain amino acids can be replaced by other amino acids with similar hydropathic indexes or scores and still produce proteins with similar biological activity, that is, still obtain biological functional equivalent proteins. When making such changes, amino acid substitutions with a hydropathic index within ±2 are preferred, amino acid substitutions with a hydropathic index within ±1 are particularly preferred, and even more particularly amino acid substitutions with a hydropathic index within ±0.5 are preferred. It should also be understood in the art that similar amino acid substitutions can be effectively carried out based on hydrophilicity. U.S. Patent No. 4,554,101 (specifically incorporated herein by reference in its entirety) points out that its maximum local average hydrophilicity, which is governed by the hydrophilicity of the adjacent amino acids of a protein, is related to the biological properties of the protein.

[0267] As detailed in U.S. Patent No. 4,554,101, the following hydrophilicity values ​​have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1); glutamate (+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 will be understood that an amino acid can be substituted for another amino acid having a similar hydrophilicity value and still obtain a biologically equivalent, and particularly immunologically equivalent, protein. In such changes, substitution of amino acids whose hydrophilicity values ​​are within ±2 is preferred, substitution of amino acids whose hydrophilicity values ​​are within ±1 is particularly preferred, and substitution of amino acids whose hydrophilicity values ​​are within ±0.5 is even more particularly preferred.

[0268] As outlined above, amino acid substitutions are therefore generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions that take into account one or more of the foregoing properties are well known to those of ordinary skill in the art and include arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine.

[0269] The section headings used throughout this document are for organizational purposes only and should not be construed as limiting the subject matter described. All documents or portions of documents cited in this application (including but not limited to patents, patent applications, papers, books, and monographs) are expressly incorporated herein by reference in their entirety. If one or more incorporated documents and similar materials define a term in a manner that contradicts the definition of the term in this application, the present application shall prevail.

[0270] Examples

[0271] The following examples are included to illustrate illustrative embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in these examples represent techniques discovered to function well in the practice of the present invention and, therefore, can be considered to constitute preferred modes for practicing the present invention. However, in light of this disclosure, it will be appreciated by those skilled in the art that 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.

[0272] Example 1

[0273] Although numerous clinical trials have been reported using NY-ESO-1 peptides with Montanide ISA-51 plus CpG or poly(I:C), clinical and immune responses have generally been weak or less pronounced. One plausible reason for this 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, resulting in a weak immune response. Previously, a DC / TAT-TRP-2 vaccine has been shown to produce robust protective, but nontherapeutic, immunity (Wang et al., 2002). In a Phase I clinical trial, TAT-ESO-1 peptide was mixed with Montanide ISA-51 for use in a vaccine that produced only a weak T cell response. During this study, two potential issues were identified: 1) TAT-ESO-1 was unable to form a stable complex with Montanide ISA-51, likely due to its positive N-terminal charge; and 2) TAT-ESO-1 formed a precipitate when mixed with CpG. Based on the charge and hydrophobic properties, a new technology of self-assembled CPP-T cell peptide nanoparticles (PEP-NANO) with TLR ligands [CpG, MPLA and poly(I:C), referred to as CMI] was designed and developed, e.g. Figure 2A and Figure 2B As schematically shown in FIG. , an amphiphilic or amphiphilic CPP-therapeutic peptide forms nanoparticles with negatively charged CpG and / or poly(I:C) through electrical interactions, and with MPLA through hydrophobicity within the particle. The CPP-therapeutic peptide is composed of a CPP such as TAT ​​with a positively charged peptide covalently linked to a therapeutic peptide (typically hydrophobic) such as NY-ESO-1 (SLLMWITQCFLPV) (SEQ ID NO: 1) and TRP-2 (SYVDFFVWL) (SEQ ID NO: 2).

[0274] Example 2

[0275] Self-assembly and characterization of nanoparticles of TAT-TRP2 peptide and TLR ligands or their combination

[0276] First, TAT-TRP2, TAT-ESO-1 and CpG are completely dissolved in sterile ultrapure water with a concentration of 10 mg / mL as a stock solution. MPLA and poly I: C are completely dissolved in sterile ultrapure water with a concentration of 1 mg / mL. In order to prepare TAT-peptide vaccine nanoparticles, indicated volumes of CpG, MPLA and poly I: C (as shown in Table 3) are thoroughly mixed in ultrapure water or buffer by strong vortexing.

[0277] Table 3

[0278] Concentration and combination of TLR ligands

[0279]

[0280] The self-assembly of TAT-peptide nanoparticles was triggered by adding TAT-peptide dropwise with ultrasonic treatment in an ice-water bath within 1 minute. TAT-TRP2 exhibits hydrophilicity / positive charge and hydrophobicity, and also promotes its electrostatic interaction with CpG ODN. The hydrophobic C-terminus of the TRP2 peptide forms a hydrophobic core together with MPLA through hydrophobic interactions. Electrostatic and hydrophobic interactions drive TAT-TRP2 to self-assemble with two or three TLR agonists to form spherical TAT-TRP2-CM nanoparticles, as demonstrated by AFM analysis of the height and DMT modulus distribution on representative cross-sections (red lines). In contrast, TRP2 peptides without TAT modification failed to form a complex with CpG-MPLA and formed long fibers.

[0281] To formulate stable and highly effective peptide vaccine nanoparticles, the formulations were optimized using different ratios of TAT-TRP2 peptide:TLR ligands, as shown in Table 3, and the size of each nanoparticle (80-150 nm) of TAT-TRP2 combined with different TLR ligands was characterized ( Figure 4 ). Grey bars indicate unstable / polydisperse complexes with large PDI (PDI>0.5).

[0282] Example 3

[0283] Zeta potential of CPP-peptide nanoparticles

[0284] To determine the surface charge of the nanoparticles, the zeta potential of complexes constructed using various nitrogen (+) to phosphate (-) (N / P) ratios (N = nitrogen from amino acid residues; P = CpG ODN phosphate group) was measured. The zeta potential of TAT-TRP2-CM nanoparticles varied with the N / P ratio ( Figure 5 ). Condition #2 of TAT-TRP2-CM and Condition #18 of TAT-TRP2-CMI (Table 3 and Figure 4 ) were used in future research. Figure 6A and Figure 6B Nanoparticle size of TAT-TRP2-CM and zeta potential of TAT-TRP2, CpG, MPLA and TAT-TRP2-CM are shown. Similar results were obtained using TAT-ESO-1 with CpG and MPLA (CM) and CpG, MPLA and poly(I:C) (CMI) ( Figure 7A 、 Figure 7B 、 Figure 7C 、 Figure 7D 、 Figure 7E and Figure 7F ).

[0285] Example 4

[0286] The formation and size of CPP-peptide nanoparticles are P H-dependent

[0287] It was demonstrated that the nanoparticles and zeta potential of TAT-TRP2-CM at pH 7.0 were destroyed and altered at pH 4.0 ( Figure 8A 、 Figure 8B and Figure 8C ).

[0288] To further characterize the self-assembly and nanoparticle size at different pH values ​​4-7, it was found that the self-assembly and nanoparticles were destroyed at different pH values. At pH 7.0, the nanoparticles of TAT-TRP2-CM were compact and 100 nm in size, but increased in size at pH 6.0 and decreased in size at pH 5.0. The nanoparticles of TAT-TRP2-CM were completely destroyed and separated ( Figure 9A Based on these results, it is inferred that TAT-TRP2-CM nanoparticles are taken up by APCs into endosomes / lysosomes during phagocytosis, where the nanoparticles are destroyed at pH 4-5. Acidification within the endo / lysosomal compartment increases the positive charge of TAT-TRP2 and neutralizes the CpG ODN. The TAT-TRP2 peptide is then released into the cytoplasm and presented by MHC class I or class II molecules in the ER, while TLR ligands bind to TLRs to trigger innate immune responses and produce cytokines, thereby enhancing antigen presentation and T cell activation ( Figure 9B ). In contrast, TAT-TRP2 alone enters APCs through cell-penetrating properties and is presented by APCs to T cells without innate immune responses and cytokine production. It was further shown that the pH-dependent properties apply to TAT-TRP2-CM, TAT-TRP2-CMI, TAT-ESO-1-CM, and TAT-ESO-1-CMI ( Figure 9C ).

[0289] Example 5

[0290] CPP-peptide nanoparticles trigger innate immune responses and cytokine production with different TLR ligand combinations

[0291] To identify the optimal combination of TLR ligands to stimulate innate immune responses, bone marrow-derived DCs were freshly isolated and then treated with different TLR ligands (single, double, or triple combinations) ( Figure 9A 、 Figure 9B 、 Figure 9C and Figure 9DFollowing treatment with various TLR ligands or combinations thereof, cytokine production (TNF-α, IL-6, IFN-α, and IFN-β) was measured in cell supernatants by ELISA. Poly(I:C) / CpG, dual CpG / MPLA combinations, and a triple CpG / poly(I:C) / MPLA combination were found to be superior to other groups in triggering innate immune cytokine production. The triple CpG / poly(I:C) / MPLA combination was the most potent activator of cytokine production ( Figure 10 ).

[0292] Example 6

[0293] The formation and size of CPP-peptide nanoparticles are P H-dependent

[0294] Although numerous clinical trials have been reported using NY-ESO-1 peptides with Montanide ISA-51 plus CpG or poly(I:C), clinical and immune responses are generally weak or less pronounced. One plausible reason for this 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, leading to a weak immune response. Previously, it has been demonstrated that DC / TAT-TRP-2 vaccines can produce strong protective, but not therapeutic, immunity (Wang et al., 2002). In a Phase I clinical trial, TAT-ESO-1 peptide was mixed with Montanide ISA-51 for use in a vaccine that produced only a weak T cell response. Overall, murine and human clinical studies have shown that current vaccine approaches have failed to achieve robust immunity and clinical responses. A major issue is that the cancer antigen peptide / protein and TLR ligand are not co-delivered to the same APCs. In most cases, only one TLR ligand is used, rather than co-delivering two or three ligands. In the following examples, we demonstrate that SAPEP-NANO technology can generate potent anti-tumor immunity in a mouse model. The technology 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) through electrical interactions, and with MPLA through hydrophobicity to form nanoparticles.

[0295] Example 7

[0296] DCs loaded with TAT-TRP2 and TLR ligand nanoparticles generate robust antitumor immunity

[0297] To enhance anti-tumor immunity, it was hypothesized that the TAT-TRP-2 peptide could form complexes with TLR ligands such as CpG and MPLA through physical properties (positive / negative charge, hydrophilicity, and hydrophobicity) and induce therapeutic immunity. To test this prediction, the B16 mouse model and tyrosinase-related protein 2 (TRP-2) peptide were used as experimental systems. The TAT-TRP-2 (YGRKKRRQRRRSYVDFFVWL) (SEQ ID NO: 17) peptide formed a tight complex with CpG / MPLA (TAT-TRP2-CM), while the TRP2 peptide failed to form a complex with CpG / MPLA (TRP2-CM) ( Figure 3A-1 、 Figure 3A-2 、 Figure 3B-1 、 Figure 3B-2 、 Figure 3C-1 and Figure 3C-2 DCs loaded with TAT-TRP2-CM or TRP2-CM were prepared and injected intravenously into mice bearing B16 tumors. Sixteen days later, lung metastases in these treated mice were examined. It was found that DC / TAT-TRP2-CM significantly suppressed the number of lung metastases compared with the DC / β-gal-CM control group, while DC / TRP2-CM failed to suppress the number of lung metastases ( Figure 11 ).

[0298] Recently, it was shown that multi-stage carrier (MSV) nanotechnology can load peptides, CpG, and MPLA into silica particles and induce a strong immune response against B16 tumor cells (Zhu et al., 2018). To compare the ability of DC / MSV vaccine to induce anti-tumor immunity and survival with DC / PEP-NANO vaccine, DC / control peptide (Group #1), DC / TRP2 / CpG / MPLA (Group #2), DC / TAT-TRP-2 / CpG / MPLA (Group #3), DC / MSV-TRP2 / CpG / MPLA (Group #4), and DC / MSV-TAT-TRP-2 / CpG / MPLA (Group #5) were prepared and then injected into tumor-bearing mice ( Figure 12A Obviously, compared with DC / TRP-2 / CpG / MPLA (#2) and DC / MSV / TRP-2 / CpG / MPLA (#4), DC / TAT-TRP2 / CpG / MPLA group (#3) and DC / MSV-TAT-TRP2 / CpG / MPLA group (#5) could induce stronger therapeutic immunity and inhibit B16 lung metastasis regardless of MSV ( Figure 12B), indicating that the TAT sequence, rather than MSV, is strictly required for the strongest immune response. More importantly, it was further shown that mice immunized with DC / TAT-TRP-2 / CpG / MPLA survived longer than mice in the DC / MSV-TAT-TRP-2 / CpG / MPLA group, all of which 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 vaccination. These results suggest that the TAT-TRP-2 / CpG / MPLA vaccine is the best among the different vaccine groups tested.

[0299] Example 8

[0300] DCs loaded with TAT-ESO-1 and TLR ligand nanoparticles generate robust antitumor immunity

[0301] To investigate whether DCs loaded with TAT-ESO-1-CM or TAT-ESO-CMI nanoparticles could induce potent antitumor immunity against RM1 / A2-ESO-1 tumor cells, experiments were performed with DC / control peptide, DC / TAT-ESO-CM, or DC / TAT-ESO-CMI vaccination. Tumor growth was monitored every two days. It was found that DC / TAT-ESO-CM vaccination significantly inhibited RM1 / HLA-A2-NY-ESO-1 tumor growth compared with the control group ( Figure 13A and Figure 13B Importantly, DC / TAT-ESO-CMI showed stronger immunity than DC / TAT-ESO-CM vaccine ( Figure 13A and Figure 13B Further analysis of immune cell responses confirmed that the antigen-specific responses in DC / TAT-ESO-CMI were better than those in DC / TAT-ESO-CM ( Figure 14A and Figure 14B These results indicate that both DC / TAT-ESO-CM and DC / TAT-ESO-CMI vaccines produced potent anti-tumor immunity.

[0302] To further demonstrate whether DC / TAT-ESO-CM can induce therapeutic anti-tumor immunity in other tumor models, breast cancer E0771 / A2-ESO tumor cells were used as a tumor model. It was shown that DC / TAT-ESO-CM vaccination completely inhibited tumor growth compared with the control ( Figure 15 ).

[0303] Example 9

[0304] Direct immunization with DC-free TAT-ESO-1 / TLR nanoparticles induces potent therapeutic antitumor immunity

[0305] Although most vaccine studies use DCs loaded with antigenic peptides, MSV particles, or a combination of CPP-peptide and TLR nanoparticles, such processes are complex and very labor-intensive, especially for clinical trials. Therefore, the inventors hypothesized whether CPP-peptide / TLR nanoparticles could be used directly for vaccination to generate potent anti-tumor immunity. To test this possibility, tumor-bearing mice were immunized three times with TAT-ESO-CMI and compared with mice immunized once with DC / TAT-ESO-CMI. The results were published in the journal Cell. Figure 16A Direct vaccination with TAT-ESO-CMI vaccine significantly inhibited tumor growth, and the degree of inhibition was much higher than that of DC / TAT-ESO-CMI ( Figure 16B and Figure 16C ). In contrast, and as expected, rapid tumor growth was observed in the control group.

[0306] Example 10

[0307] TAT-CT83 peptide vaccine

[0308] CT83 (also known as CXORF61 and KKLC1) has been shown to be highly expressed in human lung and breast cancers ( Figures 17A-17D 、 Figure 18A and Figure 18B ), consistent with previous reports (Fukuyama et al., 2006; Paret et al., 2015). Therefore, it is possible that CT83 can be used as an immune target for cancer vaccines and immunotherapy.

[0309] To test this possibility, a series of TAT-linked 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 with CMI generated a strong T cell response against the CT83-A2 peptide ( Figure 19A 、 Figure 19B 、 Figure 19C 、 Figure 19D and Figures 20A-20D ). HLA-DR13 and HLA-DP4 restricted T cells were generated after in vitro peptide stimulation.

[0310] To determine whether TAT-CT83-CMI can generate potent antitumor immunity, TAT-CT83 peptide vaccines were prepared by mixing 100 μg of 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 for each mouse. Figure 20A The experimental design using HLA-A2 transgenic mice is shown in Figure 2. It is shown that the TAT-CT83-CMI vaccine can strongly induce potent antitumor immunity against murine breast cancer E0771 / A2 / CT83 cells ( Figure 20B and Figure 20C ). In addition, this type of anti-tumor immunity can be further enhanced by anti-PD-1 blockade therapy ( Figure 20B and Figure 20C Importantly, vaccine-induced T cell infiltration into tumor tissue was higher compared to unvaccinated mice ( Figure 20D ).

[0311] Table 4

[0312] TAT-linked CT83 peptide

[0313] Peptide name TAT-linked peptides TAT-CT83 A2-1 YGRKKRRQRRRKLVELEHTL (SEQ ID NO: 18) TAT-CT83 A2-2 YGRKKRRQRRRLLASSILCA (SEQ ID NO: 19) TAT-CT83 A2-3 YGRKKRRQRRRYLLLASSIL(SEQ ID NO: 20) TAT-CT83 A2-4 YGRKKRRQRRRRILVNLSMV (SEQ ID NO: 21) CT83-DP4-TAT SILCALIVFWKYRRFQRNYGRKK (SEQ ID NO: 22) <![CDATA[CT83-LP1 10-31 ]]> SILCALIVFWKYRRFQRNTGEM (SEQ ID NO: 23) <![CDATA[CT83-LP2 66-87 ]]> ILNNFPHSIARQKRILVNLSMV (SEQ ID NO: 24) TAT-CT-83-A2-5 YGRKKRRQRRRKLVELEHTLLSKG(SEQ ID NO: 25) TAT-CT-83-A2-6 YGRKKRRQRRRKLVELEHTLLS (SEQ ID NO: 26) TAT-CT-83-A2-7 YGRKKRRQRRRKLVELEHTLL (SEQ ID NO: 27) TAT-CT-83-A2-8 YGRKKRRQRRRILNNFPHSI (SEQ ID NO: 28)

[0314] 1. TAT-ESO-CMI produces strong anti-tumor immunity in breast cancer

[0315] Similarly, experiments were conducted using E0771 / A2-ESO breast cancer cells in HLA-A2 Tg mice. Compared with the control-treated group, a single DC / TAT-ESO-CMI vaccination (1×10 6 cells / mouse) completely inhibited tumor growth ( Figure 21A 、 Figure 21B and Figure 21C ). In addition, it was shown that in a therapeutic model, vaccination with TAT-ESO-CMI vaccine without DC also produced potent anti-tumor immunity and inhibited tumor cell growth ( Figure 21D ).

[0316] 2. Combination therapy of TAT-TRP2-CMI or TAT-ESO-CMI vaccine and anti-PD-1 blockade:

[0317] To test whether the SAPNANO vaccine can be combined with immune checkpoint therapy, it was shown that TAT-TRP-2-CMI SAPNANO vaccination plus anti-PD-1 therapy can further enhance anti-tumor immunity and prolong the survival of mice compared with TAT-TRP-2-CMI SAPNANO alone ( Figure 22A、 Figure 22B and Figure 22C Specifically, TAT-TRP2-CMI plus anti-PD-1 significantly prolonged the survival of mice ( Figure 22C ).

[0318] To further test this concept, HLA-A2 transgenic mice bearing RM1-A2-ESO tumors were vaccinated with TAT-ESO-CMI alone or in combination with anti-PD-1 therapy. Figure 23A and Figure 23B ). It was shown that SAPNANO vaccine alone significantly inhibited tumor growth ( Figure 22A and Figure 22B ). Anti-tumor immunity induced by SAPNANO vaccine can be further enhanced by anti-PD-1 blockade therapy ( Figure 23A and Figure 23B ).

[0319] 3. Combination therapy of TAT-ESO-CMI vaccine and TCR-T cell immunotherapy

[0320] To test whether the novel SAPNANO vaccine could enhance A2-ESO TCR-T cell-mediated anti-breast cancer immunity, experiments were performed using E0771 / A2-ESO tumor cells, and it was found that adoptive transfer of A2-ESO TCR-T cells followed by vaccination with the TAT-ESO-CMI vaccine inhibited E0771 / A2-ESO tumor growth better than either vaccine alone ( Figure 24A Notably, the TAT-ESO-CMI vaccine induced stronger anti-tumor immunity than adoptive transfer of TCR-T cells ( Figure 24A Consistently, TAT-ESO-CMI vaccine expanded tumor-infiltrating A2-ESO TCR-T cells (25.9%) compared with A2-ESO TCR-T cells alone (5.5%) ( Figure 24B These results indicate that the TAT-ESO-CMI vaccine can expand A2-ESO TCR-T cells in vivo.

[0321] To further test 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. Although the TAT-ESO-CMI vaccine alone did not significantly inhibit tumor growth due to limited immune cells such as T cells and DCs after immune reconstitution, in the MDA-MB-231-A2-ESO breast cancer model, the TAT-ESO-CMI vaccine can be combined with ESO-specific TCR engineered T cell therapy to produce a stronger anti-tumor effect than either TCR-T cell group alone ( Figure 25A 、 Figure 25B 、 Figure 25C 、 Figure 25D and Figure 25E ).

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[0397] It should be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or variations in view of this will be suggested to those skilled in the art and are intended to be included within the spirit and scope of this application and within the scope of the appended claims. All references cited herein (including publications, patent applications, and patents) are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and are set forth in their entirety herein. Reciting ranges of values ​​herein is intended only to serve as a shorthand method of individually referring to each individual value within the range, unless otherwise indicated herein, and each individual value is incorporated into the specification as if it were individually cited herein.

[0398] Descriptions of any aspect or embodiment of the invention herein using terms such as “comprising,” “having,” “including,” or “containing” with respect to one or more elements are intended to provide support for similar aspects or embodiments of the invention “consisting of,” “consisting essentially of,” or “comprising essentially of,” the specified one or more elements, unless otherwise specified or clearly contradicted by context (e.g., descriptions of compositions herein comprising specified elements are to be understood to also describe compositions consisting of those elements, unless otherwise specified or clearly contradicted by context).

[0399] All of the compositions and methods disclosed and claimed herein can be prepared and executed without undue experimentation in light of the present disclosure. Although the compositions and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that the compositions and methods, as well as the steps or sequence of steps of the methods described herein, can be varied without departing from the concept, spirit, and scope of the present invention. More specifically, it will be apparent that certain agents of chemical and / or physiological relevance can be substituted for the agents described herein while achieving the same or similar results.

[0400] All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Claims

1. A composition comprising: (a) a population of nanoparticles that self-assemble at neutral pH and comprise a plurality of cationic cell-penetrating peptides covalently linked to a hydrophobic therapeutic peptide ligand; (b) a pharmaceutically acceptable buffer, diluent, carrier or vehicle; and (c) a first TLR ligand and a second TLR ligand, wherein the first TLR ligand is MPLA and the second TLR ligand is CpG, (d) a negatively charged molecule selected from poly(I:C), and The cationic cell-penetrating peptide interacts with the negatively charged molecule through electrical interaction, and the therapeutic peptide ligand interacts with MPLA through hydrophobic interaction.

2. The composition according to claim 1, further comprising a therapeutic peptide ligand selected from the group consisting of NY-ESO-1 of SEQ ID NO: 1, TRP-2 of SEQ ID NO: 2, or a CT83 peptide selected from the group consisting of SEQ ID NO: 18 to SEQ ID NO:

28.

3. The composition of claim 1, wherein the nanoparticles comprise poly(I:C) and are suitable and configured to increase IFN-I expression when introduced into suitable mammalian cells.

4. The composition of claim 3, wherein the composition is suitable and configured for increasing the expression of IFN-α or IFN-β.

5. The composition of claim 1, which is comprised within an isolated mammalian cell population selected from tumor cells or dendritic cells. The composition according to claim 1 , wherein the plurality of cationic cell-penetrating peptides are disclosed as any one of SEQ ID NO: 3 to SEQ ID NO:

16.

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

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

9. The composition of claim 1, further comprising an adjuvant.

10. The composition according to claim 1, further comprising an antigen, an antigenic polypeptide or an antigenic peptide fragment thereof.

11. The composition of claim 1, wherein 1) the composition is formulated with a liposome population, a nanoparticle population, or a microparticle population; or 2) the composition is mixed with one or more surfactants or vesicles.

12. The composition of claim 1, wherein the composition is mixed with one or more ethosomes, phospholipids or sphingolipids.

13. The composition of claim 1, wherein the composition is mixed with one or more trans-bodies.

14. The composition of claim 1, which is mixed with one or more pharmaceutically acceptable carriers, buffers, diluents, vehicles or excipients.

15. The composition of claim 1 formulated for systemic administration to a mammal.

16. The composition of claim 1 formulated for intravenous administration to a human.

17. The composition of claim 1, suitable and 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.

18. The composition of claim 1, for use in treating, preventing, or ameliorating one or more symptoms of a disease, disorder, or functional disorder in a mammal.

19. The composition according to claim 1, for treating, preventing or ameliorating one or more symptoms of deficiency, defect or trauma in a mammal.

20. The composition according to claim 1, for use in treating, preventing or ameliorating one or more symptoms of an injury or abnormal condition in a mammal.

21. The composition of claim 1, for use in treating, preventing or ameliorating one or more symptoms of cancer or infection in a human.

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

23. An isolated population of mammalian cells comprising a composition according to any preceding claim.

24. The isolated mammalian cell population of claim 23, characterized as human dendritic cells.

25. A kit comprising: 1) A composition according to any one of claims 1 to 22; 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 or disorder in the mammal; and 3) 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 an abnormal condition or injury in the mammal.

26. Use of a composition according to any one of claims 1 to 22 for the manufacture of a medicament for treating or ameliorating one or more symptoms of cancer or infection in a mammalian subject.

27. The use of claim 26, wherein the mammalian subject is a human, a non-human primate, a companion animal, or livestock.

28. The use of claim 26, wherein the medicament is administered for a time sufficient to treat or ameliorate the one or more symptoms of the cancer or the infection in the animal.

29. The use according to claim 28, wherein the cancer is diagnosed or identified as refractory, metastatic, recurrent or treatment-resistant cancer.

30. The use according to claim 28, wherein the subject is a human.

31. The use of claim 28, wherein the composition is administered systemically to the animal in a single administration or in a series of multiple administrations over a period of one or more days, over a period of one or more weeks, or over a period of one or more months or longer.

32. The use of claim 28, wherein the composition further comprises a second different chemotherapeutic agent or a second different population of self-assembled nanoparticles, the second different population of self-assembled nanoparticles comprising a second different therapeutic agent.

33. Use of a composition according to any one of claims 1 to 22 for the manufacture of a medicament for administration to one or more cells, tissues, organs or systems of a mammalian subject in need thereof, wherein the medicament comprises a diagnostic agent, a therapeutic agent or a prophylactic agent.

34. The use according to claim 33, wherein the one or more cells are human dendritic cells.

35. The use according to claim 33, wherein the one or more tissues are tumorous.

36. Use of the composition of any one of claims 1 to 22 or the isolated mammalian cell population of claim 23 or claim 24 for the manufacture of a medicament for therapeutically treating at least one cell, at least one tissue or at least one organ of a patient in need thereof, wherein the medicament is administered for a time effective to provide the composition to the at least one cell, at least one tissue or at least one organ of the patient.

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