Targeted Lung Delivery Compositions and Methods of Using the Same

Abstract

In one aspect, the present disclosure relates to the identification of certain peptides that enable the transport of solid particles across the air-blood barrier in the lungs. In certain embodiments, the solid particles comprise any type of solid cargo to which the peptides contemplated in the present disclosure can be attached.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority under 35 U.S.C.§119(e) to U.S. Provisional Patent Application No. 62 / 910,998, filed on October 4, 2019, which is hereby incorporated by reference in its entirety.

Background Art

[0002] Background Particularly in developing countries and disaster areas, inhalation - based aerosol vaccination for achieving rapid immunization is needle - free and, unlike oral therapy, is not exposed to first - pass metabolism. The respiratory compartment of lung tissue constitutes more than 90% of the total organ volume, which is equivalent to about 80 square meters. The thin and highly permeable lung epithelium generally defines a selective permeability for molecules that are allowed to pass through and enter the bloodstream. Small molecules, peptides or proteins, such as insulin, and viral vaccines are the most suitable candidates for inhalation therapy. Lipophilic molecules are rapidly absorbed through the lungs, probably by passive diffusion through the plasma membrane, while hydrophilic molecules can be transported by specific cell receptors or through tight junctions.

[0003] In fact, aerosol - mode phage - based vaccine introduction follows many infection routes. Over the past two decades or more, significant efforts have been concentrated on optimizing the prototype inhalation - based form of insulin. The current formulations approved by the FDA are still undergoing evaluation in large - scale population - based trials, and concerns about safety and / or efficacy are hindering their broad commercial appeal. Recently, aerosol - based vaccine - inoculation platforms have attracted particular attention for effective field protection against airborne pathogens such as tuberculosis, influenza, Ebola, and measles.

[0004] Aerosolization and pulmonary drug delivery improve drug bioavailability while reducing potential side effects by achieving a more rapid onset of action. However, this route also poses many challenges, particularly for systemic applications, and limits its use in respiratory diseases. Aerosol therapy is generally evaluated by monitoring pharmacological endpoints in vivo. The development of new and efficient therapies is further hampered by a lack of knowledge about the mechanisms of transport and fate of aerosolized agents, i.e., the actual mechanisms by which inhaled particles interact with the air-blood barrier, the physicochemical changes of aerosolized molecules upon contact with the lung surface, bioavailability, and finally, significant gaps in the clearance processes and removal of insoluble active compounds. SUMMARY OF THE INVENTION

[0005] Accordingly, there is a need in the art for new compositions that enable the pulmonary delivery of bioactive substances. In certain embodiments, such constructs can be used to facilitate targeted pulmonary vaccination. The present disclosure addresses and satisfies this need. [The present invention 1001] TIFF0007692218000001.tif26153 A transport peptide comprising at least one amino acid sequence selected from the group consisting of [The present invention 1002] The transport peptide of the present invention 1001, comprising the amino acid sequence of SEQ ID NO:2. [The present invention 1003] The transport peptide of the present invention 1001, consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs:1 to 4. [The present invention 1004] The transport peptide of the present invention 1003, consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs:1 to 4. [The present invention 1005] The surface of the solid particle presents the transport peptide of the present invention 1001, and the solid particle is selected from the group consisting of bacteriophage, engineered cells, tissue fragments, nanoparticles, vesicles, dendrimers, virus-like particles, adenovirus, adeno-associated virus (AAV), adeno-associated virus phage (referred to as AAVP), and any combination thereof. [The present invention 1006] The solid particle of the present invention 1005, wherein the transport peptide is attached to the surface of the solid particle or presented on the surface of the solid particle. [The present invention 1007] The solid particle of the present invention 1006, wherein the transport peptide is attached to at least a part of the surface of the solid particle or presented on at least a part of the surface of the solid particle. [The present invention 1008] The solid particle of the present invention 1005, which is a filamentous phage. [The present invention 1009] The solid particle of the present invention 1005 further comprises an agent selected from the group consisting of therapeutic substances, biologically active molecules, imaging agents, radioactive substances, salts, peptides, proteins, lipids, nucleic acids, gases, and any combination thereof, and the agent is attached to the solid particle and / or contained within the solid particle. [The present invention 1010] The solid particle of the present invention 1005, which is a filamentous phage. [The present invention 1011] The solid particle of the present invention 1010, wherein the filamentous bacteriophage comprises fd, fl, or M13 bacteriophage. [The present invention 1012] The solid particle of the present invention 1005, wherein the solid particle is a filamentous phage and the surface of the solid particle presents an antigen. [The present invention 1013] A method for promoting and / or increasing the transport of solid particles through the air-blood barrier in a target lung, comprising administering the solid particles of the present invention 1005 to the subject, wherein the administration is via a route including nasal, buccal, inhalation, intratracheal, intrapulmonary, or intrabronchial. [The present invention 1014] A method for promoting the systemic circulation of solid particles in a subject, comprising administering the solid particles of the present invention 1005 to the subject, wherein the administration is via a route including nasal, buccal, inhalation, intratracheal, intrapulmonary, or intrabronchial. [The present invention 1015] A method for immunizing a subject against a disease or disorder, comprising administering the solid particles of the present invention 1005 to the subject, wherein the surface of the solid particles is further derivatized with an antigen that promotes an immune response against the disease or disorder in the subject, and the administration is via a route including nasal, buccal, inhalation, intratracheal, intrapulmonary, or intrabronchial. [The present invention 1016] A method for treating, alleviating, and / or preventing a disease or disorder in a subject, comprising administering the solid particles of the present invention 1005 to the subject, wherein the surface of the solid particles is further derivatized with an antigen that promotes an immune response against the disease or disorder in the subject, and the administration is via a route including nasal, buccal, inhalation, intratracheal, intrapulmonary, or intrabronchial. [The present invention 1017] A method for treating a subject at risk of developing a disease or disorder, comprising administering the solid particles of the present invention 1005 to the subject, wherein the surface of the solid particles is further derivatized with an antigen that promotes an immune response against the disease or disorder in the subject, and the administration is via a route including nasal, buccal, inhalation, intratracheal, intrapulmonary, or intrabronchial. [The present invention 1018] Any method of the present invention 1013 - 1017, wherein the peptide comprises the amino acid sequence of SEQ ID NO:2. [The present invention 1019] Any method of the present invention 1013 - 1017, wherein the peptide consists of the amino acid sequence of SEQ ID NO:2. [The present invention 1020] Any method of the present invention 1013 - 1017, wherein the solid particles are filamentous phage. [The present invention 1021] The method of the present invention 1020, wherein the filamentous bacteriophage comprises fd, fl, or M13 bacteriophage. [The present invention 1022] Any of the methods of the present invention 1013-1017, wherein the solid particles are administered to the subject in a composition further comprising an immunogenic adjuvant. [The present invention 1023] Any of the methods of the present invention 1013-1017, wherein the subject is a mammal. [The present invention 1024] Any of the methods of the present invention 1013-1017, wherein the subject is a human. [The present invention 1025] A vaccine comprising solid particles, wherein the surface of the solid particles presents at least one of the transport peptides of the present invention 1001, and wherein the solid particles are selected from the group consisting of bacteriophages, engineered cells, tissue fragments, nanoparticles, vesicles, dendrimers, virus-like particles (VLPs), adenoviruses, adeno-associated viruses (AAV), adeno-associated virus phages (referred to as AAVP), and combinations thereof. [The present invention 1026] The vaccine of the present invention 1025, selected from the group consisting of DNA vaccines, RNA vaccines, replicative virus vector vaccines, non-replicative virus vector vaccines, inactivated virus vector vaccines, virus-like particle vaccines, and any combination thereof. [The present invention 1027] The vaccine of the present invention 1025, wherein the particles comprise a vaccine active substance selected from the group consisting of DNA, RNA, replicative virus vectors, non-replicative virus vectors, inactivated virus vectors, virus-like particles, and any combination thereof.

Brief Description of the Drawings

[0006] The following detailed description of the specific embodiments of the present disclosure will be better understood when read in combination with the accompanying drawings. For the purpose of explaining the present disclosure, exemplary embodiments are shown in the drawings. However, it should be understood that the present disclosure is not limited to the exact configurations and means of the embodiments shown in the drawings. [Figure 1] Figures 1A - 1F depict the combinatorial aerosol selection of a phage display library and the identification of new ligand peptide - mediated transport. Figure 1A: A random phage display library (CX8C) was administered via aerosol and recovered from the bloodstream at fixed time points up to 6 hours. Figure 1B: Diagram of 4 rounds of phage display library selection and collection times for each round: R1 (60 minutes), R2 (30 minutes), R3 (10 minutes), and R4 (5 minutes). (C) Phage enrichment through selection. Data are mean ± SD (*P < 0.05; ***P < 0.001). Figure 1D: Percentage of peptide motifs recovered from the 4th round of selection (R4). Cyclic peptides recovered at a frequency equal to or higher than 5% are CAINSLSRKC, CAKSMGDIVC, CGRKQVESSC, and CRGKSAEGTC. 16 peptides with a frequency lower than 5% were also identified. Figure 1E: Transport of phage particles presenting the peptide motifs identified in R4. Uninserted phage was used as a control. Data are mean ± SD (***P < 0.001; n.s. indicates not statistically significant). Figure 1F: In vivo transport of targeted CAKSMGDIVC - presenting phage particles and negative - control uninserted phage particles. Data are mean ± SEM (*P < 0.05; ***P < 0.001). [Figure 2]Figures 2A-2D depict that lung homeostasis remains normal in peptide-mediated phage transport. Figure 2A: Gross morphology and hematoxylin and eosin (H&E) staining of lung tissue sections from mice administered vehicle alone (PBS), negative control non-inserted phage particles, targeted CAKSMGDIVC-displaying phage particles, or LPS-dextran via aerosol. Figure 2B: Lung permeability, total protein recovered from BALF, and infiltrating neutrophils were measured in mice administered targeted CAKSMGDIVC-displaying phage particles, negative control (either non-inserted phage or vehicle alone), or LPS injury model (positive control: LPS-dextran) via aerosol (n = 3 mice / group). Data are representative of three independent experiments. Bars represent mean ± SEM (***P < 0.0001; **P < 0.01). Figure 2C: Immunohistochemistry of phage overlay binding assay using targeted CAKSMGDIVC-displaying phage particles or control non-inserted phage particles in lung or control organs (pancreas) of mouse tissue sections. Figure 2D: Relative quantification of the number of phage particles throughout the lung over time (***P < 0.001). Scale bar, 100 μm. [Figure 3]Figures 3A-3I depict the identification and in vitro validation of α3β1 integrin as a receptor for targeted CAKSMGDIVC-displaying phage particles. Figure 3A: Targeted CAKSMGDIVC-displaying phage particles bind to human recombinant α3β1 integrin. BSA and non-inserted phage were used as negative controls (***P<0.001). Figure 3B: Concentration-dependent inhibition of the binding of targeted CAKSMGDIVC-displaying phage particles to α3β1 integrin by its cognate synthetic peptide compared to an unrelated control peptide. Figure 3C: Immunofluorescence images of A549 cells stained with DAPI (blue) and anti-α3 chain antibody, followed by Cy3-conjugated secondary antibody (red) (scale bar, 100 μm). Figure 3D: Phage binding on the surface of A549 cells (***P<0.001). Figure 3E: Transport of targeted CAKSMGDIVC-displaying phage particles or control non-inserted phage particles through an A549 cell monolayer in a transwell assay. Figures 3F-3I: Phage internalization in A549 wild-type cells and A549 cells transduced with shRNA ITGA3 by immunofluorescence analysis using anti-phage antibody, followed by secondary anti-Cy3-conjugated antibody (red) (scale bar, 50 μm). Transwell system targeted CAKSMGDIVC-displaying phage particle transport inhibition assay: Silenced for 1 h at different concentrations for α3 chain (shRNA ITGA3) or β1 chain (shRNA ITGB1) (***P = 0.001, *P = 0.0417, ***P = 0.008, **P = 0.0136) (Figure 3F) or recombinant protein: GST (100 ng) (Figure 3G), or CAKSMGDIVC-GST (100 ng) (Figure 3H), or anti-α3 blocking antibody (Figure 3I) in A549 cells. The inhibitory effect was not observed in any of the control, wild-type A549 cells, or control cells transduced with non-targeting shRNA (pLKO) or GST alone or isotype control IgG antibody (***P<0.001). [Figure 4-1]Figures 4A - 4F depict the expression, localization, and binding of the ligand CAKSMGDIVC peptide to α3β1 integrin in lung tissue sections. Figures 4A - 4B: Immunofluorescence of sectioned paraffin - embedded lung tissue sections. Type 1 alveolar epithelial cells (AT1) were stained with anti - podoplanin antibody (purple), and type 2 alveolar epithelial cells (AT2) were stained with anti - proSPC antibody (green), anti - α3 chain antibody (red), and DAPI (blue). White arrows indicate the presence of α3β1 integrin in respiratory bronchioles. Figures 4A - B show the detection of α3β1 integrin in cells in the airway and alveolar regions of the lung, particularly in type 1 alveolar epithelial (AT1), type 2 alveolar epithelial (AT2), and respiratory bronchiole cells. Scale bar: 50 μm. Figure 4C: Immunofluorescence analysis of lung tissue sections obtained from animals administered CAKSMGDIVC - presenting phage particles or control non - insert phage particles targeted via aerosol: AT1 cells (purple), AT2 cells (green), phage (red), and DAPI (blue) were imaged by confocal microscopy. White arrows indicate the presence of targeted CAKSMGDIVC - presenting phage particles or control non - insert phage particles co - localized with AT1 and AT2 cells. Yellow arrows indicate phage particles in alveolar macrophages. [Figure 4-2] Figures 4A - 4F depict the expression, localization, and binding of the ligand CAKSMGDIVC peptide to α3β1 integrin in lung tissue sections. Figure 4D: Manders' overlap coefficients for CAKSMGDIVC - presenting phage particles or control non - insert phage particles co - localized with alveolar epithelial AT1 or AT2 cells (*P = 0.0439, **P = 0.0053). Figure 4E: Club cells were stained with anti - CCSP antibody (white) and anti - α3 integrin chain antibody (red). Individual cell nuclei were stained with DAPI (blue). Scale bar: 100 μm. Figure 4F: Shows the presence of targeted CAKSMGDIVC - presenting phage particles (red) or control non - insert phage particles co - localized with cells stained with anti - CCSP antibody (white). Scale bar: 50 μm. [Figure 5]Figures 5A-5E depict that the ligand CAKSMGDIVC peptide binds to alveolar epithelial AT1, AT2 enriched cell populations, and club cells. Figure 5A: Cell sorting by flow cytometry. Lung cells were isolated and purified from a cohort of animals (n = 5) administered aerosolized targeted CAKSMGDIVC-displaying phage particles or control non-insert phage particles and stained with the following antibodies: anti-EPCAM, anti-CD45, anti-T1-α, anti-CD31, and anti-F4 / 80. Figure 5B: Cells were gated (gate 1: CD31 and T1-α; gate 2: CD31 and F4 / 80) based on their specific phenotypes, sorted, centrifuged, and the amount of phage was determined by counting TUs. Data are representative of three independent experiments (**P = 0.0053, *P = 0.0375). Figure 5C: Schematic of a two-compartment pharmacokinetic model using extravascular (lung) administration. Blood flow and rapidly perfused organs (central compartment) and slowly perfused organs (peripheral compartment). Mononuclear phagocyte system (MPS) sequestration refers to the clearance of phage particles by MPS organs (liver, spleen). The lung, the site of administration, is compartmentalized into the alveolar airspace and mononuclear phagocytes. Aerosol phage particles from the alveolar airspace are transported to the blood stream (central compartment) or internalized by macrophages. Figure 5D: Fit of the pharmacokinetic model for targeted CAKSMGDIVC-displaying phage particles or control non-insert phage particles. Data are represented as mean ± SD (n = 3). Figure 5E: Pearson correlation coefficient R>0.99 indicates a strong correlation between the observed data and the model fit. Note: The y-axis is on a log10 scale in Figures 5D-5E. [Figure 6]Figures 6A-6I depict that intratracheal administration of CAKSMGDIVC-displaying phage particles induces a robust and specific systemic antibody response in rhesus monkeys. Figure 6A: Schematic of intratracheal administration of targeted CAKSMGDIVC-displaying phage particles or control non-inserted phage particles in rhesus monkeys. Prior to treatment, blood samples (baseline) were collected. Starting at the time point of the first dose (day 1), blood samples (1 mL) were collected every hour from 1 h to 6 h. Serum samples were collected every 14 days as indicated over the course of the study. Figure 6B: Immunofluorescence analysis of α3β1 integrin (red) in alveolar epithelial cells of lung tissue sections from rhesus monkeys. Individual cell nuclei were stained using DAPI (blue). Scale bar: 100 μm. Figure 6C: Presence of phage particles in the bloodstream of rhesus monkeys administered targeted CAKSMGDIVC-displaying phage particles. Phage load was determined by TU count. Figure 6D: Titers of total purified phage-specific serum IgG antibodies were analyzed by ELISA in 96-well plates coated with 1010 phage particles / well (**P<0.01, ***P = 0.0004). Figure 6E: Fold changes in the titers of phage-specific IgG were calculated by dividing the mean of the antibody titers from each time point by the mean of the antibody titers from the baseline. Figures 6F-6G: Fold changes in the titers of total purified phage-specific serum IgA (Figure 6F) and IgA antibodies (Figure 6G) were determined as above (*P<0.05, **P<0.01). Figures 6H-6I: CAKSMGDIVC-specific IgG (Figure 6H) and CAKSMGDIVC-specific IgA (Figure 6I) were analyzed by ELISA in 96-well plates coated with synthetic CAKSMGDIVC peptide or an unrelated control peptide (*P<0.05, ***P<0.001). Note: The y-axis is on a log2 scale in Figures 6D, 6F, 6H, and 6I. [Figure 7]Figures 7A-7D depict that α3β1 integrin mediates the transport of CAKSMGDIVC-displaying phage particles in A549 cells. Figure 7A: Western blot of the expression of α3 and β1 integrin chains in A549 cells after transduction with shRNA lentiviral particles targeting the human ITGA3 and ITGB1 genes. Anti-β-actin was used as a protein loading control. Figure 7B: Representative images of cultured cells. Functional assays were performed using A549 cells transduced with shRNA ITGA3 clone #3 and ITGB1 clone #1. Figure 7C: Saturation curve of anti-α3 integrin chain blocking antibody in adherent A549 cells. Figure 7D: Mathematical modeling of the phage particle transport kinetics in vitro. Fitting of an exponential function (red and blue lines) to the mean permeability assay data (markers) for targeted CAKSMGDIVC-displaying phage particles (red) or control non-insert phage particles (blue). Pearson correlation coefficients R > 0.96 for both indicate excellent fitness. Data are represented as mean ± SD (n = 3). Note: The y-axis is on a log10 scale. [Figure 8] Figures 8A-8C depict immunohistochemistry and single-cell RNA sequencing of α3β1 integrin transcripts in mouse lung tissue. Figure 8A: Immunohistochemical staining of sectioned paraffin-embedded lung tissue with anti-α3 integrin chain antibody or isotype control antibody. Representative images of alveoli or airways are shown. Figure 8B: Itga3 and Itgb1 transcripts of mouse α3β1 integrin by scRNA-seq in lung epithelial cell types. Figure 8C: Prediction of pharmacokinetic models for the peripheral compartment (slowly perfused organs) and MPS isolation of targeted CAKSMGDIVC-displaying phage particles or control non-insert phage particles. Note: The y-axis is on a log10 scale. [Figure 9]Figures 9A - 9F depict the expression of α3β1 integrin in lung tissue sections from human patients or non - human primates, and the humoral response upon intratracheal administration of CAKSMGDIVC - presenting phage particles. Figure 9A: Immunofluorescence analysis of the expression of α3β1 integrin (red) in alveolar epithelial cells in a lung tissue section of a rhesus monkey. The alveolar epithelial cells were co - stained with an anti - RAGE (green) antibody and DAPI (blue) for nuclear staining. Figure 9B: Expression of α3β1 integrin in a normal human lung tissue section by immunohistochemistry. Figures 9C - 9D: Fold changes in the titers of phage - specific serum IgG (Figure 9C) or serum IgA (Figure 9D) compared to control non - inserted phage particles. Figures 9E - 9F: Fold changes in the titers of CAKSMGDIVC - specific IgG (Figure 9E) and IgA (Figure 9F) were calculated by dividing the mean antibody titer at each time point by the mean antibody titer from the baseline. Scale bar: 100 μm. [Figure 10] Aerosol administration of CAKSMGDIVC - presenting phage particles is depicted to induce robust and specific lung and systemic antibody responses in mice. Serum and BALF were collected from mice after 14 days of aerosol administration of targeted CAKSMGDIVC - presenting phage particles or control non - inserted phage particles. The titers of phage - specific IgG, IgM, or IgA antibodies from serum or bronchoalveolar lavage fluid were analyzed by ELISA in 96 - well plates coated with 1010 phage particles / well (***P < 0.001). Note: The y - axis is on a log2 scale.

Mode for Carrying Out the Invention

[0007] Detailed Description The present disclosure relates, in one aspect, to the identification of certain peptides that enable the transport of solid particles across the air-blood barrier in the lung. In certain embodiments, the solid particles include any type of solid cargo to which the peptides contemplated in the present disclosure can adhere. In other embodiments, the solid particles are further derivatized with a therapeutically useful compound, for example, but not limited to, an antigen that can be used to trigger an immune response in a subject to whom the compositions of the present disclosure are administered. In yet other embodiments, the therapeutically useful compound is presented on the surface of the solid particles. In yet other embodiments, the therapeutically useful compound is attached to the surface of the solid particles. In yet other embodiments, the therapeutically useful compound is contained within the solid particles.

[0008] As described herein, a bias-free combinatorial phage display-based strategy was applied to identify ligand / receptor-mediated pathways for the safe and effective transport of particles across the air-blood barrier. The delivery strategy described herein successfully induces systemic effects with a very low risk of lung tissue damage. In certain non-limiting embodiments, the constructs of the present disclosure can be used as phage-based vaccines for treating systemic non-respiratory diseases. An aerosolized phage display ligand library was screened in vivo to isolate targeting motifs that can cross the intact lung air-blood barrier. The ligand motif CAKSMGDIVC was selected and isolated, and its cognate receptor, integrin α3β1, on the surface of lung club cells and alveolar epithelial cells was purified. Binding of targeted phage particles presenting the CAKSMGDIVC motif to α3β1 integrin promoted specific phage particle uptake and transport in vitro and in vivo. These findings were validated in a non-human primate model. This non-invasive method of lung delivery of highly stable antigen carriers (i.e., phage particles) can induce robust and specific immune responses and has unlimited applications for vaccine development. Collectively, the combinatorial selection system and results discussed herein provide new translational means for inhalation therapies and their systemic application.

[0009] To gain mechanistic insights into the physiological transport of molecules across the air-blood barrier and to explore the diversity of associated surface receptors, a combinatorial screening of an aerosolized phage display random peptide library was designed and performed in mice. From a pool of peptide-displaying phage particles recovered from the bloodstream, four potent ligand peptide candidates mediated phage transport across the lung barrier. Among these selected ligands, the index peptide CAKSMGDIVC showed one of the highest transport efficiencies in vivo, suggesting that data specific ligand-receptor interactions may be causative for lung-targeted delivery. Monitoring the distribution, transport, and clearance of CAKSMGDIVC-displaying phage particles deposited in the airways by aerosol treatment in vivo and ex vivo indicated that phage transport did not result in detectable lung injury and there was no anatomical or functional lung impairment. These results support the finding that phage particles may be suitable for safe inhaled administration.

[0010] To identify the putative receptor for the ligand CAKSMGDIVC peptide, a series of phage binding assays were performed in vitro and in vivo. Specific binding to human recombinant α3β1 integrin, followed by functional binding of CAKSMGDIVC-displaying phage particles and their transport through the cell monolayer of alveolar epithelial surrogates supported the ligand-receptor interaction. However, evidence that the targeted phage particles are key to passing through the lung hilum through a ligand-receptor-mediated mechanism was clearly demonstrated by the specific binding of CAKSMGDIVC-displaying phage particles to α3β1 integrin on the surface of AT1, AT2, and club cells in vivo. α3β1 integrin is expressed on the apical and basolateral membranes of alveolar cells. Without wishing to be limited by any theory, general mechanisms such as transcytosis (active receptor-mediated) or paracellular (passive passage between adjacent cells) transport could be potential facilitators of phage particle trafficking to the bloodstream. Therefore, ligand-directed delivery through selective targeting of α3β1 integrin represents a substantial advancement over traditional non-targeting aerosol formulations that require permeation enhancers or solubilizing carriers and thereby affect drug stability and dispersion.

[0011] To support the translational significance of the ligand peptide-directed lung delivery approach introduced herein, a targeted phage-based vaccination protocol was designed in non-human primates as a proof-of-concept for disease vaccination in non-human primates as an aerosol treatment approach for systemic humoral immunization. Selective lung transport of CAKSMGDIVC-displaying phage particles, followed by activation of specific systemic humoral responses, recapitulates long-standing principles of viral vaccinology and confers an advantage over conventional site-specific vaccination routes.

[0012] In certain non-limiting embodiments, phage particles are highly stable under harsh environmental conditions. In other non-limiting embodiments, large-scale production of phage particles is cost-effective (Bao, et al., 2018, Adv Drug Deliv Rev; Barbu, et al., 2016, Phage Therapy in the Era of Synthetic Biology. Cold Spring Harb Perspect Biol 8). In yet other non-limiting embodiments, phage-based vaccines do not induce detectable toxic side effects (Aghebati-Maleki, et al., 2016, J Biomed Sci 23:66). In yet other non-limiting embodiments, native phage particles do not have tropism for mammalian cells, do not replicate inside eukaryotic cells, and their use is generally considered safe when compared to other classical virus-based vaccination strategies (Barbu, et al., 2016, Cold Spring Harb Perspect Biol 8; Aghebati-Maleki, et al., 2016, J Biomed Sci 23:66). In yet other non-limiting embodiments, unlike conventional peptide vaccines that can often be inactivated due to a minimal temperature range (about 1°C), the systems introduced herein do not have the cumbersome and expensive requirements for maintaining a so-called "cold chain" during field application. In yet other non-limiting embodiments, the ligand-receptor discovery and vaccination properties of the phage-based systems of the present invention can also be used for the development of lung-directed gene target delivery using a library of adeno-associated virus (AAV) and phage hybrid vectors (referred to as AAVP) (Hajitou, et al., 2006, Cell 125:385-398; Suwan, et al., 2019, PNAS doi:10.1073 / pnas.1906653116). In yet other non-limiting embodiments, phage particles themselves are very strong immunogens and act as powerful adjuvants for inducing a persistent humoral response (Trepel, et al., 2001, Cancer Res 61:8110-8112).

[0013] Certain aspects of the disclosed subject matter are described in more detail below, and examples of the subject matter are partially illustrated in the accompanying drawings. The disclosed subject matter is described in combination with the recited claims, and it will be understood that the illustrated subject matter is not intended to limit the disclosed subject matter to the claims.

[0014] Throughout this document, values expressed in a range format should be interpreted in a flexible manner that includes not only the numerical values explicitly recited as the limits of the range, but also all the individual numerical values or sub-ranges subsumed within that range as if each numerical value and sub-range were explicitly recited. For example, a range of "about 0.1% to about 5%" or "about 0.1% to 5%" is to be interpreted to include not only about 0.1% to about 5%, but also the individual values within the indicated range (e.g., 1%, 2%, 3%, and 4%) and sub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%). The description "about X to Y" has the same meaning as "about X to about Y" unless otherwise indicated. Similarly, the description "about X, Y, or about Z" has the same meaning as "about X, about Y, or about Z" unless otherwise indicated.

[0015] In the methods described herein, acts may be performed in any order, except when the temporal or operational order is explicitly recited. Additionally, the recited acts may be performed in parallel unless the explicit claim language recites that they are to be performed separately. For example, the claimed acts of performing X and performing Y can be carried out simultaneously within a single operation, and the resulting method will fall within the literal scope of the claimed method.

[0016] Definitions Unless otherwise defined, all scientific and technical terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Any methods and materials similar or equivalent to those described herein can be used in the practice for testing of this disclosure, but the selected materials and methods are described herein. The following scientific terms are used in the description and claims of this disclosure.

[0017] In general, the scientific terms used herein as well as laboratory procedures in cell culture, molecular genetics, pharmacology, protein chemistry, and organic chemistry are those well known and commonly employed in the art.

[0018] Standard techniques are used for biochemical and / or biological manipulations. The techniques and procedures are generally performed according to conventional methods in the art and various general references, which are provided throughout this literature.

[0019] In this document, the terms "a", "an", or "the" are used to include one or more than one unless the context clearly dictates otherwise. The term "or" is used to refer to a non-exclusive "or" unless otherwise indicated. "At least one of A and B" or "at least one of A or B" has the same meaning as "A, B, or A and B". Additionally, it should be understood that terms or scientific terms used herein and not otherwise defined are for illustrative purposes only and not limiting. Any use of section headings is intended to aid in reading the document and should not be construed as limiting, and the information related to a particular section heading may be within or outside of that particular section. All publications, patents, and patent documents referenced in this document are hereby incorporated by reference in their entirety as if each were individually incorporated by reference.

[0020] As used herein, "about" when referring to a measurable value such as an amount, a temporal duration, and the like, means an inclusion of variations of ±20%, ±10%, more preferably ±5%, still more preferably ±1%, and even more preferably ±0.1% from the specified value, and such variations are appropriate for carrying out the disclosed methods.

[0021] "Adjuvant" refers to a substance that can enhance the immunogenicity of an antigen. An adjuvant can be a single substance or a mixture of substances and can function by acting directly on the immune system or by providing slow release of the antigen. Examples of adjuvants are aluminum salts, polyanions, bacterial glycopeptides, and slow release agents such as Freund's incomplete adjuvant.

[0022] The terms "alleviate" or "treat" mean that the clinical signs and / or symptoms associated with a disease are reduced as a result of the act being performed. The signs or symptoms to be monitored are well known to a skilled clinician.

[0023] The term "antibody" as used herein refers to an immunoglobulin molecule that specifically binds to an antigen. An antibody can be an intact immunoglobulin derived from a natural or recombinant source and can be the immunoreactive portion of an intact immunoglobulin. An antibody is typically a tetramer of immunoglobulin molecules. Antibodies in the present disclosure include, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab, and F(ab) 2In addition to these, they can exist in various forms, including single-chain antibodies (scFv) and humanized antibodies, as well as any modifications of these for enhancing or altering effector activity, such as glycosylation or mutations in the Fc domain (Harlow, et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow, et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston, et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird, et al., 1988, Science 242:423-426).

[0024] As used herein, the term "antigen" or "Ag" is defined as a molecule that elicits an immune response. This immune response can be accompanied by either, or both, antibody production or activation of specific immunocompetent cells. One of ordinary skill in the art will understand that virtually any macromolecule, including substantially all proteins or peptides, can act as an antigen. Furthermore, an antigen can be derived from recombinant or genomic DNA. One of ordinary skill in the art will understand that any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response encodes an "antigen" as the term is used herein. Furthermore, one of ordinary skill in the art will understand that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. The present disclosure includes, but is not limited to, the use of partial nucleotide sequences of one or more genes, and it is readily apparent that these nucleotide sequences can be arranged in various combinations to elicit a desired immune response. Further, one of ordinary skill in the art will understand that an antigen need not be encoded by a "gene" at all. It is readily apparent that an antigen can be produced, synthesized, or derived from a biological sample. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0025] As used herein, by "combination therapy" is meant that a first agent is administered in combination with another agent. "In combination" or "in combination with" refers to the administration of one treatment modality in addition to another treatment modality. Thus, "in combination" refers to the administration of one treatment modality before, during, or after the delivery of another treatment modality to an individual. Such combinations are considered to be part of a single treatment regimen or regime.

[0026] As used herein, the term "conservative sequence modification" is intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. The modifications can be introduced into the antibodies of the disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the CDR regions of an antibody can be replaced with other amino acid residues from the same side chain family, and the modified antibody can be tested for its ability to bind to an antigen using the functional assays described herein.

[0027] "Disease" is the health state of an animal where the animal cannot maintain homeostasis and its health continues to deteriorate if the disease is not resolved. In contrast, a "disorder" in an animal is a health state where the animal can maintain homeostasis, but its health state is less favorable than in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decline in the animal's health state.

[0028] As used herein, the terms "inducing an immune response" or "immunizing" refer to the process of generating a B cell and / or T cell response to a heterologous protein.

[0029] "Encoding" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, which serves as a template for the synthesis of other polymers and macromolecules in a biological process having either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids and the resulting biological properties. Thus, a gene encodes a protein when transcription and translation of the gene-corresponding mRNA produces the protein in a cell or other biological system. Both the nucleotide sequence that is identical to the mRNA sequence, the coding strand normally provided in the sequence listing, and the non-coding strand used as a template for transcription of the gene or cDNA can be referred to as encoding the protein or other product of that gene or cDNA.

[0030] As used herein, "endogenous" refers to any material produced from or within an organism, cell, tissue, or system.

[0031] As used herein, the term "exogenous" refers to any material introduced from or produced outside of an organism, cell, tissue, or system.

[0032] As used herein, the term "expression" is defined as the transcription and / or translation of a specific nucleotide sequence driven by its promoter.

[0033] "Expression vector" refers to a vector containing a recombinant polynucleotide that includes an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression, and other elements for expression can be supplied by the host cell or in an in vitro expression system. Examples of expression vectors include all those known in the art, such as cosmids, plasmids (e.g., those contained in naked or liposomes) into which recombinant polynucleotides have been incorporated, and viruses (e.g., Sendai virus, lentivirus, retrovirus, adenovirus, and adeno-associated virus).

[0034] "Homologous," as used herein, refers to subunit sequence identity between two polymer molecules, e.g., between two nucleic acid molecules, e.g., two DNA molecules or two RNA molecules, or between two polypeptide molecules. When the subunit positions in both of the two molecules are occupied by the same monomer subunit, e.g., when the positions in each of two DNA molecules are occupied by adenine, they are homologous at that position. The homology between two sequences is a direct function of the number of matching or homologous positions. For example, if half of the positions in two sequences (e.g., 5 positions in a polymer 10 subunits in length) are homologous, the two sequences are 50% homologous, and if 90% of the positions (e.g., 9 out of 10) match or are homologous, the two sequences are 90% homologous.

[0035] "Identity", as used herein, refers to subunit sequence identity between two polymer molecules, particularly between two amino acid molecules, such as between two polypeptide molecules. Two amino acid sequences are identical at a position if they have the same residue at the same position, e.g., if the position in each of two polypeptide molecules is occupied by arginine. The identity or degree to which two amino acid sequences have the same residue at the same position in an alignment is often expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of matching or identical positions. For example, if half of the positions in two sequences (e.g., 5 positions in a 10-subunit length polymer) are identical, the two sequences are 50% identical, and if 90% of the positions (e.g., 9 out of 10) match or are identical, the two amino acid sequences are 90% identical.

[0036] The term "immunogenicity", as used herein, refers to the innate ability of an antigen or organism to induce an immune response in an animal when administered to the animal. Thus, "enhancing immunogenicity" refers to increasing the ability of an antigen or organism to induce an immune response in an animal when administered to the animal. The increased ability of an antigen or organism to induce an immune response can be measured, in particular, by a greater number of antibodies binding to the antigen or organism, a greater diversity of antibodies against the antigen or organism, a greater number of T cells specific for the antigen or organism, a greater cytotoxic or helper T cell response against the antigen or organism, a higher expression of cytokines in response to the antigen, etc.

[0037] The term "immunoglobulin" or "Ig", as used herein, is defined as a class of proteins that function as antibodies. Antibodies expressed by B cells may be referred to as BCR (B cell receptor) or antigen receptor. The five members of this class of proteins are IgA, IgG, IgM, IgD, and IgE, as well as the subclasses within each class. IgA is the major antibody present in body secretions such as saliva, tears, breast milk, gastrointestinal secretions, and mucus secretions of the respiratory and urogenital tracts. IgG is the most common circulating antibody. IgM is the main immunoglobulin produced in the primary immune response in most subjects. It is the most efficient immunoglobulin in agglutination, complement fixation, and other antibody responses and is important in defense against bacteria and viruses. IgD is an immunoglobulin that has no known antibody function but can act as an antigen receptor. IgE is an immunoglobulin that mediates immediate hypersensitivity by causing the release of mediators from mast cells and basophils upon exposure to an allergen.

[0038] The term "immune response", as used herein, is defined as the cellular response to an antigen that occurs when lymphocytes identify an antigenic molecule as foreign and induce the formation of antibodies and / or activate lymphocytes to remove the antigen.

[0039] When an "immunologically effective amount", "autoimmune disease inhibiting effective amount" or "therapeutic amount" is indicated, the exact amount of the disclosed composition to be administered can be determined by a physician or researcher in consideration of the disease state.

[0040] "Isolated" means altered or removed from its natural state. For example, a nucleic acid or peptide that occurs naturally in a living animal is not "isolated", but the same nucleic acid or peptide that is partially or completely separated from the materials that naturally occur together in its natural state is "isolated". An isolated nucleic acid or protein can exist in a substantially purified form or can exist in a non-native environment, such as in a host cell.

[0041] As used herein, the term "modified" means an altered state or structure of a molecule or cell of the present disclosure. A molecule can be modified in many ways, including chemically, structurally, and functionally. A cell can be modified through the introduction of nucleic acids.

[0042] As used herein, the term "modulate" means to mediate a detectable increase or decrease in the level of a response in a subject as compared to the level of the response in the subject in the absence of treatment or in the absence of a compound, and / or as compared to the level of the response in an otherwise identical but untreated subject. The term encompasses disturbing and / or affecting the native signal or response, thereby mediating a beneficial therapeutic outcome in a subject, preferably a human.

[0043] "Parenteral" administration of an immunogenic composition includes, for example, subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrasternal injection, or infusion techniques.

[0044] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to a compound containing amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can constitute the sequence of a protein or peptide. A polypeptide includes any peptide or protein containing two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which are commonly referred to in the art as, for example, peptides, oligopeptides, and oligomers, and longer chains, which are commonly referred to in the art as proteins of many types. “Polypeptide” includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof. A peptide can be linear or branched, can contain modified amino acids, and can be interspersed with non-amino acids. The term also encompasses amino acid polymers that are modified either naturally or by intervention, and the modifications include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, for example, conjugation with a labeling component. For example, in addition to one or more analogs of amino acids (including, for example, non-natural amino acids, etc.), polypeptides and proteins containing other modifications known in the art are also included within the definition. A polypeptide can exist as a single chain or as associated chains.

[0045] As used herein, the term "pharmaceutical composition" refers to a mixture of at least one compound useful within the present disclosure with other chemical components, such as carriers, stabilizers, diluents, adjuvants, dispersing agents, suspending agents, thickening agents, and / or excipients. Pharmaceutical compositions facilitate the administration of compounds to organisms. A plurality of techniques for administering compounds exist in the art, including, but not limited to, intravenous, oral, aerosol, parenteral, ocular, pulmonary, and topical administration.

[0046] The description "pharmaceutically acceptable carrier" includes pharmaceutically acceptable salts, pharmaceutically acceptable materials, compositions or carriers, such as liquid or solid fillers, diluents, excipients, solvents or encapsulating materials, that are involved in carrying or transporting the compounds of the present disclosure within or to a subject so as to be able to perform their intended functions. Typically, such compounds are carried or transported from one organ, or part, of the body to another organ, or part, of the body. Each salt or carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Some examples of materials that can serve as pharmaceutically acceptable carriers include the following: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; diluents; granulating agents; lubricants; binders; disintegrants; wetting agents; emulsifiers; coloring agents; release agents; coating agents; sweetening agents; flavoring agents; fragrances; preservatives; antioxidants; plasticizers; gelling agents; thickening agents; hardeners (hardener); hardeners (setting agent); suspending agents; surfactants; wetting agents; carriers; stabilizers; and other non-toxic compatible substances used in pharmaceutical formulations, or any combination thereof. As used herein, "pharmaceutically acceptable carrier" also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, etc., that are compatible with the activity of the compound and physiologically acceptable to the subject. Auxiliary active compounds can also be incorporated into the compositions.

[0047] As used herein, the term "polynucleotide" is defined as a chain of nucleotides. Further, a nucleic acid is a polymer of nucleotides. Thus, nucleic acids and polynucleotides are interchangeable as used herein. One of ordinary skill in the art has the general knowledge that a nucleic acid is a polynucleotide and can be hydrolyzed into monomeric "nucleotides". Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotides include, but are not limited to, all nucleic acid sequences obtained by any means available in the art, including, but not limited to, recombinant means, i.e., cloning nucleic acid sequences from recombinant libraries or cell genomes using conventional cloning techniques and PCR (trademark), and synthetic means.

[0048] As used herein with respect to an antibody, the term "specifically binds" means an antibody that recognizes a particular antigen and does not substantially recognize or bind to other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species can also bind to that antigen from one or more species. However, such cross-species reactivity by itself does not change the classification of the antibody as specific. In another example, an antibody that specifically binds to an antigen can also bind to different allelic forms of the antigen. However, such cross-reactivity by itself does not change the classification of the antibody as specific. In some instances, the terms "specific binding" or "specifically binds" can be used with respect to the interaction of an antibody, protein, or peptide with a second chemical species to mean that the interaction is dependent on the presence of a particular structure on the chemical species (e.g., an antigenic determinant or epitope), e.g., the antibody recognizes and binds to a particular protein structure rather than proteins generally. If an antibody is specific for epitope "A", the presence of a molecule containing epitope A (or free, unlabeled A) will reduce the amount of labeled A bound to the antibody in a reaction containing labeled "A" and the antibody.

[0049] The term "subject" is intended to include living organisms (e.g., mammals) in which an immune response can be induced. A "subject" or "patient" can be a human or non-human mammal when used therein. Non-human mammals include, for example, non-human primates, as well as domestic and companion animals, such as ovine, bovine, porcine, canine, feline, and murine mammals. Preferably, the subject is a human.

[0050] "Target site" or "target sequence" refers to a genomic nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule can specifically bind under conditions sufficient for binding to occur.

[0051] As used herein, the term "therapeutic" means treatment and / or prevention. A therapeutic effect can be obtained by suppression, alleviation, or eradication of a disease state.

[0052] As used herein, "treating" a disease means reducing the frequency or severity of at least one sign or symptom of the disease or disorder experienced by a subject.

[0053] A "vector" is a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid into the interior of a cell. A number of vectors are known in the art and include, but are not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be construed to include non-plasmid and non-viral compounds that facilitate the entry of nucleic acids into cells, such as polylysine compounds, and liposomes. Examples of viral vectors include, but are not limited to, Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, and lentivirus vectors.

[0054] Range: Throughout this disclosure, various aspects of the disclosure may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, a description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, a description of a range, such as 1 to 6, should be considered to have specifically disclosed sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This is true regardless of the breadth of the range.

[0055] Compounds and Compositions In one aspect, the present disclosure relates to the identification of certain peptides that enable the transport of solid particles across the air-blood barrier in the lung. In certain embodiments, the solid particles are further derivatized with a therapeutically useful compound, for example, but not limited to, an antigen that can be used to trigger an immune response in a subject to whom the compositions of the present disclosure are administered. In still other embodiments, the therapeutically useful compound is presented on the surface of the solid particles. In still other embodiments, the therapeutically useful compound is contained within the solid particles.

[0056] In certain embodiments, the transport peptides contemplated within the present disclosure include, but are not limited to, CAINSLSRKC (SEQ ID NO:1), CAKSMGDIVC (SEQ ID NO:2), CGRKQVESSC (SEQ ID NO:3), and / or CRGKSAEGTC (SEQ ID NO:4). In certain embodiments, the transport peptides of the present disclosure are cyclic, with the cysteine at position n and the cysteine at position n+8 forming a disulfide bond. In other embodiments, the transport peptides of the present disclosure are not cyclic. In still other embodiments, the transport peptide consists of CAINSLSRKC (SEQ ID NO:1). In still other embodiments, the transport peptide consists of CAKSMGDIVC (SEQ ID NO:2). In still other embodiments, the transport peptide consists of CGRKQVESSC (SEQ ID NO:3). In still other embodiments, the transport peptide consists of CRGKSAEGTC (SEQ ID NO:4). In still other embodiments, the transport peptide consists essentially of CAINSLSRKC (SEQ ID NO:1). In still other embodiments, the transport peptide consists essentially of CAKSMGDIVC (SEQ ID NO:2). In still other embodiments, the transport peptide consists essentially of CGRKQVESSC (SEQ ID NO:3). In still other embodiments, the transport peptide consists essentially of CRGKSAEGTC (SEQ ID NO:4). In still other embodiments, the transport peptide comprises CAINSLSRKC (SEQ ID NO:1). In still other embodiments, the transport peptide comprises CAKSMGDIVC (SEQ ID NO:2). In still other embodiments, the transport peptide comprises CGRKQVESSC (SEQ ID NO:3). In still other embodiments, the transport peptide comprises CRGKSAEGTC (SEQ ID NO:4). In still other embodiments, the transport peptide has at least 70%, 80%, 90%, or 100% homology with the peptides of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.In yet other embodiments, the transport peptide has at least 70%, 80%, 90%, or 100% identity to the peptides of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.

[0057] In certain embodiments, the transport peptides contemplated within this disclosure include AINSLSRK (SEQ ID NO:5), AKSMGDIV (SEQ ID NO:6), GRKQVESS (SEQ ID NO:7), and / or RGKSAEGT (SEQ ID NO:8). In other embodiments, the transport peptide has at least 70%, 80%, 90%, or 100% homology to the peptides of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8. In yet other embodiments, the transport peptide has at least 70%, 80%, 90%, or 100% identity to the peptides of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.

[0058] In certain embodiments, the transport peptide contemplated in the present invention is a portion of a polypeptide, and the N-terminus of the transport peptide is the N-terminus of the polypeptide (i.e., the N-terminus of the transport peptide is not linked to other amino acids / peptides). In certain embodiments, the transport peptide contemplated in the present invention is a portion of a polypeptide, and the N-terminus of the transport peptide is not the N-terminus of the polypeptide. In certain embodiments, the transport peptide contemplated in the present invention is a portion of a polypeptide, and the N-terminus of the transport peptide is linked through an amide bond to the C-terminus of a first amino acid, which is a single amino acid or the C-terminus of a (poly)peptide. In certain embodiments, the first amino acid is aspartic acid. In certain embodiments, the first amino acid is glutamic acid. In certain embodiments, the first amino acid is lysine. In certain embodiments, the first amino acid is arginine. In certain embodiments, the first amino acid is histidine. In certain embodiments, the first amino acid is alanine. In certain embodiments, the first amino acid is valine. In certain embodiments, the first amino acid is leucine. In certain embodiments, the first amino acid is isoleucine. In certain embodiments, the first amino acid is proline. In certain embodiments, the first amino acid is phenylalanine. In certain embodiments, the first amino acid is methionine. In certain embodiments, the first amino acid is tryptophan. In certain embodiments, the first amino acid is glycine. In certain embodiments, the first amino acid is asparagine. In certain embodiments, the first amino acid is glutamine. In certain embodiments, the first amino acid is cysteine. In certain embodiments, the first amino acid is serine. In certain embodiments, the first amino acid is threonine. In certain embodiments, the first amino acid is tyrosine. In certain embodiments, the first amino acid is not aspartic acid. In certain embodiments, the first amino acid is not glutamic acid. In certain embodiments, the first amino acid is not lysine. In certain embodiments, the first amino acid is not arginine.In certain embodiments, the first amino acid is not histidine. In certain embodiments, the first amino acid is not alanine. In certain embodiments, the first amino acid is not valine. In certain embodiments, the first amino acid is not leucine. In certain embodiments, the first amino acid is not isoleucine. In certain embodiments, the first amino acid is not proline. In certain embodiments, the first amino acid is not phenylalanine. In certain embodiments, the first amino acid is not methionine. In certain embodiments, the first amino acid is not tryptophan. In certain embodiments, the first amino acid is not glycine. In certain embodiments, the first amino acid is not asparagine. In certain embodiments, the first amino acid is not glutamine. In certain embodiments, the first amino acid is not cysteine. In certain embodiments, the first amino acid is not serine. In certain embodiments, the first amino acid is not threonine. In certain embodiments, the first amino acid is not tyrosine.

[0059] In certain embodiments, the transport peptide contemplated in the present invention is a portion of a polypeptide, and the C-terminus of the transport peptide is the C-terminus of the polypeptide (i.e., the C-terminus of the transport peptide is not linked to other amino acids / peptides). In certain embodiments, the transport peptide contemplated in the present invention is a portion of a polypeptide, and the C-terminus of the transport peptide is not the C-terminus of the polypeptide. In certain embodiments, the transport peptide contemplated in the present invention is a portion of a polypeptide, and the C-terminus of the transport peptide is linked through an amide bond to the N-terminus of a second amino acid, which is a single amino acid or the N-terminus of a (poly)peptide. In certain embodiments, the second amino acid is aspartic acid. In certain embodiments, the second amino acid is glutamic acid. In certain embodiments, the second amino acid is lysine. In certain embodiments, the second amino acid is arginine. In certain embodiments, the second amino acid is histidine. In certain embodiments, the second amino acid is alanine. In certain embodiments, the second amino acid is valine. In certain embodiments, the second amino acid is leucine. In certain embodiments, the second amino acid is isoleucine. In certain embodiments, the second amino acid is proline. In certain embodiments, the second amino acid is phenylalanine. In certain embodiments, the second amino acid is methionine. In certain embodiments, the second amino acid is tryptophan. In certain embodiments, the second amino acid is glycine. In certain embodiments, the second amino acid is asparagine. In certain embodiments, the second amino acid is glutamine. In certain embodiments, the second amino acid is cysteine. In certain embodiments, the second amino acid is serine. In certain embodiments, the second amino acid is threonine. In certain embodiments, the second amino acid is tyrosine. In certain embodiments, the second amino acid is not aspartic acid. In certain embodiments, the second amino acid is not glutamic acid. In certain embodiments, the second amino acid is not lysine. In certain embodiments, the second amino acid is not arginine.In certain embodiments, the second amino acid is not histidine. In certain embodiments, the second amino acid is not alanine. In certain embodiments, the second amino acid is not valine. In certain embodiments, the second amino acid is not leucine. In certain embodiments, the second amino acid is not isoleucine. In certain embodiments, the second amino acid is not proline. In certain embodiments, the second amino acid is not phenylalanine. In certain embodiments, the second amino acid is not methionine. In certain embodiments, the second amino acid is not tryptophan. In certain embodiments, the second amino acid is not glycine. In certain embodiments, the second amino acid is not asparagine. In certain embodiments, the second amino acid is not glutamine. In certain embodiments, the second amino acid is not cysteine. In certain embodiments, the second amino acid is not serine. In certain embodiments, the second amino acid is not threonine. In certain embodiments, the second amino acid is not tyrosine.

[0060] Conservative amino acid substitutions, i.e., substitution of one amino acid with another amino acid having a related side chain, are also contemplated herein. Genetically encoded amino acids are generally grouped into four families: (1) acidic, i.e., aspartic acid, glutamic acid; (2) basic, i.e., lysine, arginine, histidine; (3) nonpolar, i.e., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar, i.e., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine may be grouped together as aromatic amino acids. Generally, substitution of a single amino acid within these families will not have a major effect on biological activity. A polypeptide can have one or more (e.g., one, two, three, etc.) single amino acid deletions compared to the exemplified sequence. A polypeptide can also include one or more (e.g., one, two, three, etc.) insertions compared to the exemplified sequence.

[0061] The present disclosure further contemplates any cell comprising any vector comprising any nucleic acid sequence encoding any of the transport peptides of the present disclosure, in addition to any nucleic acid sequence encoding any of the transport peptides of the present disclosure, in addition to any nucleic acid sequence encoding any of the transport peptides of the present disclosure. The present disclosure further contemplates nucleic acid sequences having about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequences provided herein.

[0062] In certain embodiments, at least one residue within the transport peptide and / or at the carboxy terminus of the transport peptide and / or at the amino terminus of the transport peptide is substituted with methylation, amidation, acylation (such as, but not limited to, acetylation), and / or any other chemical group without adversely affecting the activity of the transport peptide within the compositions and / or methods of the present disclosure. In other embodiments, the N-terminus of the transport peptide is acylated, such as, but not limited to, acetylated. In other embodiments, the C-terminus of the transport peptide is amidated.

[0063] In certain embodiments, the present disclosure provides solid particles with a transport peptide presented on the surface of the solid particles. In other embodiments, the transport peptide is attached to the surface of the solid particles. In yet other embodiments, the transport peptide is covalently attached to the surface of the solid particles. In still other embodiments, the solid particles are selected from the group consisting of phage, engineered cells, tissue fragments, nanoparticles, vesicles, dendrimers, virus-like particles (VLPs), adenoviruses, adeno-associated viruses (AAVs), adeno-associated virus phages (referred to as AAVPs), and any combination thereof. In some cases, the nanoparticles have a diameter on the nanometer scale and can vary from a diameter of about 1 nm to a diameter of about 1,000 nm. In some cases, the phage has a diameter shorter than about 10 nm, such as, but not limited to, about 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, or 10 nm. In some cases, the phage has a length shorter than 1,000 nm, such as, but not limited to, about 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, or 1,000 nm. In yet another embodiment, the composition further comprises an agent selected from the group consisting of therapeutic substances, biologically active molecules, imaging agents, radioactive substances, salts, peptides, proteins, lipids, nucleic acids, gases, and any combination thereof, wherein the agent is attached to and / or contained within the solid particles. In certain embodiments, the transport peptide is attached to and / or presented over the entire surface of the solid particles. In other embodiments, the transport peptide is attached to at least a portion of the surface of the solid particles and / or presented on at least a portion of the surface of the solid particles. The solid particles can be prepared using methods known to those skilled in the art or purchased from commercial suppliers.

[0064] In some embodiments, the present disclosure provides a vaccine composition comprising a transport peptide as described elsewhere herein. In some embodiments, the vaccine composition is a live attenuated vaccine, an inactivated vaccine, a subunit, recombinant, polysaccharide, or conjugated vaccine, and / or a toxoid vaccine. In some embodiments, the vaccine composition is a DNA vaccine, an RNA vaccine, a replicating viral vector vaccine, a non-replicating viral vector vaccine, an inactivated viral vector vaccine, and / or a virus-like particle vaccine that is known to be useful for nasal, buccal, inhaled, intratracheal, intrapulmonary, and / or bronchial delivery. In some embodiments, the vaccine composition comprises an adjuvant. Exemplary adjuvants are described elsewhere herein.

[0065] The transport peptides of the present disclosure can be synthesized using chemical and biochemical methods known to those of skill in the art of chemical synthesis or peptide synthesis. The transport peptides can be attached to the surface of solid particles using any method known to those of skill in the art. In certain embodiments, the transport peptides can be attached to the surface of solid particles via a covalent bond. In a non-limiting example, a free amino group in the transport peptide can be attached to a free carboxylate group on the surface of the solid particle via a covalent amide bond. In a non-limiting example, a free carboxylic acid group in the transport peptide can be attached to a free amino group on the surface of the solid particle via a covalent amide bond. In other embodiments, the transport peptides can be attached to the surface of solid particles via a non-covalent bond.

[0066] In certain embodiments, the solid particles are bacteriophages, such as, but not limited to, filamentous phages. Filamentous bacteriophages can include, but are not limited to, fd, fl, or M13 bacteriophages. In certain embodiments, the bacteriophage is an fd bacteriophage. In other embodiments, the bacteriophage is a T4, T7, or λ phage. In some embodiments, the filamentous phage has a diameter equal to or less than about 10 nm. In some embodiments, the filamentous phage has a diameter equal to or less than about 9 nm. In some embodiments, the filamentous phage has a diameter equal to or less than about 8 nm. In some embodiments, the filamentous phage has a diameter equal to or less than about 7 nm. In some embodiments, the filamentous phage has a diameter equal to or less than about 6 nm. In some embodiments, the filamentous phage has a diameter equal to or less than about 5 nm. In some embodiments, the filamentous phage has a diameter equal to or less than about 4 nm. In some embodiments, the filamentous phage has a diameter equal to or less than about 3 nm. In some embodiments, the filamentous phage has a diameter equal to or less than about 2 nm. In some embodiments, the filamentous phage has a diameter equal to or less than about 1 nm. In some embodiments, the filamentous phage has a diameter equal to or greater than about 9 nm. In some embodiments, the filamentous phage has a diameter equal to or greater than about 8 nm. In some embodiments, the filamentous phage has a diameter equal to or greater than about 7 nm. In some embodiments, the filamentous phage has a diameter equal to or greater than about 6 nm. In some embodiments, the filamentous phage has a diameter equal to or greater than about 5 nm. In some embodiments, the filamentous phage has a diameter equal to or greater than about 4 nm. In some embodiments, the filamentous phage has a diameter equal to or greater than about 3 nm. In some embodiments, the filamentous phage has a diameter equal to or greater than about 2 nm. In some embodiments, the filamentous phage has a diameter equal to or greater than about 1 nm.

[0067] Typically, filamentous phages (M13, fd, fl) have a filamentous capsid with a circular ssDNA molecule. The genome typically contains 10 genes but does not contain a gene for a lysis protein. The virion is envelope-coated. Filamentous phages typically infect only Escherichia coli (E. coli) cells that have an F plasmid, because the phage must adsorb to the F pilus to gain entry into the cell. Their life cycle involves an intracellular dsDNA intermediate replication form, which is converted to an ssDNA molecule prior to capsid formation. The phages provide an easy means for preparing ssDNA for DNA sequencing. The best-known example is bacteriophage M13, which has been adapted for use as a cloning and sequencing vector. The wild-type Ml3 genome is 6,407 bp in length. Other relatives of Ml3 are fd and fl. The phage-modified cloning vector fUSE5 has a length of approximately 9,200 pb.

[0068] Various methods of phage display and methods of producing diverse populations of peptides are well known in the art. For example, U.S. Patent Nos. 5,223,409; 5,622,699; 5,866,363; and 6,068,829; and Japanese Patent No. 4875497 B2, each of which is incorporated herein by reference, describe methods of preparing phage libraries. Phage display technology involves genetically manipulating bacteriophages such that small peptides can be expressed on their surfaces [Smith, 1985, Science 228(4705):1315-1317]. In this technology, a gene encoding a protein of interest is inserted into a phage coat protein gene, such that the phage "presents" a protein on its outside while containing the gene for the protein on the inside, resulting in a link between genotype and phenotype. In the case of M13 filamentous phage display, DNA encoding a protein or peptide of interest is ligated into the pIII or pVIII gene encoding the minor or major coat protein, respectively. Multiple cloning sites may be used to ensure that the fragment is inserted in all three possible reading frames, such that the cDNA fragment is translated in the appropriate frame. The phage gene and insert DNA hybrid is then inserted into Escherichia coli bacterial cells, such as TG1, SS320, ER2738, or XL1-Blue E. coli (a process known as "transduction"). When a "phagemid" vector is used, phage particles are not released from the E. coli cells until they are infected with a helper phage, which allows for the packaging of the phage DNA and the assembly of mature virions having the associated protein fragment as part of the outer coat on the minor (pIII) or major (pVIII) coat protein.

[0069] It should be noted that the phage display method can be applied not only to transport peptides but also to any peptide and / or protein (such as, but not limited to, biologically active peptides and / or antigens) to be presented on the surface of the phage.

[0070] The peptides and proteins contemplated in the present disclosure can be prepared in several known ways, such as, for example, by chemical synthesis (entirely or in part), by digesting longer polypeptides using proteases, by translation from RNA, from cell cultures (e.g., from recombinant expression), by purification from the organism itself (e.g., after bacterial culture or directly from a patient), and others. Methods for producing the proteins of the present disclosure are known to those skilled in the art. For example, protein production can include culturing the host cells of the present disclosure under conditions that induce protein expression.

[0071] Non-limiting methods for the production of peptides with a length of less than 40 amino acids involve in vitro chemical synthesis (Raddrizzani, et al., 2000, Briefs in Bioinformatics 14(2):121-130; Fields, et al., 1997, Principles of Peptide Synthesis. ISBN:0387564314). Solid-phase peptide synthesis is available, which is a method based on, for example, tPoc or Fmoc chemistry (Chan, et al., 2000, Fmoc solid phase peptide synthesis. ISBN:0849368413). Enzymatic synthesis can also be used in part or in whole. As an alternative to chemical synthesis, biological synthesis can be used, for example, polypeptides can be produced by translation. This can be carried out in vitro or in vivo. Biological methods are generally limited to the production of polypeptides based on L-amino acids, but manipulations of the translation machinery (e.g., aminoacyl tRNA molecules) can be used to introduce D-amino acids (or other non-natural amino acids such as iodotyrosine or methylphenylalanine, azidohomoalanine, etc.) (Ibba, 1996, Biotechnology and Genetic Engineering Review 13:197-216). However, chemical synthesis can be used when D-amino acids are included. The proteins of the present disclosure can have covalent modifications at the C-terminus and / or N-terminus.

[0072] The proteins useful within the present disclosure can take on various forms (e.g., native, fusions, glycosylated, non-glycosylated, lipidated, non-lipidated, phosphorylated, non-phosphorylated, myristoylated, non-myristoylated, monomeric, multimeric, particulate, denatured, etc.). The proteins of the present disclosure can be provided in a purified or substantially purified form, i.e., in a form substantially free of other polypeptides (e.g., free of naturally occurring polypeptides), and generally are at least about 50 (weight)% pure, usually at least about 90% pure, i.e., less than about 50% of the composition, more preferably less than about 10% (e.g., 5%) is composed of other expressed proteins.

[0073] The polypeptides of the present disclosure can include a detectable label (e.g., a radioactive or fluorescent label, or a biotin label). The proteins of the present disclosure can be glycosylated either naturally or non-naturally (i.e., the polypeptide has a glycosylation pattern different from the glycosylation pattern found in the corresponding naturally occurring polypeptide).

[0074] Various assays can be used to evaluate the in vivo immunogenicity of the proteins of the present disclosure. For example, the polypeptide can be recombinantly expressed and used to screen patient sera by immunoblot. A positive reaction between the polypeptide and the patient sera indicates that the patient has previously initiated an immune response, particularly an antibody response, to the protein in question, i.e., the protein is an immunogen. This method can also be used to identify immunodominant proteins.

[0075] Method In one aspect, the present disclosure provides a method for promoting or increasing the transport of solid particles through the air-blood barrier in the lung of a subject. In certain embodiments, the method includes administering to the subject solid particles to which a transport peptide of the present disclosure is attached. In other embodiments, the administration is via a route including nasal, buccal, inhalation, intratracheal, intrapulmonary, or intrabronchial.

[0076] In one aspect, the present disclosure provides a method for promoting systemic circulation of solid particles in a subject. In certain embodiments, the method includes administering to the subject solid particles having a transport peptide of the present disclosure attached to their surface. In other embodiments, the administration is via a route including nasal, buccal, inhalation, intratracheal, intrapulmonary, or intrabronchial.

[0077] In one aspect, the present disclosure provides a method for immunizing a subject against a disease or disorder. In certain embodiments, the method includes administering to the subject solid particles having a transport peptide of the present disclosure attached to their surface, wherein the surface of the solid particles is further derivatized with an antigen that promotes an immune response against the disease or disorder. In other embodiments, the administration is via a route including nasal, buccal, inhalation, intratracheal, intrapulmonary, or intrabronchial.

[0078] In another aspect, the present disclosure provides a method for vaccinating a subject against a disease or disorder. In certain embodiments, the method includes administering to the subject a vaccine comprising solid particles having a transport peptide of the present disclosure attached to their surface, wherein the surface of the solid particles is further derivatized with an antigen that promotes an immune response against the disease or disorder. In other embodiments, the administration is via a route including nasal, buccal, inhalation, intratracheal, intrapulmonary, or intrabronchial.

[0079] In one aspect, the present disclosure provides a method for treating and / or preventing a disease or disorder in a subject. In certain embodiments, the method includes administering to the subject solid particles having a transport peptide of the present disclosure attached to their surface, wherein the surface of the solid particles is further derivatized with an antigen that promotes an immune response against the disease or disorder. In other embodiments, the administration is via a route including nasal, buccal, inhalation, intratracheal, intrapulmonary, or intrabronchial.

[0080] In one aspect, the present disclosure provides a method of treating a subject at risk of developing a disease or disorder. In certain embodiments, the method comprises administering to the subject solid particles to which a transport peptide of the present disclosure is attached, the surface of the solid particles being further derivatized with an antigen that promotes an immune response against the disease or disorder. In other embodiments, the administration is via a route including nasal, buccal, inhalation, intratracheal, intralung, or intrabronchial.

[0081] The compositions of the present disclosure can be administered in a manner appropriate for the disease or disorder to be treated (or prevented). The amount and frequency of administration are determined by factors such as the condition of the patient and the type and severity of the patient's disease, but appropriate dosages and schedules can be determined by clinical trials.

[0082] The compositions of the present disclosure can generally be administered directly to a patient. Direct delivery can be achieved by parenteral injection (e.g., subcutaneous, intraperitoneal, intravenous, intramuscular, or injection into the interstitial space of a tissue), or by rectal, oral, vaginal, topical, transdermal, intranasal, sublingual, ocular, aural, pulmonary, or other mucosal administration. In certain embodiments, the administration is via a route including nasal, buccal, inhalation, intratracheal, intralung, or intrabronchial.

[0083] The dosing regimen can be a single-dose schedule or a multiple-dose schedule. For example, multiple doses can be used in a primary immunization schedule and / or a booster immunization schedule. A booster dose schedule can be carried out following a primary dose schedule. The timing between priming doses (e.g., between 4 and 16 weeks), as well as the preferred timing between priming and boosting, can be determined by conventional methods.

[0084] Solid forms suitable for dissolution or suspension in a liquid vehicle prior to injection can also be prepared (e.g., lyophilized compositions). The compositions can be prepared for pulmonary administration, for example, as an inhaler using, e.g., fine powders or sprays. The compositions can be prepared for nasal, ear or eye administration, for example, as sprays, droplets, gels or powders. See, e.g., Almeida, et al., 1996, J. Drug Targeting 3:455-467.

[0085] The antigens in the composition can typically be present at a concentration of at least 1 μg / ml each. Generally, the concentration of any given antigen is sufficient to induce an immune response against that antigen.

[0086] Pharmaceutical composition Certain aspects of the present disclosure are directed to prophylactically treating an individual in need thereof. As used herein, the term "prophylactic treatment" refers to the administration of an antigen to a subject who does not exhibit signs or symptoms of a disease, pathology, or medical disorder, or who exhibits only early signs or symptoms of a disease, pathology, or disorder, such that the treatment is carried out for the purpose of reducing, preventing, or lessening the risk of the disease, pathology, or medical disorder. Prophylactic treatment functions as a preventative treatment against a disease or disorder.

[0087] Certain aspects of the present disclosure are directed to therapeutically treating an individual in need thereof. As used herein, the term "therapeutically" includes, but is not limited to, the administration of an antigen to a subject who exhibits signs or symptoms of a pathology, disease, or disorder, where the treatment is carried out on the subject for the purpose of reducing or eliminating those signs or symptoms of the pathology, disease, or disorder.

[0088] Aspects of the present disclosure are directed to compositions and methods for enhancing an immune response of a subject to one or more antigens. As used herein, the terms "subject" and "host" are intended to include living organisms, such as mammals. Examples of a subject or host include non-mammals, such as non-mammalian vertebrates, such as birds (e.g., chickens or ducks), fish or frogs (e.g., Xenopus), and non-mammalian invertebrates, as well as transgenic species thereof, including but not limited to horses, cows, sheep, pigs, goats, dogs, cats, rabbits, guinea pigs, rats, mice, hamsters, non-human primates, and humans. Preferably, the subject is a human.

[0089] The compositions of the present disclosure can include one or more pharmaceutically or physiologically acceptable carriers. A pharmaceutically acceptable carrier is a compound that does not induce a harmful effect by itself on the individual to whom the composition is administered. Suitable carriers are typically large, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, sucrose, trehalose, lactose, and lipid aggregates (e.g., oil droplets or liposomes). Such carriers are well known to those skilled in the art. Vaccines can also contain diluents, such as water, saline, and glycerol. Additionally, adjuvant substances, such as wetting or emulsifying agents, and pH buffering substances can be present. Sterile, pyrogen-free, phosphate-buffered saline is a typical carrier.

[0090] The compositions of the present disclosure can include an antimicrobial agent, particularly in the case of packages in a multiple-dose format. The compositions of the present disclosure can include a surfactant, such as Tween (polysorbate), such as Tween 80. The surfactant is generally present at a low level, such as <0.1%. The compositions of the present disclosure can include a sodium salt (e.g., sodium chloride) to provide tonicity. A concentration of sodium chloride of 10±2 mg / ml is typical. The compositions of the present disclosure can generally include a buffer. A phosphate buffer is typical. The compositions of the present disclosure can include a sugar alcohol (e.g., mannitol) or a disaccharide (e.g., sucrose or trehalose) at, for example, about 15 to 30 mg / ml (e.g., 25 mg / ml), which is particularly applicable when they are lyophilized or when they include a material reconstituted from the lyophilized material. The pH of the composition for lyophilization can be adjusted to about 6.1 prior to injection.

[0091] The compositions of the present disclosure can include an immunogenic adjuvant. An adjuvant is a pharmacological or immunological agent that modifies the effect of other agents. Adjuvants can be added to vaccines to boost the immune response to produce more antibodies and longer-lasting immunity and to minimize the dose of antigen required. Adjuvants can also be used to enhance the effectiveness of vaccines, for example, by modifying the immune response to specific types of immune system cells, such as by activating T cells instead of antibody-secreting B cells, depending on the purpose of the vaccine. Immunogenic adjuvants include, but are not limited to, alum, MF59, AS03, virosomes, AS04, aluminum hydroxide, and paraffin oil.

[0092] Mineral-containing compositions suitable for use as adjuvants in the present disclosure include mineral salts such as aluminum salts and calcium salts. The present disclosure includes mineral salts such as hydroxides (e.g., oxyhydroxides), phosphates (e.g., hydroxylated phosphates, orthophosphates), and sulfates, etc., or mixtures of different mineral compounds, and the compounds can take any suitable form (e.g., gel, crystalline, and amorphous, etc.), and adsorption is preferred. The mineral-containing composition can also be formulated as particles of a metal salt.

[0093] Aluminum phosphate is useful, especially in compositions containing oligosaccharide antigens, and a typical adjuvant is amorphous aluminum hydroxylated phosphate with a PO / Al molar ratio of 0.84 - 0.92, contained at 0.6 mg Al / ml. Adsorption using low doses of aluminum phosphate can be used, for example, at 50 - 100 μg / conjugate / dose.

[0094] Oil emulsion compositions suitable for use as adjuvants in the present disclosure include squalene-water emulsions such as MF59 (5% squalene, 0.5% Tween 80, 0.5% Span 85, formulated into submicron particles using a microfluidizer). Complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA) can also be used.

[0095] Saponin compositions can also be used as adjuvants in the present disclosure. Saponins are a heterogeneous group of sterol glycosides and triterpenoid glycosides found in the bark, leaves, stems, roots, and flowers of a wide range of plant species. Saponins from the bark of the tree Quillaja saponaria Molina have been widely studied as adjuvants. Saponins can also be obtained commercially from Smilax ornata (sarsaprilla), Gypsophilla paniculata (brides Veil), and Chlorogalum pomeridianum (soap plant). Saponin adjuvant formulations include purified formulations such as QS21, as well as lipid formulations such as ISCOM. QS21 is commercially available as Stimulon (trademark).

[0096] Additional adjuvants suitable for use in the present disclosure include bacterial or microbial derivatives such as non-toxic derivatives of enterobacterial lipopolysaccharide (LPS), lipid A derivatives, immunostimulatory oligonucleotides, and ADP ribosylation toxins and their detoxified derivatives. Non-toxic derivatives of LPS include monophosphoryl lipid A (MPL) and 3-O-deacylated MPL (3dMPL). 3dMPL is a mixture of 3 de-O-acylated monophosphoryl lipid A with 4, 5, or 6 acyl chains. "Small particle" forms of 3 de-O-acylated monophosphoryl lipid A are also available. Such "small particles" of 3dMPL are small enough to be sterile filtered through a 0.22um membrane. Other non-toxic LPS derivatives include monophosphoryl lipid A mimics such as aminoalkyl glucosaminide phosphate derivatives, such as RC-52950,51. Lipid A derivatives include derivatives of lipid A from Escherichia coli, such as OM-174.

[0097] Immunostimulatory oligonucleotides suitable for use as adjuvants in the present disclosure include nucleotide sequences containing CpG motifs (dinucleotide sequences containing unmethylated cytosine linked by a phosphate bond to guanosine). Double-stranded RNAs and oligonucleotides containing palindrome or poly(dG) sequences have also been shown to be immunostimulatory. CpG can include nucleotide modifications / analogs, such as phosphorothioate modifications, and can be double-stranded or single-stranded. The CpG sequence can be directed to TLR9, such as the motif GTCGTT or TTCGTT. The CpG sequence can be specific for inducing a Th1 immune response, such as CpG-A ODN, or more specific for inducing a B cell response, such as CpG-B ODN. Preferably, CpG is CpG-A ODN.

[0098] Preferably, the CpG oligonucleotide is constructed such that the 5' end is accessible for receptor recognition. Optionally, two CpG oligonucleotide sequences can be attached at their 3' ends to form an "immunomer". Bacterial ADP-ribosylating toxins and their detoxified derivatives can be used as adjuvants in the present disclosure. Preferably, the protein is derived from Escherichia coli (E. coli heat-labile enterotoxin "LT"), Vibrio cholerae ("CT"), or Bordetella pertussis ("PT"). The toxin or toxoid is preferably in the form of a holotoxin containing both the A and B subunits. Preferably, the A subunit contains a detoxifying mutation, and preferably, the B subunit is not mutated. Preferably, the adjuvant is a detoxified LT mutant, such as LT-K63, LT-R72, and LT-G192. ADP-ribosylating toxins and their detoxified derivatives, particularly LT-K, can be used. Numerical references for amino acid substitutions are preferably based on the alignment of the A and B subunits of the ADP-ribosylating toxin.

[0099] Administration / Dosage / Formulation The dosing regimen can affect what constitutes an effective amount. The therapeutic formulations can be administered to a subject either before or after the onset of a disease or disorder contemplated in the present disclosure. Further, in addition to several divided dosages, variable dosages can be administered daily or sequentially, or the dosage can be administered by continuous infusion, or the dosage can be a bolus injection. Further, the dosage of the therapeutic formulation can be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.

[0100] Administration of the compositions of the present disclosure to a patient, such as a mammal, such as a human, can be carried out in an effective dosage and time period to treat a disease or disorder contemplated in the present disclosure using known procedures. The effective amount of the therapeutic compound required to achieve a therapeutic effect can vary depending on factors such as the condition of the disease or disorder in the patient; the age, sex, and weight of the patient; and the ability of the therapeutic compound to treat a disease or disorder contemplated in the present disclosure. The dosing regimen can be adjusted to provide optimal therapeutic efficacy. For example, several divided doses can be administered daily, or the dosage can be proportionally reduced as indicated by the exigencies of the therapeutic situation. A non-limiting example of the effective dosage range of the therapeutic compounds of the present disclosure is from about 1 to 5,000 mg per kg of body weight per day. One of ordinary skill in the art can study the relevant factors and determine the effective amount of the therapeutic compound without undue experimentation.

[0101] In certain embodiments, the effective dosage range is measured in units known to those of skill in the art as being suitable for the description of phage dosages. In some embodiments, the effective dosage range of the vaccines or therapeutic compounds of the present disclosure is measured in transduction units (TU) / kg / day or particles / kg / day. In some embodiments, the dosage provided to a patient is about 10 6 ~10 12It is TU / kg / day. In some embodiments, the effective dosage range is measured by plaque forming units (PFU), colony forming units (CFU), 50% tissue culture infective dose (TCID50), plaque reduction neutralization test (PRNT), and combinations thereof.

[0102] The actual dosage level of the active ingredient in the pharmaceutical compositions of the present disclosure can be varied so as to obtain an amount of the active ingredient effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient.

[0103] The therapeutically effective amount or therapeutically effective dosage of the compounds of the present disclosure depends on the age, sex and weight of the patient, the current medical condition of the patient, and the progression of the disease or disorder contemplated in the present disclosure.

[0104] A medical doctor having ordinary skill in the art, such as a physician or veterinarian, can readily determine and prescribe an effective amount of the pharmaceutical composition required. For example, a physician or veterinarian can start with a dosage of the compound of the present disclosure used in the pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0105] In certain embodiments, the compositions of the present disclosure are administered to a patient at a dosage of 1 to 5 times per day or more. In other embodiments, the compositions of the present disclosure are administered to a patient within a dosage range including, but not limited to, once a day, every two days, every three days to once a week, and once every two weeks. It will be readily apparent to those skilled in the art that the frequency of administration of the various combination compositions of the present disclosure will vary from individual to individual depending on many factors including, but not limited to, age, the disease or disorder to be treated, sex, general health status, and other factors. Therefore, the present disclosure should not be construed as limited to any particular dosage regimen, and the exact dosage and composition to be administered to any patient will be determined by the attending physician taking into account all other factors for that patient.

[0106] The amount of the compound administered per day can be administered, in non-limiting examples, daily, every other day, every two days, every three days, every four days, every five days, weekly, every two weeks, every three weeks, every four weeks, or monthly. For example, in the case of administration every other day, a dose of 5 mg / day can be started on Monday, the first subsequent dose of 5 mg / day can be administered on Wednesday, the second subsequent dose of 5 mg / day can be administered on Friday, and so on. As a second example, in the case of administration every four weeks for immunization purposes, each dose can be administered every 28 days. In certain embodiments where the disclosed formulation or composition is administered every 28 days for immunization purposes, serum is collected every 14 days.

[0107] If the patient's condition improves, at the discretion of the physician, administration of the inhibitor of the present disclosure can be optionally continued, or alternatively, the dose of the drug administered can be temporarily reduced or temporarily discontinued for a certain period of time (i.e., a "drug holiday"). The length of the drug holiday can optionally vary from 2 days to 1 year, and by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. Examples of dose reduction during the drug holiday include 10% to 100%, and by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0108] If the patient's condition improves, a maintenance dose is administered if necessary. Thereafter, the dosage or the frequency of administration, or both, is reduced to a level at which the improvement of the disease is maintained, as a function of the disease or disorder. In certain embodiments, the patient requires intermittent treatment based on any recurrence of symptoms and / or infections over a long period.

[0109] The compounds for use in the methods of the present disclosure can be formulated into unit dosage forms. The term "unit dosage form" refers to physically discrete units suitable as unit dosages for a patient being treated, each unit containing a predetermined amount of the active material calculated to produce the desired therapeutic effect, optionally in association with a suitable pharmaceutical carrier. The unit dosage form can be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day), and when multiple daily doses are used, the unit dosage forms can be the same or different for each dose.

[0110] The toxicity and therapeutic efficacy of such therapeutic regimens are optionally determined in cell cultures or experimental animals, which includes, but is not limited to, the determination of LD 50 (the dose lethal to 50% of the population) and ED 50 (the dose therapeutically effective in 50% of the population). The dose ratio between the toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio between LD 50 and ED 50 . Data obtained from cell culture assays and animal studies are optionally used in formulating the dosage range for use in humans. The dosage of such compounds is, in certain embodiments, within a range of circulating concentrations that includes the ED 50 with minimal toxicity. The dosage optionally varies within this range depending on the dosage form employed and the route of administration utilized.

[0111] In certain embodiments, the compositions of the present disclosure are formulated using one or more pharmaceutically acceptable excipients or carriers. In certain embodiments, the pharmaceutical compositions of the present disclosure comprise a therapeutically effective amount of a compound of the present disclosure and a pharmaceutically acceptable carrier.

[0112] The carrier can also be a solvent or dispersion medium containing, for example, saline, buffered saline, water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycols, etc.), suitable mixtures thereof, and vegetable oils. Appropriate fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal, etc. In many cases, it is advisable to include in the composition isotonic agents, such as sugars, sodium chloride, or polyalcohols, such as mannitol and sorbitol.

[0113] In certain embodiments, the present disclosure is directed to a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound of the present disclosure, alone or in combination with a second pharmaceutical agent; and instructions for using the compound for treating, preventing, or reducing one or more symptoms of a disease or disorder contemplated in the present disclosure.

[0114] The formulations can be used in combination with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances known in the art and suitable for any suitable mode of administration. The pharmaceutical preparations can be sterilized and, if desired, can be mixed with auxiliaries, such as lubricants, preservatives, stabilizers, wetting agents, emulsifying agents, salts for influencing the osmotic pressure, buffers, colorants, flavoring agents, and / or aromatic substances, etc. They can also be combined, if desired, with other active agents, such as analgesics.

[0115] The routes of administration of any composition of the present disclosure include oral, nasal, pulmonary, rectal, vaginal, parenteral, buccal, sublingual, or topical. The compounds for use in the present disclosure can be administered by any suitable route, such as oral or parenteral routes, for example, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., transvaginal and perivaginal), nasal (intra) and (trans)rectal), intravesical, intralung, intraduodenal, intragastric, intrathecal, subcutaneous, intramuscular, intradermal, intraarterial, intravenous, intrabronchial, inhalation, and can be formulated for topical administration. In certain embodiments, the routes of administration of any composition of the present disclosure include nasal, buccal, inhalation, intratracheal, intralung, and intrabronchial.

[0116] Suitable compositions and dosage forms include, for example, dispersions, suspensions, solutions, syrups, granules, beads, powders, pellets, liquid sprays for nasal or oral administration, dry powders or aerosolized formulations for inhalation, etc. It should be understood that the formulations and compositions useful in the present disclosure are not limited to the specific formulations and compositions described herein.

[0117] The powdered and granule formulations of the pharmaceutical preparations of the present disclosure can be prepared using known methods. Such formulations can be administered directly to a subject and can be used, for example, to form materials suitable for administration to a subject. Each of these formulations can further include one or more of a dispersing or wetting agent, a suspending agent, and a preservative. Additional excipients, such as fillers and sweetening agents, flavoring agents, or coloring agents, can also be included in these formulations.

[0118] Oral administration For oral administration, tablets, dragees, liquids, drops, suppositories, or capsules, caplets and gelcaps are particularly suitable. Compositions intended for oral use can be prepared according to any method known in the art, and such compositions can contain one or more agents selected from the group consisting of inert and non-toxic pharmaceutical excipients suitable for the manufacture of tablets. Such excipients include, for example, inert diluents such as lactose; granulating and disintegrating agents such as corn starch; binders such as starch; and lubricants such as magnesium stearate. Tablets can be uncoated or can be coated by known techniques for elegance or to delay release of the active ingredient. Preparations for oral use can also be presented as hard gelatin capsules in which the active ingredient is mixed with an inert diluent.

[0119] Parenteral administration As used herein, "parenteral administration" of a pharmaceutical composition includes any route of administration characterized by a physical breaching of the tissue of a subject and administration of the pharmaceutical composition through an entry in the tissue. Parenteral administration thus includes, but is not limited to, administration of the pharmaceutical composition by injection of the composition, administration by application of the composition through a surgical incision, and administration by application of the composition through a tissue-permeable non-surgical wound. In particular, parenteral administration is envisioned to include, but is not limited to, subcutaneous, intravenous, intraperitoneal, intramuscular, intracardiac injection, and kidney dialysis infusion techniques.

[0120] Buccal, pulmonary, inhalation, and intranasal administration, etc. The pharmaceutical compositions of the present disclosure can be prepared, packaged, or sold in formulations suitable for pulmonary administration via the buccal cavity. Such formulations can be liquid or dry / powder formulations that include one or more targeting peptides of the present disclosure. In some embodiments, the formulations include the active ingredients described elsewhere herein. In some embodiments, the particles of the dry / powder formulation have a diameter in the range of about 0.5 to about 7 micrometers, and in certain embodiments about 1 to about 6 micrometers. Such compositions are advantageously in the form of dry powder for administration using a device that includes a dry powder reservoir that can direct a stream of propellant to disperse the powder, or for administration using a self-injectable solvent / powder dispensing container, e.g., a device that includes an active ingredient dissolved or suspended in a low-boiling propellant in a sealed container. In certain embodiments, such powders include particles having a diameter such that at least 98% of the particles by weight are greater than 0.5 micrometers and at least 95% of the particles by number are less than 7 micrometers. In certain embodiments, at least 95% of the particles by weight have a diameter greater than 1 micrometer and at least 90% of the particles by number have a diameter less than 6 micrometers. The dry powder composition can include a solid fine powder diluent, e.g., sugar, and is advantageously provided in unit dosage form. See also EP Patent Nos. EP 02 12 753B1 and 1 370 318B1.

[0121] Low-boiling propellants generally include liquid propellants having a boiling point of less than 65 o °F at atmospheric pressure. Generally, the propellant can constitute 50 to 99.9% (w / w) of the composition, and the active ingredient can constitute 0.1 to 20% (w / w) of the composition. The propellant can further include additional ingredients, e.g., liquid nonionic or solid anionic surfactants or solid diluents (in certain embodiments having a particle size on the same order as the particles containing the active ingredient).

[0122] The pharmaceutical compositions of the present disclosure formulated for pulmonary delivery can also provide the active ingredient in the form of droplets of a solution or suspension. Such formulations can be prepared, packaged, or sold as an optionally sterile, aqueous or dilute alcoholic solution or suspension containing the active ingredient, and can conveniently be administered using any nebulizing or atomizing device. Such formulations can further contain one or more additional ingredients including, but not limited to, flavoring agents such as sodium saccharin, volatile oils, buffering agents, surfactants, or preservatives such as methyl hydroxybenzoate. In certain embodiments, the droplets provided by this route of administration have an average diameter in the range of about 0.1 to about 200 micrometers.

[0123] The pharmaceutical compositions of the present disclosure can be delivered using an inhaler, such as those described in U.S. Patent No. US 8,333,192 B2, which is hereby incorporated by reference in its entirety.

[0124] The formulations described herein as being useful for pulmonary delivery are also useful for intranasal delivery of the pharmaceutical compositions of the present disclosure.

[0125] Another formulation suitable for intranasal administration is a coarse powder containing the active ingredient and having an average particle size of about 0.2 to 500 micrometers. Such formulations are administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nose from a powder container held near the nostrils. Formulations suitable for transnasal administration can, for example, contain from about 0.1% (w / w) to 100% (w / w) of the active ingredient and can further contain one or more of the additional ingredients described herein.

[0126] Additional dosage forms Additional dosage forms of the present disclosure include dosage forms as described in U.S. Patent Nos. 6,340,475; 6,488,962; 6,451,808; 5,972,389; 5,582,837; and 5,007,790. Additional dosage forms of the present disclosure also include dosage forms as described in U.S. Patent Application Nos. 20030147952; 20030104062; 20030104053; 20030044466; 20030039688; and 20020051820. Additional dosage forms of the present disclosure also include dosage forms as described in PCT Application Nos. WO 03 / 35041; WO 03 / 35040; WO 03 / 35029; WO 03 / 35177; WO 03 / 35039; WO 02 / 96404; WO 02 / 32416; WO 01 / 97783; WO 01 / 56544; WO 01 / 32217; WO 98 / 55107; WO 98 / 11879; WO 97 / 47285; WO 93 / 18755; and WO 90 / 11757.

[0127] Controlled Release Formulations and Drug Delivery Systems In certain embodiments, the formulations of the present disclosure can be, but are not limited to, controlled release, such as sustained release, delayed release, and pulsatile release formulations, in addition to short-term rapid disappearance.

[0128] The term sustained release is used in its conventional meaning and refers to a pharmaceutical formulation that provides a gradual release of a drug over a long period of time and, although not necessarily, can result in a substantially constant blood level of the drug over a long period of time. The time period can be months or longer and should be a longer release than the same amount of the agent administered in bolus form.

[0129] For sustained release, the compounds can be formulated with a suitable polymer or hydrophobic material that provides the compounds with sustained release properties. Thus, the compounds for use in the methods of the present disclosure can be administered in the form of microparticles, for example, by injection, or in the form of wafers or disks by implantation.

[0130] In certain embodiments of the present disclosure, the compounds of the present disclosure are administered to a patient, alone or in combination with another pharmaceutical agent, using a sustained release formulation.

[0131] The term delayed release is used in its conventional meaning and refers to a pharmaceutical formulation that provides an initial release of a drug after some delay following drug administration and that may, but does not necessarily, include a delay of from about 10 minutes to about 12 hours.

[0132] The term pulsed release is used in its conventional meaning and refers to a pharmaceutical formulation that provides release of a drug in such a manner as to produce a pulsed plasma profile of the drug following drug administration.

[0133] The term immediate release is used in its conventional meaning and refers to a pharmaceutical formulation that provides release of a drug immediately following drug administration.

[0134] As used herein, short term refers to any time period up to about 8 hours, up to about 7 hours, up to about 6 hours, up to about 5 hours, up to about 4 hours, up to about 3 hours, up to about 2 hours, up to about 1 hour, up to about 40 minutes, up to about 20 minutes, or up to about 10 minutes following drug administration, and all or any partial increments thereof, and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes and all or any partial increments thereof.

[0135] As used herein, rapid disappearance refers to any time period up to about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes after drug administration and up to and including all and any whole or partial increments of these, as well as about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes and all and any whole or partial increments of these.

[0136] One of ordinary skill in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures, aspects, claims, and examples described herein. Such equivalents are considered to be within the scope of this disclosure and are covered by the claims appended hereto. For example, modifications in reaction conditions using alternatives recognized in the art and no more than routine experimentation, including but not limited to, for example, reaction time, reaction size / volume, and experimental reagents such as solvents, catalysts, pressure, atmospheric conditions such as nitrogen atmosphere, and reducing / oxidizing agents, are to be understood as being within the scope of this application.

[0137] It is to be understood that whenever values and ranges are provided herein, all values and ranges subsumed within these values and ranges are meant to be subsumed within the scope of this disclosure. Further, in addition to all values falling within these ranges, the upper or lower limit of a range of values is also contemplated by this application.

[0138] The following examples further illustrate aspects of the disclosure with examples. However, they are in no way intended to limit the teachings or disclosure of the disclosure as presented herein.

[0139] The practice of the present disclosure, unless otherwise indicated, uses conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the knowledge of those of ordinary skill in the art. Such techniques are well explained in the literature, such as, for example, "Molecular Cloning: A Laboratory Manual", 4th edition (Sambrook, 2012); "Oligonucleotide Synthesis" (Gait, 1984); "Culture of Animal Cells" (Freshney, 2010); "Methods in Enzymology", "Handbook of Experimental Immunology" (Weir, 1997); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Short Protocols in Molecular Biology" (Ausubel, 2002); "Polymerase Chain Reaction: Principles, Applications and Troubleshooting", (Babar, 2011); "Current Protocols in Immunology" (Coligan, 2002). These techniques are applicable to the production of the polynucleotides and polypeptides of the present disclosure and can therefore be considered in the production and practice of the present disclosure.

[0140] It should be understood that the methods and compositions useful in the present disclosure are not limited to the specific formulations shown in the examples. The following examples are presented to provide those of ordinary skill in the art with a complete disclosure and description of the methods of making and using the cells, expansion and culture methods, and treatment methods of the present disclosure, and are not intended to limit the scope of what the inventors regard as their disclosure.

Examples

[0141] Experimental Examples The present disclosure will now be described with reference to the following examples. These examples are provided for illustrative purposes only and the present disclosure is not limited to these examples, but rather includes all variations that are obvious as a result of the teachings provided herein.

[0142] Materials and Methods Animals: BALB / c mice were purchased from The Jackson Laboratory (Sacramento, CA). All animal experiments were approved by the Institutional Care and Use Committees (IACUCs) of the University of Texas M.D. Anderson Cancer Center (UTMDACC), the University of New Mexico Health Sciences Center, and Rutgers Cancer Institute of New Jersey. Adult rhesus monkeys used in the vaccination studies were housed at the Michale E. Keeling Center for Comparative Medicine and Research, an Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)-accredited animal facility at the University of Texas M.D. Anderson Cancer Center (UTMDACC). For aerosol administration, trained mouse and primate handlers followed the National Research Council's Guide for the Care and Use of Laboratory Animals.

[0143] Tissue culture: Human alveolar epithelial adenocarcinoma A549 cells were purchased from the American Type Culture Collection (ATCC) and maintained in Dulbecco's Modified Eagle's Minimum Essential Medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS), vitamins, non-essential amino acids, penicillin / streptomycin, and L-glutamine at 37 °C in a 5% CO 2 humidified incubator.

[0144] In vivo selection of an aerosolized phage display library: To identify ligand peptide sequences that mediate the delivery of phage particles into the bloodstream through the pulmonary hilum, a random phage peptide library presenting insert CX 8 C (where X is any amino acid residue and C is a cysteine residue) was used for in vivo screening. Six- to eight-week-old BALB / c females were used. The phage input in mice was 10 9 TU / mouse. Animals were aerosolized via the intratracheal route with 50 μL of phosphate-buffered saline (PBS) containing the phage library using a high-pressure syringe (Penn-Century) and a MICROSPRAYER® Aerosolizer connected to a small animal laryngoscope (Penn-Century). Using this device, a air-free liquid aerosol was administered directly to the trachea of animals deeply anesthetized with isoflurane (1%). Four rounds of selection were performed as described. In round 1 (R1), animals were aerosolized with 10 9 TU of CX 8A C library was provided (see the scheme in Fig. 1B). After 1 h, phage particles were recovered from the bloodstream, amplified, and pooled as R1. In round 2 (R2), the phage pooled in R1 was administered and recovered 30 min after aerosol treatment. The subsequent R2 was amplified and pooled for administration in round 3 (R3). After 10 min, the phage pooled in R3 was recovered and processed for aerosol treatment in the final round 4 (R4). After 5 min, the phage particles were recovered from the bloodstream, amplified, and sequenced. Purification of phage particles and DNA sequencing of phage were performed as described (Arap, et al., 1998, Science 279:377-380).

[0145] Antibodies and reagents: The anti-mouse integrin α3 / CD49c antibody was from R&D System. The anti-human integrin α3 (ASC-1) blocking antibody was from Merck-Millipore. Human ITGA3 (clones: 002204.2-1161s21c1; 002204.1-2356s1c1 and 002204.1-2887s1c1), ITGB1 (clones: TRCN0000275133; TRCN0000275134 and TRCN0000275135), and shRNA lentiviral transduction particles for control pLKO.1 non-mammalian shRNA were purchased from MISSION® shRNA (Sigma-Aldrich).

[0146] LPS-induced lung injury and lung permeability assay: Mice were randomly divided into the following groups (n = 3 - 5 each): vehicle only (PBS), negative control (non - inserted phage particles), targeted phage (phage particles presenting CAKSMGIDVC), and positive control (LPS - dextran treatment). To induce acute lung injury, mice were anesthetized with 1% isoflurane and aerosolized with LPS (0.5 mg / kg in 50 μL of PBS; Klebsiella pneumoniae, Sigma) in 50 μL of PBS. Five hours later, the animals were aerosolized with 50 μL of a solution containing 10 mg / kg body weight of high - molecular - weight dextran (70 kDa, Invitrogen) dissolved in sterile PBS. The remaining groups were aerosolized with 50 μL of PBS only (vehicle), 10 9 TU of non - inserted phage (negative control) or 10 9 TU of targeted phage (presenting CAKSMGIDVC). Mice were sacrificed 1 h later. Thirty minutes before the end - point, Evans blue dye (20 mg / kg) was administered intravenously (IV). Lungs were perfused and homogenized in PBS for Evans blue dye extraction measurement. Tissue homogenates were quantified at an absorbance of 620 nm and corrected for the presence of heme pigment. The concentration of Evans blue dye was determined according to a standard calibration curve and expressed as total protein (μg / mL of protein). Neutrophils in BALF were counted using Trypan Blue (ThermoFisher Scientific) in a cell counter. Total protein in BALF was determined by bicinchoninic acid (BCA) calorimetric assay (ThermoFisher Scientific). Fluorescent dextran was purchased from Invitrogen, and Evans blue dye was purchased from Sigma - Aldrich. Anti - fd bacteriophage antibody was purchased from Sigma - Aldrich.

[0147] Phage binding assay: Phage binding to recombinant proteins (α3β1, α6β1, α6β4, NRP-1, and SDC-1) and BSA (Sigma) was performed as described (Cardo-Vila, et al., 2008, PLoS One 3:e3452). Briefly, each of 100 ng of the indicated proteins dissolved in 50 μL of PBS was immobilized overnight (ON) at 4°C in microtiter wells. The wells were washed twice with PBS, blocked with PBS containing 3% BSA for 1 h at room temperature (RT), and incubated with targeted CAKSMGDIVC-display phage particles or control no-insert phage particles in 50 μL of PBS containing 1.5% BSA. After 2 h at RT, the wells were gently washed 10 times with PBS, the phage was recovered by host bacteria infection, and represented as “relative TU” as empirical measurements of biological replicates and controls when compared to each other as described (Arap, W. et al., Nat. Med., 2002, 8:121-127; Cardo-Vila, et al., 2008, PLoS One 3:e3452). All human recombinant proteins α3β1, α6β1, α6β4, NRP-1, and SDC-1 were obtained commercially from R&D Systems. Recombinant GST and CAKSMGDIVC-GST were produced in Escherichia coli transformed with the pGEX4T-1 plasmid (Amersham, GE Healthcare) and purified using standard protocols. All synthetic peptides were custom-made by Merrifield synthesis and quality-controlled against the required specifications (Biomatik and PolyPeptide Laboratories).

[0148] Cell Barrier Permeability Assay: Cells were grown to complete confluence on TRANSWELL® inserts (0.4-μm pore size). After equilibration (30 min, 37°C) in pre-warmed serum-free DMEM, either targeted CAKSMGDIVC-display phage particles or control no-insert phage particles (10 each) 9Dose solutions containing TU) were added to the upper chamber (donor) of the TRANSWELL® system, respectively. Phage particles transported through the cell monolayer were collected by sampling the lower chamber (receiver) at predetermined intervals throughout the experiment along with replacement of the receiver chamber fluid with warm medium. Phage particle transport was determined by TU count. shRNA lentiviral transduction particles for human ITGA3, ITGB1, and control pLKO.1 non-mammalian shRNA were purchased from MISSION® shRNA (Sigma-Aldrich), and cell transduction was performed as indicated by the manufacturer.

[0149] Fluorescence microscopy imaging: Cells were seeded in complete medium on circular cover glasses (1.5 × 10 5 cells / cover glass) in 24-well plates and grown overnight at 37 °C in 5% CO 2 2. Cells were washed three times with PBS and fixed for 10 min at RT in PBS containing 4% paraformaldehyde (PFA) (Electron Microscopy Science), followed by incubation for 30 min in 50 mM ammonium chloride buffer and 1 h in a blocking solution of PBS containing 1% BSA. For intracellular staining of phage particles, cells were blocked for 1 h with DMEM containing 30% FBS at 37 °C, followed by 10 9The TU phage particles were incubated with the cells for 1 h. The cells were washed 5 times with PBS containing 10% BSA, followed by 5 washes for 3 min each with glycine buffer at pH 2.8 containing 50 mM glycine and 150 mM NaCl to remove the adhered phage particles. The cells were then washed with PBS, fixed in PBS containing 4% PFA for 10 min, permeabilized with 0.2% Triton X-100 for 10 min, washed with PBS, and then blocked with PBS containing 5% normal serum and 1% BSA for 30 min. The primary anti-fd bacteriophage (Sigma-Aldrich) and anti-mouse α3 integrin / CD49c (R&D System) antibodies were diluted in PBS containing 1% BSA, incubated with the cells for 2 h at RT, washed 5 times with PBS, and then incubated with the secondary antibody for 1 h at RT. For nuclear staining, VECTASHIELD mounting medium containing 4′,6-diamino-2-phenylindole (DAPI, Vector Laboratories) was used. Fluorescent images were acquired on a Nikon Eclypse Ti2 inverted fluorescence microscope (Nikon). For tissue immunofluorescence, paraffin-embedded lung tissue sections (10 μm thick) were incubated with Histochoice (Sigma-Aldrich), followed by removal of paraffin using xylene and ethanol. After antigen retrieval with Dako Target Retrieval Solution at pH 6.0 (Agilent Dako), the slides were washed and blocked with 10% donkey serum in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h, and incubated with antibodies: anti-proSPC (1:50) (Millipore, AB3786), anti-podoplanin (1:100) (Thermo-Invitrogen, eBio8.1.1), anti-CCSP antibody (Millipore-Merck, 07-623), anti-fd bacteriophage antibody (1:500) (Sigma-Aldrich), and anti-α3 integrin / CD49c (R&D System), followed by incubation with the conjugated secondary antibody.High-resolution images were obtained by two-photon confocal microscopy at the Advanced Light Microscopy Core Facility, University of Colorado (Denver). Pixel colocalization was analyzed using Fiji ImageJ Software.

[0150] Phage overlay and tissue immunohistochemistry: Lung tissue sections (5 μm) were deparaffinized, rehydrated, and blocked for endogenous peroxidase and non-specific protein binding (Agilent Dako). For the phage overlay assay, tissue sections were incubated with targeted CAKSMGDIVC-display phage particles or negative control non-insert phage particles (each 2×10 9 TU) at RT for 2 h. After washing with Tris-buffered saline containing 0.1% Tween 20 (TBST), slides were incubated with primary anti-fd bacteriophage antibody (1:800), followed by incubation with rabbit horseradish peroxidase (HRP)-conjugated secondary antibody. Integrin α3 was detected using anti-α3 chain antibody (1:500), followed by the appropriate HRP-conjugated secondary antibody. Images were acquired on a Nikon Eclypse Ti2 inverted microscope.

[0151] Isolation and cell sorting of lung cells: Six- to eight-week-old female BALB / c mice were used. After aerosol treatment, the animals were sacrificed, perfused with 10 mL of PBS, and then the lungs were gently inflated with air. The lungs were washed with PBS solution containing 5 mM EDTA and 5 mM EGTA, followed by RPMI medium and then RPMI containing 25 mM HEPES and elastase (4.5 U / mL), and then immediately, the lungs were inflated with a solution of water containing 1% low melting point agarose (Promega) and tissue digestion was performed at 37 °C for 45 minutes. After the tissue was gently minced, the cells were filtered through 100 μm (Falcon #352360), 40 μm (Falcon #352340), and 20 μm (Pluriselect #43-50020-01) filters and centrifuged with a 150 μL cushion layer of 100% Percoll (Sigma-Aldrich) in a 15 mL conical tube. For cell sorting, the cells were blocked with Fc block anti-mouse CD16 / CD32 antibody (BD Pharmigen, 553142) and stained with rat anti-mouse EPCAM brilliant violet 421 conjugated (BioLegend, 118225), rat anti-mouse CD45 Alexa fluor 700 conjugated (eBioscience, 56-0451-80), Syrian hamster anti-mouse podoplanin monoclonal antibody PE-Cyanine 7 conjugated (eBioscience, 25-5382-80), rat anti-CD31 FITC conjugated (BD Pharmigen, 553372), and rat anti-mouse F4 / 80 PE-conjugated (BD Pharmigen, 565410). The cells were sorted on an iCyt sy3200 (Sony) cell sorter previously calibrated with compensation beads (UltraComp eBeads, Thermo Scientific). The AT1, AT2 enriched cell populations and macrophages were centrifuged and the number of phage particles per cell type was determined by TU count.

[0152] Mathematical modeling: To investigate the transport of phage particles through cell monolayers, data from in vitro transwell assays (Figure 3E) were used to quantify the parameters of an empirical mathematical model (Equation 1) of phage accumulation in the lower chamber. The mass of phage particles transported through the cell barrier at time t [measured experimentally as N(t), TU] is given by Equation (Equation 1): N(t)=N s (1 - e -kt )(Equation 1) where N s represents the mass (TU) of phage particles in the lower chamber at saturation; k is the transport rate constant, and its reciprocal gives the characteristic time of the transport process (τ = 1 / k). The first derivative of Equation 1 TIFF0007692218000002.tif7128 provides the time-dependent transport rate of phage particles through the in vitro cell barrier. The least-squares fitting of the model to the experimental data was performed in MATLAB. The transport rate decays exponentially with time at rate k, and its maximum value can be determined as TIFF0007692218000003.tif7128 at time t = 0.

[0153] To understand the whole-body disposition kinetics of phage particles in vivo, a two-compartment pharmacokinetic model was developed (Figure 5C). This model is based on the laws of mass conservation and mass action and consists of the following system of ordinary differential equations (Equations 2 - 5): Alveolar subcompartment TIFF0007692218000004.tif7165 Mononuclear phagocyte subcompartment TIFF0007692218000005.tif7165 Central compartment (blood flow) TIFF0007692218000006.tif7165 Peripheral compartment (slowly perfused organs) TIFF0007692218000007.tif7165 where N L,a、 N C , and N Prepresents the mass (TU) of phage particles in the alveolar airspace, central, and peripheral compartments; N 0 is the mass of the inhaled phage; N L,mac is the mass of phage particles in the alveolar macrophage subcompartment; k 1,2 represents the first-order phage transfer rate constant from the central compartment to the peripheral compartment, k 2,1 represents the first-order phage transfer rate constant from the peripheral compartment to the central compartment. The system of ordinary differential equations (ODEs) was numerically solved as an initial value problem in MATLAB using the built-in non-stiff ODE solver ode45, and then the least-squares fitting of the model to the in vivo data was performed in MATLAB.

[0154] Intratracheal administration of CAKSMGDIVC-displayed phage particles: Phage input in mice was administered via the intratracheal route with 50 μL of PBS using a MicroSprayer® Aerosolizer connected to a high-pressure syringe (Penn-Century) and a small animal laryngoscope (Penn-Century) at 10 9 TU of targeted CAKSMGDIVC-displayed phage particles or negative control non-inserted phages. Intratracheal aerosol administration of two consecutive doses of targeted CAKSMGDIVC-displayed phage particles or control non-inserted phage particles was performed in paired rhesus monkeys. In addition to the anatomical morphology of the airways from mice to non-human primates and humans, due to species-specific differences in body size, breathing pattern, inhalation method, and devices, the dose of phage administration was 10 per individual monkey 12Increased in TU. Monkeys were anesthetized using intramuscular injection of Telazol (tiletamine and zolazepam) for induction, followed by endotracheal intubation and maintenance with inhaled isoflurane (percentage adjusted based on monitoring of depth of anesthesia - heart rate, respiratory rate, response to stimuli). Sterile IV tubing was cut to a length approximately 4 cm longer than the endotracheal tube and attached to a syringe containing CAKSMGDIVC targeting phage or control non - inserted phage. The tubing was inserted into the endotracheal tube and the phage was slowly administered into the trachea over 60 seconds. For blood collection, monkeys were anesthetized with intramuscular injection of ketamine. Blood samples were collected hourly until 6 h after aerosol treatment at the time of administration of dose 1 (designated as day 1). The second dose was administered 28 days later while collecting serum every 14 days. Endotoxin removal was performed for each phage preparation prior to administration of each endotracheal dose. Phage solution containing endotoxin was treated on ice for 10 minutes with 10% Triton X - 114 in endotoxin - free water. The solution was then warmed to 37°C for 10 minutes, followed by removal of the Triton X - 114 phase by centrifugation at 14,000 rpm for 1 minute. Endotoxin levels were measured using Lonza's Limulus Amebocyte Lysate (LAL) Kinetic - QCL kit. Phage preparations with < 0.05 EU / mL endotoxin were used in this study.

[0155] Serological analysis in non - human primates: IgG from monkeys was purified from serum using Protein G agarose resin (Sigma Aldrich). Flow - through was used to purify IgA with jacalin agarose resin (Thermo Fisher Scientific). Coated overnight at 4°C on 96 - well plates (Nunc MaxiSorp flat bottom, Thermo Scientific) with 10 10ELISA was performed using 50 μL of particles. For this assay, phage titration was carried out by quantitative qPCR using fUSE primers (fUSE5 forward: 5’-TGAGGTGGTATCGGCAATGA-3’ and fUSE5 reverse: 5’-GGATGCTGTATTTAGGCCGTTT-3’ as follows). ELISA was also performed using 96-well plates coated with synthetic peptide (10 μg / mL) CAKSMGDIVC or an irrelevant control peptide (CGRRAGGSC) overnight at 4°C unless otherwise specified. The coated plates were blocked with PBS containing 5% low-fat milk and 1% BSA (Sigma-Aldrich) for 1 h at 37°C. Two-fold serial dilutions (starting at 1:4) of purified IgG and IgA were applied to the wells and incubated at 37°C for 2 h. After three washes with PBS and PBST, the bound antibodies were detected with anti-sal IgG (KPL; 074-11-021) or IgA (KPL: 074-11-011) HRP conjugate. Purified polyclonal IgG anti-CAKSMGDIVC antibody (Biomatik USA, Delaware) and anti-fd bacteriophage antibody (Sigma) were used as positive controls. The plates were read at an absorbance of 450 nm.

[0156] Statistical analysis: Differences between groups were tested for statistical significance by Student's t-test or analysis of variance (two-way ANOVA). Statistical significance was set at p < 0.05. Analyses were performed in GraphPad Prism 8 and MATLAB R2015b.

[0157] Selected Results In particular, in developing countries and disaster areas, inhaled-based vaccination to achieve rapid immunization is needle-free and, unlike the oral route, is not exposed to undesirable first-pass metabolism. The lung surface area varies depending on the measurement technique and the degree of inflation, and an approximate value can vary from 70 to 130 square meters in the inflated lung. The thin and highly permeable alveolar region of the lung, including alveolar type 1 (ATI) and type 2 (AT2) cells, as well as associated microvascular endothelium, generally defines the selective permeability of molecules allowed to pass through and enter the bloodstream. Low molecular weight drugs, peptides, or proteins such as insulin, small viruses, and even immunogens are suitable candidates for inhalant administration. More recently, inhaled-based vaccination platforms have received particular attention for effective field use as well as protection from airborne pathogens such as tuberculosis, influenza, Ebola virus, and measles. Indeed, the ongoing pandemic of coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) provides prima facie evidence of the magnitude of unmet public health needs in the global pandemic situation.

[0158] In theory, lung delivery improves therapeutic bioavailability while reducing potential side effects by achieving a more rapid onset of action. However, inhalation also presents inherent challenges, particularly for systemic applications, and limits its use at this point for respiratory diseases. Generally, inhaled-based therapies are evaluated through the monitoring of pharmacologic endpoints in vivo. However, surprisingly, the actual mechanisms by which inhaled particles interact with the air-blood barrier, the physicochemical changes of molecules upon contact with the lung surface, bioavailability, uptake by the local immune system, and the clearance or removal processes of insoluble active compounds remain largely unknown.

[0159] In certain embodiments, the lipophilic moiety can be rapidly absorbed through the lungs by passive diffusion across the alveolar cell plasma membrane, while the hydrophilic moiety tends to be transported by specific surface receptors or through cell tight junctions. Elucidating the physiological mechanisms that allow for the selective transport of active particles through the lungs while preserving respiratory function and homeostasis is important for the design of a general pulmonary delivery system for multiple applications.

[0160] As described in the present disclosure, a phage display-based combinatorial random peptide platform has been developed and applied to identify unique ligand / receptor-mediated pulmonary transport pathways for the safe and effective absorption of particles passing through the air-blood barrier for phage-based targeted applications, including but not limited to vaccines.

[0161] In one aspect, an aerosol phage display random peptide library was screened in vivo to select and isolate targeting peptides that can pass through the intact lung air-blood barrier and enter the bloodstream. A new ligand peptide motif, CAKSMGDIVC, was verified, and its corresponding receptor, integrin α3β1, was biochemically purified via affinity chromatography. Integrin α3β1 is expressed on the surface of club cells, which are epithelial secretory cells found in the lung termini and respiratory bronchioles in addition to alveolar epithelial cells. Specific binding of targeted phage particles presenting the CAKSMGDIVC motif to α3β1 was found to promote phage particle uptake and transport to circulation in vivo. In another aspect, a two-compartment pharmacokinetic mathematical model was developed to understand and predict the systemic disposition kinetics of phage particles in pulmonary targeted administration. In yet another aspect, evaluation of a ligand / receptor-based aerosol system in a non-human primate model found that the system is useful for pulmonary targeted delivery and potential development of phage-based vaccines. Collectively, the combinatorial selection system and discoveries reported herein provide a versatile enabling platform for ligand-directed pulmonary aerosol delivery with broad translational applications.

[0162] Example 1: Continuous Screening of Phage Particles Passing Through the Intact Lung Barrier In Vivo Identification of ligand peptide motifs that are physiologically transported through intact lung epithelium was performed using a phage display library containing approximately 10 10 transducing units (TU) of random cyclic peptides (10 9This was done by administering (unique arrays of) phage particles to mice by in vivo intratracheal aerosol treatment. This method utilizes a microspray aerosolization device for aqueous preparations based on the generation of high pressure for particles smaller than 2.5 μm in size, which is predicted to reach the distal airspaces as shown in preclinical studies of pulmonary drug deposition (Guillon, et al., 2018, Int J Pharm 536:116-126). In the initial round of selection, passage through the pulmonary hilum in mice was confirmed by detection of phage particles in fixed time points after aerosol treatment and in blood samples collected up to 6 hours (Figure 1A). In vivo screening was performed in consecutive cohorts of mice (n = 3 each). After each subsequent round of selection, the recovered phage particles were pooled, amplified, and re-aerosolized (Figure 1B) to select ligand peptides that efficiently mediate the transport of phage particles across the pulmonary epithelial-endothelial layer, and the collection time was progressively reduced from 60 minutes in Round 1 (R1) to 5 minutes in Round 4 (R4) (Figure 1B). Progressive enrichment was observed (Figure 1C), and the corresponding DNA encoding the individual peptides recovered from R4 was sequenced. While depicting the percentage of each enriched peptide, notably, nearly half of the total number of sequences was composed of only four dominant peptides, while the other half of the sequences (n = 16) were below a frequency of 5%, arbitrarily set as an experimental threshold for further studies and development in this study (Figure 1D). When these peptide-displaying phage particles were individually administered to mice via the airspaces, all four dominant ligand candidates reached the systemic circulation by passing through the pulmonary hilum within 1 h after administration (50 - 200-fold range; mean of approximately 110-fold) compared to non-inserted phage particles that served as negative controls (Figure 1E).

[0163] While not wishing to be limited by any theory, the functional analysis focused on the index ligand peptide sequence CAKSMGDIVC (phage clone 2) because it demonstrated one of the highest transport efficiencies to the systemic circulation (Figure 1E). Indeed, the CAKSMGDIVC-displaying phage particle peptide was transported most efficiently through the lung and was present in the bloodstream at very high concentrations up to 2 h after aerosol treatment, while non-targeting (no insert) negative control phage particles were hardly detectable (Figure 1F). Phage clearance from the bloodstream was observed 8 h after aerosol administration. While not wishing to be limited by any theory, this may occur through non-specific clearance mediated by the reticuloendothelial system (Staquicini, F.I. et al., J Clin Invest., 2011, 121:161-173; Pasqualini, R. et al., Nature, 1996, 380:364-366; Hajitou, A. et al., Cell, 2006, 125:385-398). Collectively, these data indicate that the CAKSMGDIVC-displaying phage particles are deposited and selectively transported from the lung to the systemic circulation. Therefore, phage uptake and transport of the peptide-displaying phage particles may be mediated by specific ligand-receptor interactions.

[0164] Example 2: Distribution, Clearance, and Lung Homeostasis by Peptide-Mediated Phage Transport To exclude the possibility that enhanced transport of index-targeting phage particles was caused by tissue damage induced during intratracheal aerosol treatment, the lung morphology and homeostasis (Figures 2A-2D) were evaluated in animals aerosolized with CAKSMGDIVC-presenting phage particles compared to animals aerosolized with vehicle only or negative control non-inserted phage particles. No detectable evidence of lung tissue damage (including pulmonary edema and / or inflammation) was observed by gross morphological analysis or histopathological analysis up to 24 h after aerosol treatment (Figure 2A). Lung permeability was evaluated by quantification of extravascular leakage of Evans blue (an azo dye with very high affinity for serum albumin) in lung tissue in lung injury or protein content in bronchoalveolar lavage fluid (BALF). No detectable differences were observed between targeted phage particles and non-inserted control phage particles. Furthermore, no evidence of acute inflammation was detected by neutrophil counts in BALF of mice treated with CAKSMGDIVC-presenting phage particles or negative control non-inserted phage particles.

[0165] The data were then compared with those of aerosolized phage particles and aerosol-administered lipopolysaccharide (LPS), a canonical inducer of lung injury, followed by high-molecular-weight dextran (hereinafter referred to as LPS-dextran), as a positive control for tissue damage characterized by leukocyte infiltration and extravascular leakage of vascular fluid. In LPS-dextran-treated mice, alveolar damage, marked vascular congestion, and microvascular injury were confirmed by gross morphological analysis and histopathological analysis (Figure 2A). Additionally, mice treated with LPS-dextran (positive control) showed marked signs of lung injury, disruption of lung tissue integrity, and marked neutrophil infiltration (Figure 2B).

[0166] In certain non-limiting embodiments, the transport of index-targeting phages from the lung to the bloodstream can be mediated by specific binding of the ligand CAKSMGDIVC to cell surface receptors. A series of phage binding assays were designed to evaluate the binding of CAKSMGDIVC-displaying phage particles to target cells in vitro and in vivo. First, when using the phage overlay assay (Staquicini, et al., 2011, Proc Natl Acad Sci U S A 108:18637-18642), phage binding to cells in lung tissue sections was shown (Figure 2C), while no binding was detected in tissue sections from control organs (pancreas shown). Control non-inserted phage particles showed only background staining (Figure 2C).

[0167] Finally, the binding and transport into the bloodstream of targeted phage particles and control phage particles as a function of time were quantified in the lungs of aerosol-treated mice (Figure 2D). A significant reduction in the number of CAKSMGDIVC-displaying phage particles present in the lungs was observed starting at 1 h and continuing until 8 h after aerosol treatment. After 24 h, very few targeted phage particles were still detected in the lungs (Figure 1F). In contrast, the levels of negative control non-inserted phage particles remained unchanged during the same time frame. These results recapitulate the kinetics of CAKSMGDIVC-displaying phage particle transport to the systemic circulation, which showed a high amount of phage particles in the bloodstream between 1 h and 4 h after aerosol treatment (Figure 1F). Phage clearance from the bloodstream was observed 8 h after aerosol treatment. Without wishing to be bound by any theory, this may occur through reticuloendothelial system non-specific clearance. Taken together, these data indicate that CAKSMGDIVC-displaying phage particles are deposited and selectively transported from the lung to the systemic circulation.

[0168] Example 3: Identification and Validation of Receptors In Vitro Using peptide affinity chromatography, the corresponding candidate receptor targeted by the CAKSMGDIVC ligand was identified. Total protein extracts from the human lung adenocarcinoma cell line (A549 cells) were loaded onto a CAKSMGDIVC peptide-conjugated column, and the interacting proteins were eluted with an excess amount of soluble CAKSMGDIVC synthetic peptide (produced through Merrifield synthesis). The eluted proteins were then identified by mass spectrometry (Table 1).

[0169] Five major potential receptor candidates were selected: in addition to integrin α3β1, α6β1, and α6β4, neuropilin-1 (NRP-1) and syndecan-1 (SDC-1). Cell-free binding assays against immobilized recombinant proteins in vitro showed that the targeted CAKSMGDIVC-displaying phage particles preferentially bound to α3β1 integrin compared to other receptor candidates (Figure 3A). Bovine serum albumin (BSA) and non-inserted phage served as negative controls and showed minimal background-level binding. Competition assays in the presence of increasing molar concentrations of either the targeted synthetic peptide or an unrelated negative control synthetic peptide (sequence CGRRAGGSC; Cardo-Vila, M. et al., PLoS One, 2008, 3: e3452) confirmed the binding specificity of the CAKSMGDIVC-displaying phage to α3β1 integrin (Figure 3B).

[0170] The interaction with endogenous α3β1 integrin expressed on the surface of human alveolar epithelial adenocarcinoma cells (A549) was evaluated. The presence of α3β1 integrin on the A549 cell surface has been reported and was also confirmed by immunofluorescence (Figure 3C). Using the Biopanning and Rapid Analysis of Selective Interactive Ligand (termed BRASIL) methodology (Giordano, et al., 2001, Nat Med 7:1249-1253), binding of CAKSMGDIVC-displaying phage particles to A549 cells was demonstrated. Higher binding than background was not observed with control non-insert phage particles (Figure 3D).

[0171] The uptake and transport of CAKSMGDIVC-displaying phage particles through cell monolayers were evaluated. A549 cells were seeded onto the upper chamber of a transwell chamber and exposed to either CAKSMGDIVC-displaying phage particles or control non-inserted phage particles. Phage transport through the A549 cell monolayer from the upper chamber was determined by the TU count recovered from the lower chamber. Transport of the targeted CAKSMGDIVC-displaying phage particles was detected as early as 1 h after addition, and the highest accumulation occurred at 8 - 24 h (Figure 3E). Minimal transport of non-inserted phage particles was observed throughout the experiment. The integrity of the cell monolayer was not affected by either the targeted phage particles or the control phage particles, as demonstrated by the absence of fluorescent dextran transport (Table 2). Finally, to confirm binding specificity, α3 or β1 integrin chains were genetically depleted from A549 cells. Knockdown of α3β1 integrin was achieved by transducing A549 cells with shRNA lentiviral particles targeting the human ITGA3 gene encoding the α3 integrin chain and the human ITGB1 gene encoding the β1 integrin chain. Non-targeting shRNA (pLKO) lentiviral particles were used as a control (Figure 7A - 7B). Binding, internalization, and transport of the targeted phage were significantly reduced in A549 cells in which the α3 integrin chain was silenced, while no effect was observed in cells transduced with the negative control shRNA (Figure 3F). Only partial binding inhibition was observed when the β1 integrin chain was silenced (Figure 3G). Biochemically, competition assays using either recombinant CAKSMGDIVC-GST peptide (Figure 3H) or anti-α3 integrin chain antibody (Figure 3I and Figure 7C) supported concentration-dependent ligand-receptor specificity, suggesting that binding of CAKSMGDIVC-displaying phage particles may target a site within the α3 chain of the α3β1 integrin heterodimer.

[0172] To characterize the receptor-mediated phage transport process, an empirical mathematical function (Equation 1) was fitted to in vitro transwell data to determine the model parameters for phage particle transport through cell monolayers (Figure 7D). Evaluation by Pearson correlation coefficients (R > 0.96 for both cases) provided confidence in the mathematical model as a strong correlation was observed between the model fits and their corresponding experimental data. The characteristic time τ of the transport process was shown to be smaller for the control non-inserted phage (about 1.5 h) than for the targeted CAKSMGDIVC-displayed phage (about 4.8 h) (Table 3). Unlike the non-inserted phage, the targeted CAKSMGDIVC-displayed phage undergoes an additional step of engaging α3β1 integrin to pass through the cell monolayer, which may account for its longer characteristic time for transport. However, specific targeting enables a greater number of phage particles N s to pass through the cell barrier. Combining the two model parameters τ and N s , the overall transport process can be characterized by the initial rate of transport, which has a value about four orders of magnitude greater for the targeted CAKSMGDIVC-displayed phage particles than for the control non-inserted phage particles (Table 3). Taken together, the data indicate that CAKSMGDIVC-displayed phage particles bind to and are transported through cell monolayers by a receptor-dependent mechanism mediated by α3β1 integrin.

[0173] Example 4: CAKSMGDIVC-displayed phage particles target α3β1 integrin in vivo Since α3β1 integrin was identified as the corresponding membrane receptor that specifically mediates the observed peptide-induced transport of CAKSMGDIVC-presenting phage particles in cell-free and cell-based assays, immunohistochemistry and immunofluorescence were used to study the cellular expression and tissue localization of α3β1 in lung tissue sections. The presence of α3β1 integrin was detected in cells in the airway and alveolar regions of the lung (Figures 4A - 4B, 4E and Figure 8A). In particular (Figure 4A), the expression of α3β1 integrin (red) was detected in type 1 (AT1, purple) and type 2 (AT2, green) alveolar epithelial cells, as well as in cells of the respiratory bronchioles (Figure 4B). Some variation in the levels of α3β1 integrin was observed by immunofluorescence analysis, but the presence of α3β1 integrin was confirmed in these cell populations by single-cell RNA sequencing (scRNA-seq) in mouse lung tissue. Transcriptome analysis of flow cytometry-sorted cell populations demonstrated that the Itga3 and Itgb1 transcripts encoding mouse α3β1 integrin were present in alveolar epithelial cells, as well as in basal cells, airway epithelial ciliated and non-ciliated cells, and at a higher degree in AT1 (about 6-fold more Itga3 and about 2-fold more Itgb1 than in AT2 cells) (Figure 8B). Since AT1 cells cover more than 95% of the alveolar surface, the high expression of α3β1 integrin in these cells can promote efficient phage transport from the lung tissue into the bloodstream.

[0174] To determine whether α3β1 integrin-expressing cells are actually associated with the transport of CAKSMGDIVC-displaying phage particles through the lung hilum in vivo, tests were conducted. Either targeted phage particles or control phage particles were administered via aerosol. One hour later, the mice were sacrificed and perfused through the heart with phosphate-buffered saline (PBS). The lungs were fixed, embedded, and sectioned for immunofluorescence analysis. Lung tissue sections from mice administered either CAKSMGDIVC-displaying phage particles or control non-inserted phage particles via aerosol were immunostained with specific markers for each cell population and anti-phage antibodies. Confocal microscopy analysis shows that CAKSMGDIVC-displaying phage particles target alveolar epithelial AT1 and AT2 in the alveoli. Co-localization of CAKSMGDIVC-displaying phage particles with AT1 cells (purple) and AT2 cells (green) is indicated by white arrows (Figure 4C), and relative quantification is also presented (Figure 4D), with only background staining of control non-inserted phage particles against alveolar cells being observed. Notably, not all of the AT1 and AT2 cells were positive for phage staining. Without wishing to be limited by theory, this suggests that variations in α3β1 integrin expression may determine the binding and transport of CAKSMGDIVC-displaying phage particles in these cell populations. High concentrations of targeted CAKSMGDIVC-displaying phage particles or control non-inserted phage particles were detected in macrophages (yellow arrows, Figure 4C).

[0175] Prominent expression of α3β1 integrin was also observed in the cells of the bronchioles (Figure 4B, white arrow), mainly non-ciliated club cells (Figure 4E). In mice, ciliated and non-ciliated cells of the lung are the main components of the bronchioles. Club cells are the main source of club cell secretory protein (referred to as CCSP) into the extracellular fluid lining the airspace. By using a specific antibody against CCSP, it was confirmed that club cells constitute most of the cells expressing α3β1 integrin in the bronchioles. Also, CAKSMGDIVC-displaying phage particles compared to negative control non-inserted phage particles, and high co-localization with club cells were present in the bronchiole region, which was also confirmed using an antibody against CCSP (Figure 4F). Taken together, these experimental results establish that CAKSMGDIVC-displaying phage particles bind to α3β1 integrin expressed on the plasma membranes of AT1 and AT2 alveolar epithelial cells as well as club cells.

[0176] Example 5: CAKSMGDIVC-displaying phage particles target α3β1 integrin in vivo To further evaluate the bioavailability of CAKSMGDIVC-displaying phage particles after aerosol administration, the binding ability of phage particles to specific lung cells was determined by flow cytometry. After aerosolization of targeted and non-targeted phage particles, the lungs were harvested and digested for single cell collection. Specific lung cell populations were isolated and flow cytometry sorted in three main cell populations (Figure 5A): AT1-enriched population (EPCAM + , CD45 - , CD31 - , T1α high ); AT2-enriched population (EPCAM + , CD45 - , CD31 - , T1α low ) and mononuclear phagocytes (EPCAM - , CD45 + , CD31 - , F4 / 80 +)。The number of either targeted phage particles or control phage particles bound to each cell population was determined by TU counts after infection of the host bacteria. When CAKSMGDIVC-presenting phage particles were recovered from both the AT1-enriched population and the AT2-enriched population, binding to the AT1-enriched population was approximately two-fold higher than binding to the AT2-enriched cells. These results also coincide with scRNA-seq analysis showing that transcripts of α3β1 integrin are approximately two-fold higher in AT1 cells than in AT2 cells (Figure 8B). Negative control phage particles showed only background binding to both cell populations. Large amounts of either targeted phage or control phage were recovered from the mononuclear phagocyte-enriched cell population, which was consistent with non-specific phagocytosis in the alveolar airspace of the lung (Figure 5B).

[0177] To understand and predict the in vivo disposition kinetics of phage particles with transport from the lung to the systemic circulation and clearance by the mononuclear phagocyte system (MPS), a non-limiting two-compartment pharmacokinetic model was developed (Figure 5C). The non-limiting model consists of (i) the systemic blood pool and rapidly perfused organs (referred to as the central compartment) and (ii) slowly perfused organs, namely adipose and muscle (referred to as the peripheral compartment). Transport of phage particles from the alveolar airspace to the systemic blood pool (i.e., the central compartment) is characterized by a first-order absorption rate constant k a The alveolar airspace contains a mononuclear phagocyte population that can internalize phage particles at a rate characterized by a first-order macrophage uptake rate constant k mac The central and peripheral compartments exchange phage particles at rates characterized by first-order transfer rate constants k 1,2 and k 2,1 Finally, clearance of particles from the blood by the liver-spleen pathway or MPS is characterized by a first-order elimination rate constant k exIt is characterized by. The pharmacokinetic model is represented by a system of ordinary differential equations (Equations 2 - 5) based on the law of conservation of mass and the law of mass action. As shown in Figure 5D, the model is fitted to data corresponding to the phage distribution (lung and blood), and the estimated kinetic parameters are shown (Table 4). The strong correlation (R > 0.99, P < 0.0001) between the mathematical model fit and the experimental observations supports the modeling approach and provides confidence in the estimated kinetic parameter values (Figure 5E). The systemic bioavailability of CAKSMGDIVC-displayed phage particles is quantified by the area under the curve (AUC 0-inf ) of the central compartment kinetic curve, which is approximately two orders of magnitude greater than that of the control non-inserted phage. Furthermore, as predicted by the model, both the targeted phage particles and the non-targeted phage particles are rapidly cleared from the systemic circulation due to sequestration in the MPS organs (e.g., liver, spleen) as shown (Table 4; Figure 8C). Considering that a high proportion of the control non-inserted phage remains restricted to the lung compartment, the presence of CAKSMGDIVC-displayed phage particles in the bloodstream (i.e., the central compartment) and the slowly perfused organs (i.e., the peripheral compartment) is at least one order of magnitude greater as shown (Figure 5E; Figure 8C), supporting the excellent systemic bioavailability of the targeted CAKSMGDIVC-displayed phage particles upon pulmonary administration.

[0178] Example 6: CAKSMGDIVC promotes the transport of targeted phage particles and induces a systemic, robust, and specific humoral response in non-human primates Considering that ligand-mediated transport of CAKSMGDIVC-displaying phage particles through the lung hilum was efficient and safe in mice, translational experiments were extended to large animal models to validate aerosol phage-based applications for immunization towards vaccination strategies. The non-limiting goal of this approach was to explore the unique mechanism underlying the properties of the CAKSMGDIVC ligand peptide and its functional interaction with the corresponding receptor α3β1 integrin expressed on lung epithelial cells, and to develop a targeted immunization system based on aerosol delivery. In certain embodiments, ligand-directed phage particles can preferentially target lymph nodes to induce specific systemic humoral responses.

[0179] To design a vaccination protocol in non-human primates, earlier analyses were extended to determine humoral responses in the lung and systemic circulation in mice. After 14 days of phage aerosol administration, an overall increase in IgG, IgA, and IgM immune responses reactive to phage particles was observed in the sera and BALF of mice administered CAKSMGDIVC-displaying phage particles. This increase was seen compared to control, pre-immunized, or sham-phage particle-immunized mice (Figure 10). Anticipating that ligand-mediated transport of CAKSMGDIVC-displaying phage particles through the lung hilum was safe in mice, a vaccination protocol was applied to rhesus macaques (Macaca mulatta), a well-known species of Old World monkeys, as an experimental model much more similar to human patients.

[0180] A preclinical trial protocol for immunization of rhesus macaques to mimic phage aerosol treatment was designed, which consisted of two consecutive doses of 10 via the intratracheal route 12It included administration of either targeted CAKSMGDIVC-displaying phage particles or control non-inserted phage particles of TU. The immunization schedule is depicted in Figure 6A. The presence of α3β1 integrin in lung tissue sections from rhesus monkeys was confirmed by confocal microscopy (Figure 6B). AT1 cells were identified by positive protein staining of the Receptor for Advanced Glycation Endproduct (RAGE) that is abundantly expressed on alveolar epithelial cells. α3 integrin chain and RAGE were co-localized throughout AT1 cells (Figure 9A). Additionally, the expression of α3β1 integrin in alveolar and airway epithelial cells on lung tissue sections from healthy human patients was confirmed by immunohistochemistry (Figure 9B), which was a result supporting the translational effort of this technology.

[0181] Each dose was administered every 28 days and serum was collected every 14 days. The transport of CAKSMGDIVC-displaying phage particles to the systemic circulation passing through the lung hilum was demonstrated in blood samples collected hourly after the first dose. CAKSMGDIVC-displaying phage particles were first detected in peripheral blood 3 h after administration and further accumulated until 5 h. As determined by TU, a decrease in phage particles was observed at the 6 h time point (Figure 6C). In sharp contrast, negative control non-inserted phage particles were detected only at minimal levels in the blood stream at all time points. Without wishing to be limited by any theory, the transport of targeted CAKSMGDIVC-displaying phage particles to the blood stream passing through the lung hilum can enhance the systemic immune response. Targeted CAKSMGDIVC-displaying phage particles generated higher titers of phage-specific IgG serum antibodies than control non-inserted phage, as indicated by ELISA analysis of the antibody response that started on day 28 after administration and increased significantly after the second dose, and on days 42 and 56 after administration (Figure 6D).

[0182] To evaluate the extent of the antibody response generated by pulmonary delivery of CAKSMGDIVC-displaying phage particles, IgG and IgA serum antibodies were further analyzed and represented as fold changes in titer. Humoral responses generated by intratracheal administration of targeted CAKSMGDIVC-displaying phage particles or control non-insert phage compared to baseline were compared. On day 28, the phage-specific IgG antibody response induced by targeted CAKSMGDIVC-displaying phage particles showed an approximately 6,000-fold increase in titer compared to baseline (Figure 6E). This represents an approximately 4-fold increase (approximately 1,000-fold relative to baseline) in phage-specific IgG titer compared to control non-insert phage (Figure 6E and Figure 9C). Administration of a second dose of CAKSMGDIVC-displaying phage particles resulted in a substantial increase in antibody levels by days 42 and 56, with the highest difference observed on day 56 post-administration, at which time the mean titer of phage-specific serum IgG antibodies was approximately 200,000-fold higher than baseline with a persistent approximately 4-fold difference relative to non-insert control phage (Figure 6E and Figure 9C).

[0183] Similar results were observed for IgA serum antibodies. Administration of CAKSMGDIVC-displaying phage particles generated higher titers of phage-specific IgA serum antibodies, which started on day 14 post-administration and increased significantly by days 42 and 56 after the second dose (Figure 6F). On day 28, the mean titer of IgA antibodies showed an approximately 50-fold increase compared to baseline (Figure 6G) and an approximately 3-fold higher increase compared to non-insert control phage (Figure 9D).

[0184] The observation that the targeted CAKSMGDIVC-displaying phage particles generated a strong specific systemic humoral response was further demonstrated using ELISA to detect CAKSMGDIVC-specific IgG and IgA serum antibodies with its cognate synthetic peptide and a control irrelevant peptide. The detection of CAKSMGDIVC-specific IgG and IgA serum antibodies was observed to start on day 14 for serum IgG (Figure 6H), and on day 28 for serum IgA (Figure 6I). Both CAKSMGDIVC-specific IgG and IgA antibodies showed an approximately 8- to 12-fold increase in titer compared to baseline (Figure 9E-9F), and there was no substantial change in titer after the second dose. Only minimal background cross-reactivity was detected against the unrelated synthetic negative control peptide. In certain non-limiting embodiments, there is a low quality expression of the recombinant minor coat protein encoding the peptide sequence (CAKSMGDIVC), with only about 3-5 copies of pIII per phage particle, and is tended to be overridden by proteins with high copy numbers (e.g., the major coat protein pVIII estimated to be several hundred copies). In certain embodiments, the specificity of the antibody response to the peptide indicates the epitope recognition and activation of a specific cellular immune response. Together, these results indicate that the selective transport of CAKSMGDIVC-displaying phage particles across the air-blood barrier into the bloodstream significantly increases the specific humoral response to the phage particles and their selected ligand peptide, thus representing an advance in the development of aerosol phage-based vaccines.

[0185] In certain embodiments, these findings support the utility and efficiency of the ligand CAKSMGDIVC peptide in lung-targeted delivery for multiple applications and reveal a new molecular mechanism of lung epithelial-endothelial transport mediated by internalizing α3β1 integrin.

[0186] (Table 1) Candidate receptors identified by mass spectrometry TIFF0007692218000008.tif78153

[0187] (Table 2) Cell integrity by transport of targeted phage particles or control phage particles A549 cell barrier integrity TIFF0007692218000009.tif76155

[0188] (Table 3) Phage particle transport through cell monolayers obtained from empirical mathematical modeling TIFF0007692218000010.tif45163

[0189] (Table 4) In vivo pharmacokinetics of phage particles obtained from mathematical modeling analysis TIFF0007692218000011.tif38166

[0190] Selected Comments Aerosol-based routes of administration have been developed over the past several years, but have achieved relatively little acceptance, probably due to a lack of mechanistic insight into fate and biodistribution, as well as potentially adverse pulmonary side effects of non-targeting aerosol agents. As described in the present disclosure, a ligand-directed lung delivery system that successfully induces systemic effects without detectable lung injury was identified and validated using an unbiased combinatorial approach. In addition to the unique and specific role of α3β1 integrin, the application of predictive mathematical modeling for the uptake and transport of targeted phage particles presenting a new index ligand peptide (i.e., CAKSMGDIVC) across the lung gate into the bloodstream in vivo was shown. As a proof of concept, an aerosol phage-based application was tested in non-human primates as an initial step towards the development of an aerosol phage-based vaccine for application to human patients. This targeted method of lung delivery of highly stable and immunogenic antigen carriers (i.e., viral phage particles) induces a robust and specific humoral immune response and has field applications for vaccine and / or other therapeutic development in a non-limiting manner.

[0191] To gain mechanistic insights into the physiological transport of molecules across the air-blood barrier and to explore the diversity of surface receptors associated with it, combinatorial screening of an aerosol phage display random peptide library was performed in mice. From a pool of peptide-displaying phage particles recovered from the bloodstream, four potent ligand peptide candidates mediated phage transport across the lung barrier. Among these selected ligands, the index peptide CAKSMGDIVC showed one of the highest transport efficiencies in vivo, and the data suggested that specific ligand-receptor interactions may be causative for lung target delivery. Monitoring the distribution, transport, and clearance of CAKSMGDIVC-displaying phage particles deposited in the airways by aerosol administration in vivo and ex vivo indicated that phage transport caused no detectable lung injury and there was no anatomical or physiological lung impairment, supporting the result that phage particles are suitable for safe inhalation administration to humans.

[0192] To identify the receptor for the ligand CAKSMGDIVC peptide, a series of phage binding assays were performed in vitro and in vivo. Specific binding to human recombinant α3β1 integrin, followed by functional binding of CAKSMGDIVC-presenting phage particles and their transport across the alveolar epithelial surrogate cell monolayer supported the ligand-receptor interaction. Evidence that passage of the targeted phage particles through the lung gate is mediated through a ligand-receptor-mediated mechanism was clearly established by specific binding of the CAKSMGDIVC-presenting phage particles to α3β1 integrin on the surface of lung AT1, AT2, and club cells in vivo. Although the expression of α3β1 in club cells is high, their functional involvement in phage transport has remained unclear to date. Therefore, ligand-directed delivery through selective targeting of α3β1 integrin represents a substantial advance over conventional non-targeting aerosol formulations (Liang, Z. et al., Drug Discov. Today, 2015, 20:380-389) that require permeation enhancers or solubilizing carriers for drug stability and dispersion. The present disclosure establishes the mechanism of internalization of a selective ligand by α3β1 integrin in the lung, which is useful, for example, for in vivo lung delivery and resultant immunization applications, among others.

[0193] To support the translational application of the ligand peptide-directed pulmonary delivery approach introduced in the present disclosure, a targeted phage display-based protocol was designed in mice and non-human primates as an aerosol strategy for pulmonary and systemic humoral immunization as a proof-of-concept for pulmonary vaccination against multiple diseases. Considering a constant immunogenic exposure to pathogens through the airways, the lung tissue is a highly active site of host defense where efficient antigen presentation occurs (but in many cases has not been evaluated or has been underestimated). Therefore, pulmonary delivery of aerosolized antigens has many advantages over other routes of administration, especially for the development of candidate vaccines or therapies against respiratory infections (including but not limited to SARS-CoV-2). Furthermore, the selective pulmonary transport of CAKSMGDIVC-presenting phage particles, followed by the activation of specific local and systemic humoral responses, recapitulates long-standing principles of vaccinology. The pulmonary delivery system studied herein has unique translational relevance for the development of vaccines against airborne pathogens.

[0194] The translational benefits of phage-based aerosol vaccines are multifaceted. In one aspect, phage particles are highly stable under harsh environmental conditions, and their large-scale production is extremely cost-effective compared to traditional methods used for vaccine manufacturing (Barbu, E.M. et al., Cold Spring Harb. Perspect. Biol., 2016, 8(10):a023879; Bao, Q. et al., Adv. Drug Deliv. Rev., 2019, 145:40-56). In another aspect, phage therapy and phage-based vaccines do not induce detectable toxic side effects. In fact, phage particles have been used for nearly a century as antibiotics against multidrug-resistant bacteria or as immunogenic vaccine carriers and have proven to be safe and effective (Schmidt, C., Nat. Biotechnol., 2019, 37:581-586; Barbu, E.M. et al., Cold Spring Harb. Perspect. Biol., 2016, 8(10):a023879). Indeed, phage administration has been exploited for discovery and transgene delivery applications, including the administration of phage libraries in mice, pet dogs, non-human primates, and even patients (Staquicini, F.I. et al., J. Clin. Invest., 2011, 121:161-173; Pasqualini, R. et al., Nature, 1996, 380:364-366; Hajitou, A. et al., Cell, 2006, 125:385-398; Arap, W. et al., Nat. Med., 2002, 8:121-127). In yet another aspect, native phage particles do not have tropism for mammalian cells and do not replicate inside eukaryotic cells, and their use is generally considered to be safe compared to other classical virus-based vaccination strategies.In yet another aspect, unlike conventional peptide-based vaccines that can often be inactivated due to a minimal temperature range of movement (about 1 °C), the system introduced herein does not have the cumbersome and expensive requirements for maintaining a strict so-called "cold chain" during field application, especially in developing countries. In yet another aspect, the ligand-receptor discovery and vaccination properties of the disclosed phage display-based system can also be used for the development of lung-targeted delivery of other viral antigens, or phage chimeras presenting entire transgenes, by using a hybrid vector of adeno-associated virus (AAV) and phage (referred to as AAVP). In yet another aspect, phage particles have been used for decades as immunogenic vaccine carriers, are themselves very strong immunogens, and act as powerful adjuvants for inducing a persistent humoral response (Trepel, M. et al., Cancer Res., 2001, 61:8110-8112; de la Cruz, V.F. et al., J. Biol. Chem., 1988, 263:4318-4322; Aghebati-Maleki, L. et al., J. Biomed. Sci., 2016, 23:66; Barbu, E.M. et al., Cold Spring Harb. Perspect. Biol., 2016, 8(10):a023879). Indeed, in certain embodiments, phage particles as antibacterial agents in the context of multi-drug resistant bacterial infections or the ongoing SARS-CoV-2 coronavirus pandemic may be well-suited for use within the targeted aerosol strategies described herein.

[0195] Enumerated embodiments: The following enumerated embodiments are provided, but their numbering should not be construed as specifying a level of importance. Embodiment 1 provides the following: An isolated transport peptide comprising at least one amino acid sequence selected from the group consisting of TIFF0007692218000012.tif26161. Aspect 2 provides the following: The transport peptide of Aspect 1, comprising the amino acid sequence of SEQ ID NO:2. Aspect 3 provides the following: The transport peptide of any one of Aspects 1 to 2, consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs:1 to 4. Aspect 4 provides the following: The transport peptide of any one of Aspects 1 to 3, consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs:1 to 4. Aspect 5 provides the following: The surface of the solid particle presents the transport peptide of any one of Aspects 1 to 4, and the solid particle is selected from the group consisting of bacteriophage, engineered cells, tissue fragments, nanoparticles, vesicles, dendrimers, virus-like particles, adenovirus, adeno-associated virus (AAV), adeno-associated virus phage (referred to as AAVP), and any combination thereof. Aspect 6 provides the following: The solid particle of Aspect 5, wherein the transport peptide is attached to the surface of the solid particle or presented on the surface of the solid particle. Aspect 7 provides the following: The solid particle of any one of Aspects 5 to 6, wherein the transport peptide is attached to at least a part of the surface of the solid particle or presented on at least a part of the surface of the solid particle. Aspect 8 provides the following: The solid particle of any one of Aspects 5 to 7, which is a filamentous phage. Aspect 9 provides the following: The solid particle of any one of Aspects 5 to 8, further comprising an agent selected from the group consisting of a therapeutic substance, a biologically active molecule, an imaging agent, a radioactive substance, a salt, a peptide, a protein, a lipid, a nucleic acid, a gas, and any combination thereof, wherein the agent is attached to the solid particle and / or contained within the solid particle. Aspect 10 provides the following: The solid particle of Aspects 5 to 9, which is a filamentous phage. Aspect 11 provides the following: The solid particles of aspect 10, wherein the filamentous bacteriophage comprises fd, fl, or M13 bacteriophage. Aspect 12 provides the following: The solid particles of any one of aspects 10 - 11, wherein the solid particles are filamentous phage and the surface of the solid particles presents an antigen. Aspect 13 provides the following: A method of promoting and / or increasing the transport of solid particles through the air - blood barrier in the lungs of a subject, comprising administering to the subject the solid particles of any one of aspects 5 - 12, wherein the administration is via a route including nasal, buccal, inhalation, intratracheal, intrapulmonary, or intrabronchial. Aspect 14 provides the following: A method of promoting the systemic circulation of solid particles in a subject, comprising administering to the subject the solid particles of any one of aspects 5 - 12, wherein the administration is via a route including nasal, buccal, inhalation, intratracheal, intrapulmonary, or intrabronchial. Aspect 15 provides the following: A method of immunizing a subject against a disease or disorder, comprising administering to the subject the solid particles of any one of aspects 5 - 12, wherein the surface of the solid particles is further derivatized with an antigen that promotes an immune response against the disease or disorder in the subject, and the administration is via a route including nasal, buccal, inhalation, intratracheal, intrapulmonary, or intrabronchial. Aspect 16 provides the following: A method of treating, alleviating, and / or preventing a disease or disorder in a subject, comprising administering to the subject the solid particles of any one of aspects 5 - 12, wherein the surface of the solid particles is further derivatized with an antigen that promotes an immune response against the disease or disorder in the subject, and the administration is via a route including nasal, buccal, inhalation, intratracheal, intrapulmonary, or intrabronchial. Aspect 17 provides the following: A method of treating a subject at risk of developing a disease or disorder, comprising administering to the subject solid particles of any one of aspects 5 to 12, wherein the surface of the solid particles is further derivatized with an antigen that promotes an immune response against the disease or disorder in the subject, and the administration is via a route including nasal, buccal, inhalation, intratracheal, intrapulmonary, or intrabronchial, said method. Aspect 18 provides the following: Any method of aspects 13 to 17, wherein the peptide comprises the amino acid sequence of SEQ ID NO:2. Aspect 19 provides the following: Any method of aspects 13 to 18, wherein the peptide consists of the amino acid sequence of SEQ ID NO:2. Aspect 20 provides the following: Any method of aspects 13 to 19, wherein the solid particles are filamentous phage. Aspect 21 provides the following: The method of aspect 20, wherein the filamentous bacteriophage comprises fd, fl, or M13 bacteriophage. Aspect 22 provides the following: Any method of aspects 13 to 21, wherein the solid particles are administered to the subject in a composition further comprising an immunogenic adjuvant. Aspect 23 provides the following: Any method of aspects 13 to 22, wherein the subject is a mammal. Aspect 24 provides the following: Any method of aspects 13 to 23, wherein the subject is a human. Aspect 25 provides the following: A vaccine comprising solid particles, wherein the surface of the solid particles presents at least one of the transport peptides of claim 1, and the solid particles are selected from the group consisting of bacteriophage, engineered cells, tissue fragments, nanoparticles, vesicles, dendrimers, virus-like particles (VLPs), adenovirus, adeno-associated virus (AAV), adeno-associated virus phage (referred to as AAVP), and combinations thereof. Aspect 26 provides the following: The vaccine of embodiment 25 selected from the group consisting of DNA vaccines, RNA vaccines, replicative viral vector vaccines, non-replicative viral vector vaccines, inactivated viral vector vaccines, virus-like particle vaccines, and any combination thereof. Embodiment 27 provides the following: The vaccine of any one of embodiments 25-26, wherein the particle contains a vaccine active substance selected from the group consisting of DNA, RNA, replicative viral vector, non-replicative viral vector, inactivated viral vector, virus-like particle, and any combination thereof.

[0196] Other embodiments: The recitation of elements in any definition of a variable element herein includes the definition of that variable element as any single element or combination (or sub-combination) of the recited elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0197] The disclosure of each and every patent, patent application, and publication referenced herein is hereby incorporated by reference in its entirety. Although the disclosure has been made with reference to specific embodiments, it will be apparent that other embodiments and variations of the disclosure may be developed by those skilled in the art without departing from the true spirit and scope of the disclosure. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

1. A cyclic transport peptide comprising the amino acid sequence of CAKSMGDIVC (SEQ ID NO:2), wherein the cysteine at position 1 and the cysteine at position 10 form a disulfide bond, said cyclic transport peptide.

2. The cyclic transport peptide according to claim 1, consisting of the amino acid sequence of SEQ ID NO:

2.

3. A solid particle whose surface presents the cyclic transport peptide according to claim 1, said solid particle being selected from the group consisting of bacteriophage, engineered cells, tissue fragments, nanoparticles, vesicles, dendrimers, virus-like particles, adenovirus, adeno-associated virus (AAV), adeno-associated virus phage (referred to as AAVP), and any combination thereof.

4. The solid particle according to claim 3, wherein the cyclic transport peptide is attached to the surface of the solid particle or presented on the surface of the solid particle.

5. The solid particle according to claim 4, wherein the cyclic transport peptide is attached to at least a portion of the surface of the solid particle or presented on at least a portion of the surface of the solid particle.

6. The solid particle according to claim 3, further comprising a substance selected from the group consisting of therapeutic substances, biologically active molecules, imaging agents, radioactive substances, salts, peptides, proteins, lipids, nucleic acids, gases, and any combination thereof, said substance being attached to the solid particle and / or contained within the solid particle.

7. The solid particle according to claim 3, which is a filamentous phage.

8. The solid particle according to claim 7, wherein the filamentous phage comprises fd, fl, or M13 bacteriophage.

9. The solid particle according to claim 3, wherein the solid particle is a filamentous phage and the surface of the solid particle presents an antigen.

10. A pharmaceutical composition comprising the solid particle according to claim 3 for promoting and / or increasing the transport of the solid particle through the air-blood barrier in the lung of a subject, said pharmaceutical composition being for administration through a route including nasal, buccal, inhalation, intratracheal, intrapulmonary, or bronchial administration.

11. A pharmaceutical composition comprising the solid particles according to claim 3 for promoting systemic circulation of solid particles in a subject, which is for administration through a route including nasal, buccal, inhalation, intratracheal, intrapulmonary, or intrabronchial routes.

12. A pharmaceutical composition comprising the solid particles according to claim 3 for immunizing a subject against a disease or disorder, wherein the surface of the solid particles is further derivatized with an antigen that promotes an immune response against the disease or disorder in the subject, and the pharmaceutical composition is for administration through a route including nasal, buccal, inhalation, intratracheal, intrapulmonary, or intrabronchial routes.

13. A pharmaceutical composition comprising the solid particles according to claim 3 for treating, remitting, and / or preventing a disease or disorder in a subject, wherein the surface of the solid particles is further derivatized with an antigen that promotes an immune response against the disease or disorder in the subject, and the pharmaceutical composition is for administration through a route including nasal, buccal, inhalation, intratracheal, intrapulmonary, or intrabronchial routes.

14. A pharmaceutical composition comprising the solid particles according to claim 3 for treating a subject at risk of developing a disease or disorder, wherein the surface of the solid particles is further derivatized with an antigen that promotes an immune response against the disease or disorder in the subject, and the pharmaceutical composition is for administration through a route including nasal, buccal, inhalation, intratracheal, intrapulmonary, or intrabronchial routes.

15. The pharmaceutical composition according to any one of claims 10 to 14, wherein the cyclic transport peptide consists of the amino acid sequence of SEQ ID NO:

2.

16. The pharmaceutical composition according to any one of claims 10 to 14, wherein the solid particles are filamentous phage.

17. The pharmaceutical composition according to claim 16, wherein the filamentous phage includes fd, fl, or M13 bacteriophage.

18. The pharmaceutical composition according to any one of claims 10 to 14, further comprising an immunogenic adjuvant.

19. The pharmaceutical composition according to any one of claims 10 to 14, wherein the subject is a mammal.

20. The pharmaceutical composition according to any one of claims 10 to 14, wherein the subject is a human.

21. A vaccine comprising solid particles, wherein the surface of the solid particles presents the cyclic transport peptide according to claim 1, and the solid particles are selected from the group consisting of bacteriophages, engineered cells, tissue fragments, nanoparticles, vesicles, dendrimers, virus-like particles (VLPs), adenoviruses, adeno-associated viruses (AAVs), adeno-associated virus phages (referred to as AAVPs), and combinations thereof, said vaccine.

22. The vaccine according to claim 21, selected from the group consisting of DNA vaccines, RNA vaccines, replicative virus vector vaccines, non-replicative virus vector vaccines, inactivated virus vector vaccines, virus-like particle vaccines, and any combination thereof.

23. The vaccine according to claim 21, wherein the particles comprise a vaccine active substance selected from the group consisting of DNA, RNA, replicative virus vectors, non-replicative virus vectors, inactivated virus vectors, virus-like particles, and any combination thereof.

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