Nanosatellite complex

Abstract

The present invention provides methods, compositions, systems and kits comprising nanosatellite complexes, the nanosatellite complexes comprising: a core-nanoparticle complex comprising a biocompatible coating surrounding a nanoparticle core; 3-25 satellite particles attached to or absorbed into the biocompatible coating; a plurality of antigenic peptides conjugated to or absorbed into the satellite particles; and at least one additional characteristic: i) the weight ratio of all the satellite particles to the nanoparticle core is 10-40%; the diameter of each satellite particle is 2-20 nm; iii) the satellite particles are present at a density of 500-20,000 or 15,000-30,000 per square micron; iv) the plurality of antigenic peptides is 100-4000 antigenic peptides; v) 10-300 of the plurality of antigenic peptides are present on each satellite particle; and / or vi) the average distance between each satellite particle is 5-20 nm.

Description

[0001] This application claims priority to U.S. Provisional Application Serial No. 62 / 746,755, filed October 17, 2018, which is incorporated herein by reference in its entirety. Technical Field

[0002] The present invention provides methods, compositions, systems and kits comprising nanosatellite complexes, the nanosatellite complexes comprising: a core-nanoparticle complex comprising a biocompatible coating surrounding a nanoparticle core; 3-25 satellite particles attached to or absorbed into the biocompatible coating; a plurality of antigenic peptides conjugated to or absorbed into the satellite particles; and at least one additional characteristic: i) the weight ratio of all the satellite particles to the nanoparticle core is 10-40%; the diameter of each of the satellite particles is 2-20 nm; iii) the satellite particles are present at a density of 500-20,000 or 15,000-30,000 per square micron; iv) the plurality of antigenic peptides is 100-4000 antigenic peptides; v) 10-300 of the plurality of antigenic peptides are present on each of the satellite particles; and / or vi) the average distance between each of the satellite particles is 5-20 nm. Background Art

[0003] Viruses are known to be highly efficient delivery vehicles, mediators of cellular uptake, and effective immunizing agents. Therefore, the use of viruses and viral properties has become desirable in a variety of biotechnological and medical applications. However, traditional attenuated live viruses or inactivated viruses are still too dangerous to be used in this manner. To address this issue, virus-like particles (VLPs) have emerged.

[0004] Virus-like particles are protein-based nanoparticles that contain viral capsid proteins that can self-assemble into geometrically rigid nanostructures that are directly similar to viral structures and can be confirmed without the need for a viral genome. Therefore, virus-like particles are considered to be a viable and safe alternative to traditional viruses. Despite this advantage, many obvious shortcomings of virus-like particle technology (including reliance on protein self-assembly, manufacturing difficulties, limited application versatility, and significant anti-carrier reactions) still exist, which limits the potential for re-administration in vivo. Due to these challenges, there is an increasing interest in developing alternative nanoparticle systems inspired by viruses.

[0005] These so-called virus-mimicking nanoparticles are rationally designed and engineered based on an understanding of the physical and chemical material properties of viruses. The viral material properties most commonly used to inform virus-mimicking nanoparticle design include particle size, particle shape, charge, hydrophobicity, antigen presentation, antigen organization, antigen density, and surface topology. Despite numerous advances in the design, engineering, and application of virus-mimicking nanoparticles, a universally applicable nanoparticle system has yet to emerge. Summary of the Invention

[0006] The present invention provides methods, compositions, systems and kits comprising nanosatellite complexes, the nanosatellite complexes comprising: a core-nanoparticle complex comprising a biocompatible coating surrounding a nanoparticle core; 3-25 satellite particles attached to or absorbed into the biocompatible coating; a plurality of antigenic peptides conjugated to or absorbed into the satellite particles; and at least one additional characteristic: i) the weight ratio of all the satellite particles to the nanoparticle core is 10-40%; the diameter of each of the satellite particles is 2-20 nm; iii) the satellite particles are present at a density of 500-20,000 or 15,000-30,000 per square micron; iv) the plurality of antigenic peptides is 100-4000 antigenic peptides; v) 10-300 of the plurality of antigenic peptides are present on each of the satellite particles; and / or vi) the average distance between each of the satellite particles is 5-20 nm. In certain embodiments, the nanosatellite complex has a diameter of about 20-70 nm (eg, about 25 nm, about 40-50 nm, or about 60 nm).

[0007] In certain embodiments, provided herein are compositions comprising a nanosatellite complex, wherein the nanosatellite complex comprises: a) a core-nanoparticle complex comprising a biocompatible coating surrounding a nanoparticle core; b) 3-25 (e.g., 3, 7, 13, 17, 21, or 25) satellite particles attached to or absorbed into the biocompatible coating; c) a plurality of antigenic peptides (e.g., from Table 4 or Table 1, Table 2, or Table 3) or a plurality of haptens conjugated to or absorbed into the satellite particles; and d) wherein the nanosatellite complex comprises at least one (e.g., 1, 2, 3, 4, 5, or 6) of the following properties: i) the weight ratio of the satellite particles to the nanoparticle core is 10-40% (e.g., 10%, 20%, 30%, or 40%); ii) each of the satellite particles has a diameter of 2-20 nm (e.g., 2 nm, 5 nm, 8 nm, 1 nm, or 2 nm); 3 nm, 17 nm, 20 nm); iii) the satellite particles are present at a density of 500-20,000 per square micron (e.g., 500, 1000, 4000, 8000, 13,000, 17,000 or 20,000 per square micron); iv) the plurality of antigenic peptides or the plurality of haptens are 100-4000 (e.g., 100, 500, 1000, 2000, v) 10-300 (e.g., 10, 40, 100, 175, 225, or 300) of the plurality of antigenic peptides or the plurality of haptens are present on each of the satellite particles; and vi) the average distance between each of the satellite particles is 5-20 nm (e.g., 5.0 nm, 6.5 nm, 7.5 nm, 10 nm, 13 nm, 17 nm, or 20 nm).

[0008] In certain embodiments, provided herein are compositions, kits, and systems comprising a nanosatellite complex, wherein the nanosatellite complex comprises: a) a core-nanoparticle complex comprising a biocompatible coating surrounding a nanoparticle core (e.g., wherein the nanoparticle core has a diameter of about 12-18 nm); b) 10-20 satellite particles attached to or absorbed into the biocompatible coating; c) a plurality of antigenic peptides (e.g., from Table 4 or Table 1, Table 2, or Table 3) conjugated to or absorbed into the satellite particles; and d) wherein the nanosatellite complex comprises at least one of the following properties: i) a weight ratio of all the satellite particles to the nanoparticle core of 10-40% (e.g., 10%, 20%, 30%, 40%); ii) a diameter of each of the satellite particles of 1-5 nm (e.g., about 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 12 nm, 13 nm, 14 nm, 15 nm, 16 nm, 17 nm, 18 nm, 19 nm, 20 nm, 21 nm, 22 nm, 23 nm, 24 nm, 25 nm, 26 nm, 27 nm, 28 nm, 29 nm, 30 nm, 31 nm, 32 nm, 33 nm, 34 nm, 35 nm, 36 nm, 37 nm, 38 nm, 39 nm, 40 nm, 41 nm, 42 nm, 43 nm, 44 nm, 45 nm, 46 nm, 47 nm, 48 nm, 49 nm, 50 nm, 51 nm, 52 nm, 53 nm, 54 nm, 55 nm, 56 nm, 57 nm, 58 nm, 59 wherein the satellite particles are present at a density of about 15,000 to about 30,000 (e.g., about 15,000, 18,000, 21,000, 25,000, 28,000, or 30,000) per square micron; wherein the plurality of antigenic peptides is about 1500 to about 3000 (e.g., about 1500, 1900, 2200, 2300, 2600, or 3000) antigenic peptides; wherein the plurality of antigenic peptides is about 100 to about 400 (e.g., about 100, 200, 250, 300, or 400) antigenic peptides; and wherein the plurality of antigenic peptides is about 100 to about 400 (e.g., about 100, 200, 250, 300, or 400) present on each satellite particle; and wherein the average distance between each satellite particle is about 4 to about 7 nm (e.g., about 4.0 nm, 5.0 nm, 5.2 nm, 5.9 nm, 6.1 nm, or 7.0 nm).

[0009] In some embodiments, provided herein are kits and systems comprising: a) a core-nanoparticle complex comprising a biocompatible coating surrounding a nanoparticle core; b) 3-25 satellite particles configured to be attached to or absorbed into the biocompatible coating; c) a plurality of antigenic peptides or a plurality of haptens configured to be conjugated to or absorbed into the satellite particles; and d) at least one of: i) a weight ratio of all of the satellite particles to the nanoparticle core of 10-40%; ii) a diameter of each of the satellite particles of 2-20 nm; and iii) the plurality of antigenic peptides or the plurality of haptens is 100-4000 antigenic peptides.

[0010] In certain embodiments, provided herein is a method for eliciting an immune response in a subject, comprising administering a composition as described herein to the subject so as to produce antibodies against an antigenic peptide or hapten. In certain embodiments, the subject is a human. In other embodiments, the subject is an animal (e.g., dog, cat, pig, horse, etc.). In additional embodiments, the method further comprises obtaining a sample from the subject and purifying some antibodies from the sample. In additional embodiments, there is no adjuvant as part of the composition or administered in other ways. In some embodiments, the subject administers a type I interferon agonist in the form of a composition or alone. In other embodiments, the subject administers an immune checkpoint inhibitor in the form of a composition or alone. In certain embodiments, the antigenic peptide comprises a B cell epitope, or a T cell epitope, or both (see, e.g., Table 4 or Table 1, Table 2 or Table 3). In other embodiments, the nanosatellite complex does not produce detectable nonspecific antibodies against the nanosatellite complex in the subject. In other embodiments, the nanosatellite complex (e.g., at a level equal to that of the virus) home to the lymph nodes of the subject. In other embodiments, the nanosatellite complexes home to the B cell compartment or T cell compartment of the subject's lymph nodes. In other embodiments, the nanosatellite complexes are taken up by the subject's subcapsular sinus macrophages at a rate equal to that of the virus.

[0011] In certain embodiments, the satellite particle comprises gold. In other embodiments, the core nanoparticle comprises Fe3O4. In some embodiments, the biocompatible coating comprises polysiloxane. In other embodiments, the nanoparticle core comprises Fe3O4, the biocompatible coating comprises polysiloxane, and at least one satellite particle comprises a plurality of satellite particles composed of gold.

[0012] In some embodiments, the 3-25 satellite particles are 10-15 satellite particles. In other embodiments, at least one characteristic is that the weight ratio of all satellite particles to the nanoparticle core is 10-40% (e.g., about 30%). In additional embodiments, the weight ratio of all satellite particles to the nanoparticle core is 25-35%. In certain embodiments, the weight ratio of all satellite particles to the nanoparticle core is 29-31%.

[0013] In some embodiments, at least one characteristic is that each satellite particle has a diameter of 2-20 nm. In certain embodiments, each satellite particle has a diameter of 5-15 nm. In other embodiments, each satellite particle has a diameter of 4-6 nm.

[0014] In certain embodiments, at least one characteristic is that the satellite particles are present at a density of 500-20,000 per square micron. In other embodiments, the satellite particles are present at a density of 13,000 to 17,000 per square micron.

[0015] In some embodiments, at least one characteristic is that the plurality of antigenic peptides is 100-4000 antigenic peptides or 100-4000 haptens. In other embodiments, the plurality of antigenic peptides is 1500-2500 antigenic peptides, or the plurality of haptens is 1500-2500 haptens.

[0016] In other embodiments, the at least one characteristic is that 10-300 of the plurality of antigenic peptides or haptens are present on each satellite particle. In other embodiments, 225-275 of the plurality of antigenic peptides or haptens are present on each satellite particle.

[0017] In some embodiments, at least one characteristic is that the average distance between each satellite particle is 5-20 nm. In certain embodiments, the average distance between each satellite particle is 6-8 nm.

[0018] In other embodiments, the at least one characteristic is at least two or three of the characteristics. In some embodiments, the at least one characteristic is at least four or five of the characteristics. In additional embodiments, the at least one characteristic is all six of the characteristics.

[0019] In certain embodiments, the antigenic peptide comprises: i) a neoantigenic determinant; ii) at least one epitope from a tumor antigen; iii) at least one epitope from a viral oncoprotein; iv) at least one epitope from an infectious virus; v) at least one epitope from a parasite; or vi) at least one epitope from an infectious bacterium. In other embodiments, the compositions, systems, and kits further comprise a physiologically compatible aqueous solution and / or cancer cells and / or antigen-presenting cells.

[0020] In some embodiments, multiple antigenic peptides are unevenly distributed on the satellite particles. In other embodiments, the diameter of the nanosatellite complex is 50-100nm (e.g., 55-65nm). In other embodiments, the surface of the nanosatellite complex is negatively charged (e.g., -10mV to -20mV). In other embodiments, the diameter of the core nanoparticle is 10-25nm (e.g., 15-20nm).

[0021] In some embodiments, the composition further comprises a type I interferon agonist. In other embodiments, the type I interferon agonist is electrostatically attracted or absorbed to i) an antigenic peptide or hapten, ii) multiple satellite particles and / or iii) a core nanoparticle. In additional embodiments, the composition does not contain an adjuvant. In additional embodiments, the composition further comprises an immune checkpoint inhibitor.

[0022] In other embodiments, the antigenic peptide comprises at least one new antigenic determinant, including, for example, an oncogenic viral antigenic determinant. In some embodiments, the antigenic peptide comprises at least one epitope from a tumor antigen, including a viral oncoprotein. In certain embodiments, the antigenic peptide comprises at least one epitope from an infectious virus, at least one epitope from a parasite, and / or at least one epitope from an infectious bacterium. Suitable antigens from viruses, parasites, and bacteria for immunizing subjects (e.g., human subjects) are well known in the art (see, for example, Tables 2 and 3). Additional antigens are being developed for use in vaccines, including, for example, adenovirus vaccines, Coxsackie B virus vaccines, cytomegalovirus vaccines, dengue fever vaccines, Eastern equine encephalitis virus vaccines, Ebola vaccines, enterovirus 71 vaccines, Epstein-Barr vaccines, hepatitis C vaccines, HIV vaccines, HTLV-1 T-lymphocytic leukemia vaccines, Marburg virus disease vaccines, norovirus vaccines, respiratory syncytial virus vaccines, severe acute respiratory syndrome (SARS) vaccines, West Nile virus vaccines, Zika fever, dental caries vaccines, ehrlichiosis vaccines, leprosy vaccines, Lyme disease vaccines, Staphylococcus aureus vaccines, Streptococcus pyogenes vaccines, syphilis vaccines, tularemia vaccines, Yersinia pestis vaccines, and leprosy vaccines. pestis vaccine, malaria vaccine, schistosomiasis vaccine, Chagas disease vaccine, hookworm vaccine, human onchocerciasis and river blindness vaccine, trypanosomiasis vaccine, and visceral leishmaniasis vaccine.

[0023] In certain embodiments, the method of administering the nanosatellite complex herein to a subject kills at least some cancer cells and / or modulates the antigen-specific immune response of the subject. In other embodiments, the cancer cells are from a cancer type selected from the group consisting of head and neck squamous cell carcinoma (HNSCC), HPV-positive cancers, odontogenic tumors, bladder cancer, breast cancer, cervical cancer, colorectal cancer, leukemia, melanoma, non-small cell lung cancer (NSCLC), ovarian cancer, pancreatic cancer, and prostate cancer. In additional embodiments, the cancer cells are part of a tumor in the subject. In other embodiments, the tumor is a low immunogenic "cold" tumor characterized by insufficient induction of tumor-specific immunity and resistance to immunogenic cytotoxicity.

[0024] In certain embodiments, nanosatellite complexes can also be used as photothermal agents and / or MRI contrast agents.

[0025] In certain embodiments, type I interferon agonists include activators of type I interferon signaling adaptor proteins, stimulators of interferon genes (STING) (including cyclic dinucleotides selected from c-di-GMP, c-di-AMP and cGAMP) or their analogs. In other embodiments, STING agonists are selected from the group consisting of: c-di-IMP, c-di-UMP and 5,6-dimethylxanthone-4-acetic acid (DMXAA), 2'3'-cGAM (PS) 2 (Rp / Sp) and 2'3'-c-di-AM (PS) 2 (Rp, Rp). In other embodiments, type I interferon agonists include Toll-like receptor (TLR) family protein agonists, such as TLR9 agonist CpG. In specific embodiments, the kits, compositions and systems further comprise a physiologically compatible aqueous solution and / or cancer cell lysate.

[0026] In certain embodiments, the subject is a human or other mammal. In some embodiments, the method includes combining the aforementioned nanosatellite complex with administering an immune checkpoint inhibitor to the subject. These immune checkpoint inhibitors may include monoclonal antibodies, such as anti-PD-L1, anti-CLTA-4, or anti-PD-1. In other embodiments, the immune checkpoint inhibitor is selected from: YERVOY (ipilimumab), KEYTRUDA (pembrolizumab), OPDIVO (nivolumab), and TECENTRIQ (atezolizumab).

[0027] In some embodiments, the core comprises a material selected from the group consisting of a near-infrared photothermal material and an MRI contrast agent material, and at least one satellite particle comprises a near-infrared photothermal material, an MRI contrast agent material, and a near-infrared optical dye material. In additional embodiments, the nanoparticle core comprises a material selected from the group consisting of Fe3O4, silicon, gold, copper, and carbon. In some embodiments, at least one satellite particle comprises a material selected from the group consisting of gold sulfide (Au2S), copper sulfide (Cu2S), carbon nanotubes, and graphene. In certain embodiments, there is no shell surrounding the core, but rather one or more satellite particles that are clearly visible as discrete particles (e.g., as observed by tunneling electron microscopy).

[0028] In embodiments, the nanoparticle core comprises Fe3O4, and / or the biocompatible coating comprises polysiloxane, and / or at least one satellite particle comprises a plurality of satellite particles composed of gold. In certain embodiments, the core particle has a diameter of 15-20 nm. In other embodiments, the satellite particles have an average diameter of 2-6 nm. In certain embodiments, the core particle is spherical or cubic in shape.

[0029] In another embodiment, the core nanoparticle comprises a first type of material selected from the group consisting of: Fe3O4, silicon, gold, copper, and carbon. In a specific embodiment, the first type of material comprises Fe3O4. In additional embodiments, the Fe3O4 is highly crystalline and the brightest diffraction ring in its X-ray diffraction (XRD) pattern is from the 440 plane. In other embodiments, the Fe3O4 has a preferred lattice orientation along the 400 plane and the 440 XRD diffraction plane. In other embodiments, the satellite particles comprise a second type of material selected from the group consisting of: gold, gold sulfide (Au2S), copper, copper sulfide (Cu2S), carbon, carbon nanotubes, and graphene. In certain embodiments, the second type of material comprises gold sulfide (Au2S). In other embodiments, the near-infrared optical dye material is selected from the group consisting of: IR820, ICG, and 5'-aminolevulinic acid (5-ALA). The present invention is not limited by the shape of the core or satellite particles. Examples of shapes include, but are not limited to, spherical, cubic, rod-shaped, disc-shaped, and the like.

[0030] In some embodiments, the diameter of each satellite particle is between 0.5 nm and 25 nm (e.g., 0.5 nm, 1.5 nm, 10 nm, 15 nm, 20 nm, 23 nm, and 25 nm). In other embodiments, the diameter of the satellite particle is between 2 nm and 7 nm (e.g., about 5 nm or about 2-4 nm). In other embodiments, the diameter of the nanoparticle core is between 35 nm and 100 nm. In other embodiments, the nanosatellite complex is present in the composition at a concentration between 1.0 mg / mL and 5.0 mg / mL (e.g., 1.0 mg / ml, 3.3 mg / ml, and 5.0 mg / ml). In other embodiments, the biocompatible coating comprises a material selected from the group consisting of human serum albumin (HSA), polyethylene glycol, a triblock copolymer, PEO-b-PPO-b-PEO (F121), PEO-b-PVP, glycosylated poly (pentafluorostyrene), chitosan, silica and gum arabic, gluconic acid, lactobionic acid, polyacrylic acid, apatite and casein. In additional embodiments, the biocompatible coating is functionalized with thiol or amine groups. Specifically, siloxane molecules can be used, such as (3-mercaptopropyl) trimethoxysilane (MPTMS) to generate thiol groups on the nanoparticle surface, or (3-aminopropyl) triethoxysilane to generate amine groups on the nanoparticle surface, to functionalize the polymer-coated nanoparticles.

[0031] In some embodiments, administering the nanosatellite complex to a subject produces a plurality of core-satellite nanocomposite-impregnated cancer cells in the subject. In further embodiments, the method comprises subjecting the subject to photothermal therapy and / or imaging, wherein the photothermal therapy: A) comprises use of a therapeutic device that emits electromagnetic radiation, and B) causes at least a portion of the core-satellite nanocomposite-impregnated cancer cells to be destroyed or killed; and wherein the imaging: A) comprises use of an imaging device configured for MRI / NMR detection and / or optical detection, and B) causes at least a portion of the core-satellite nanocomposite-impregnated cancer cells to be visualized ex vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0033] Figure 1 The upper portion shows a simplified schematic diagram for generating exemplary inorganic virus-like nanoparticle (IVLN) blanks and IVLN-peptide complexes. Figure 1 The lower part shows a simplified schematic diagram for the production of lipid-coated iron oxide nanoparticles (lipid-IONPs), which were used as a control in Example 1.

[0034] Figure 2 A shows the formulation conditions of IVLN and the loading efficiency of satellite particles on the iron oxide nanoparticle core. Figure 2 B shows TEM imaging of IVLN under different formulation conditions. Figure 2 C shows the mathematical modeling of the distance between satellites under different configuration conditions. Figure 2 D shows the mathematical modeling of the satellite density on the nanoparticle surface under different formulation conditions. Figure 2 E shows peptide loading on the IVLN surface and loading specificity to satellites. Figure 2 F shows the volume-weighted hydrodynamic particle distribution of IVLN at different stages of formulation.

[0035] Figure 3 The results of Example 1 for producing antigen-specific antibodies in mice using IVLN-peptide are shown. Figure 3 A shows the experimental timeline and immunization schedule. Figure 3 B shows the antigen-specific IgG titers at day 10 after the first boost at different IVLN formulation conditions and peptide densities. Figure 3 C shows antibody quantification and antigen-specific IgG titers of IVLN against soluble and nanoparticle controls.

[0036] Figure 4 A shows the delivery efficiency and kinetics of nanoparticles to lymph nodes quantified as a percentage of the initial iron dose based on ex vivo quantification using ICP-MS. Figure 4 B shows semi-quantitative analysis of peptide delivery to lymph nodes at 3 hours based on ex vivo IVIS imaging. Figure 4 C shows the distribution of nanoparticles to lymphocytes and antigen-presenting cells in lymph nodes at 3 hours in vivo. Figure 4 D shows the cellular uptake of nanoparticles in vitro.

[0037] Figure 5 The amino acid sequence of the ERBB2 / HER2 protein is shown (SEQ ID NO: 4), with identified T cell epitopes or HLA ligands highlighted in grey shading, as provided by the tumor T cell antigen database TANTIGEN.

[0038] Figure 6 An exemplary nanosatellite complex is shown with various exemplary parameters labeled.

[0039] Figure 7Figure 2 shows that, in certain embodiments, inorganic virus-like nanosatellites (IVLNs) have three important features similar to the viral spike antigen S protein (peplomer): spike antigen cluster topology, optimal distance between antigen clusters (5 nm), and local high antigen density on the spike. (A) Schematic diagram of the step-by-step production of peptide-functionalized inorganic virus-like nanoparticles (IVLN-HER2) by (1) self-assembly of AuNPs onto polymer-coated IONP surfaces via gold-siloxane interactions (IVLNs) and then (2) conjugation of terminal cysteine-modified HER2 peptides to IVLNs via gold-thiol bonds. (B) Gold nanoparticle (AuNP) loading per iron oxide nanoparticle (IONP) core measured by ICP-MS. Data represent mean ± SD, n ≥ 6; curves were fitted using a linear regression model, R 2 =0.998, p<0.001. (C) STEM HAADF images of IVLNs at increasing AuNP / IONP ratios from 0 to 30%; Scale bar: 0 wt% condition (50 nm); Scale bar: 5-30 wt% condition (20 nm). (C-Inset) STEM image of a single IVLN. (D) As shown by mathematical modeling ( Figure 7 D) Calculated distance between AuNPs on the IVLN surface. (E) AuNP density (per unit area) on the IVLN surface compared to the known antigen density on the viral capsid as calculated by mathematical modeling. (F) Peptide loading on IVLN with variable AuNPs (0 AuNPs - black; 4 AuNPs - blue; 12 AuNPs - red) as determined by modified fluorescamine fluorescence detection assay; data represent mean ± SD, n = 3; curves were fitted using a linear regression model (0 AuNPs: R 2 =0.904, p<0.01; 4 AuNPs: R 2 =0.962, p<0.01; 12 AuNPs: R 2 =0.977, p<0.001). (G) Peptide loading on lipid-coated iron oxide nanoparticles (IONP-HER2); data represent mean ± SD, n = 3; curves were fitted using a linear regression model (R 2 =0.989, p<0.001). (H) Image and volume-weighted particle size of IVLN-HER2 obtained by dynamic light scattering (DLS). (I) Image and volume-weighted particle size of lipid-coated iron oxide nanoparticles-HER2 (IONP-HER2).

[0040] Figure 8Data from Example 2 are shown, which show that the IVLN-HER2 tested enhances antigen-specific antibody production. (A) Animal study immunization and analysis sampling timeline. (B) At the 38th day and with 5 μg HER2 peptide + 10 μg cGAMP as adjuvant, the quantification of nonspecific total IgG and antigen-specific antibody titers (IgG, IgG1 and IgG2a) from the serum of BALB / c mice. (C) At the 38th day and with 50 μg HER2 peptide + 10 μg cGAMP as adjuvant, the quantification of nonspecific total IgG and antigen-specific antibody titers (IgG, IgG1 and IgG2a) from the serum of BALB / c mice; Data represent mean ± SE, n=5. Data represent mean ± SE, n=5. Statistical comparisons are based on single-factor ANOVA, followed by post-hoc Tukey's paired comparisons. Asterisks indicate statistical significance at *p<0.05, **p<0.01, ***p<0.001 levels. ANOVA, analysis of variance; SE, standard error; ns, not statistically significant.

[0041] Figure 9 The results of Example 2 are shown, including those for IVLNs with the following characteristics: antigen clusters (14 clusters) generated against HER2-specific IgG, the distance between antigen clusters (5-6 nm), and the local antigen density (2000 peptides / IVLN, approximately 150 peptides / AuNP). (A) Immunization schedule for mice. (B) Quantification of antigen-specific IgG antibodies by ELISA, expressed as antibody titers; data represent mean ± SE, n = 5. Statistical comparisons were based on one-way ANOVA followed by post hoc Tukey's pairwise comparisons. Asterisks indicate statistical significance at the *p < 0.05, **p < 0.01, ***p < 0.001 levels. ANOVA, analysis of variance; SE, standard error.

[0042] Figure 10 It was shown that IVLN-HER2 tested in Example 2 increased Ag-specific B cell activation and GC formation by 6-fold compared to IONP-HER2. (A) Identification of CD19 using B cell receptor tetramer staining + Representative FACS plots of the gating strategy for HER2-specific B cells of the tetramer+ population. (B) Quantification of the percentage of HER2-specific B cells induced in the total viable cells 10 days after primary immunization with a dose of 50 μg HER2 peptide + 10 μg cGAMP as adjuvant; data represent mean ± SE, n ≥ 3. (C) Representative FACS plots of the gating strategy for GC cells. GC cells were identified as B220 + IgD 低(D) Total B220 cells induced 10 days after primary immunization at a dose of 50 μg HER2 peptide + 10 μg cGAMP as adjuvant. + Quantification of the percentage of GC-type cells in the B cell population; Data represent mean ± SE, n ≥ 3. Statistical comparisons were based on one-way ANOVA followed by post hoc Tukey's pairwise comparisons. Asterisks indicate statistical significance at the *p < 0.05, ***p < 0.001 levels. ANOVA, analysis of variance; SE, standard error.

[0043] Figure 11 The result of embodiment 2 is shown, it shows that the CyTOF analysis of immune cells shows that IVLN-HER2 promotes Tfh-dependent B cell activation in lymph nodes. 38 days after the first immunization (10 days after the second boost), the immune cells in the lymph nodes are subjected to CyTOF analysis. (A, B) overall analysis using SPADE unsupervised cluster analysis. Nodes include cells with similar marker expression. Based on whether the relative cell number in that node in the IVLN-HER2 sample is higher (blue) or lower (red) compared with IONP-HER2 or HER2, the node is colored. (D) The frequency of germinal center B cells (CD19+ / GL7+ or B220+ / GL7+) in the lymph nodes of mice immunized with INLN-HER2, INOP-HER2 and HER2 peptides alone. (D) The frequency of CD4+T follicular helper T cells (CD4+ / CXCR5+ / PD-1+) in the lymph nodes of mice immunized with INLN-HER2, INOP-HER2 and HER2 peptides. (E) Frequency of plasma cells in lymph nodes of mice immunized with INLN-HER2, INOP-HER2, and HER2 peptide (50 μg HER2 peptide, 10 μg cGAMP).

[0044] Figure 12 Results from Example 2 are shown, which show that IVLN-HER2 improved lymph node delivery and B cell zone distribution compared to IONP-HER2. (A) Quantification of nanoparticle delivery to the ipsilateral lymph nodes (popliteal + inguinal) of the administration site at the specified time intervals, expressed as the initial iron oxide percentage delivered using ICP-MS; data represent mean ± SE, n = 3. (B) Representative ex vivo IVIS fluorescence images and semi-quantitative analysis of peptide delivery to lymph nodes obtained 3 hours after administration of Cy5.5-labeled soluble HER2 peptide, IONP-HER2-Cy5.5 and IVLN-HER2-Cy5.5 (popliteal (top) + inguinal (bottom)) (Ex / Em = 675 / 720nm, exposure = 0.5s). The colored bars represent the average radiant efficiency (p / s / cm 2 / sr) / (μW / cm 2 ); Data represent mean ± SD, n = 3. (C) Quantification of in vivo nanoparticle distribution of specific immune cell populations in lymph nodes as identified by flow cytometry (Cy5.5-labeled nanoparticles); subcapsular sinus macrophages were identified as CD11b + CD169 高 ; B cells were identified as B220 + ; Data represent mean ± SD, n = 3. (D) Quantification of in vitro cellular uptake of nanoparticles in RAW264.7 macrophages and murine primary B cells by ICP-MS quantification of total Fe (pg Fe per cell) normalized by cell count. Data represent mean ± SD, n = 3. Statistical comparisons were based on one-way ANOVA followed by post hoc Tukey's pairwise comparisons or Student's unpaired T-test. Asterisks indicate statistical significance at the **p < 0.01, ***p < 0.001 levels. ANOVA, analysis of variance; SD, standard deviation; SE, standard error.

[0045] Figure 13 Results from Example 2 are shown, which demonstrate that IVLN-HER2 induced HER2-specific antibodies have the function of inhibiting HER2+ cancers. (A) Animal Studies Immunization and HER2 + Breast cancer (D2F2 / E2) tumor inoculation timeline. (B) Tumor volume growth curve of D2F2 / E2 tumors implanted subcutaneously in the flanks of BALB / c mice at 250,000 cells per mouse, treated with a 50 μg HER2 peptide dose + 10 μg cGAMP. (C) Tumor volume growth curve of D2F2 / E2 tumors implanted subcutaneously in the flanks of BALB / c mice at 250,000 cells per mouse, treated with a 5 μg HER2 peptide dose + 10 μg cGAMP. Data represent mean ± SE, n = 5. Statistical comparisons are based on one-way ANOVA followed by post hoc Tukey's pairwise comparisons. Asterisks indicate statistical significance at the *p < 0.05, **p < 0.01, ***p < 0.001 levels. ANOVA, analysis of variance; SE, standard error. DETAILED DESCRIPTION

[0046] The present invention provides methods, compositions, systems and kits comprising nanosatellite complexes, the nanosatellite complexes comprising: a core-nanoparticle complex comprising a biocompatible coating surrounding a nanoparticle core; 3-25 (or 2-35) satellite particles attached to or absorbed into the biocompatible coating; a plurality of antigenic peptides (or haptens with a carrier) conjugated to or absorbed into the satellite particles; and at least one additional characteristic: i) the weight ratio of all the satellite particles to the nanoparticle core is 10-40%; the diameter of each of the satellite particles is 2-20 nm; iii) the satellite particles are present at a density of 500-20,000 or 15,000-30,000 per square micron; iv) the plurality of antigenic peptides is 100-4000 antigenic peptides; v) 10-300 of the plurality of antigenic peptides are present on each of the satellite particles; and / or vi) the average distance between each of the satellite particles is 5-20 nm. In certain embodiments, the nanosatellite complex has a virus-like topology with a virus-like antigenic plaque distance and a 3D plaque topology.

[0047] Viruses are the most efficient delivery vehicles and effective immunizing agents in nature. Therefore, for decades, viral material properties have been a source of inspiration for nanoparticle design and engineering. These so-called virus-mimicking nanoparticles have the potential to be widely used, including drug delivery, molecular imaging, cancer immunotherapy, and gene transfection. However, to date, this potential has been limited by the selective material property methods of virus mimicking. Here, we demonstrate that the overall approach to virus-mimicking nanoparticle design is crucial for functional efficacy. Specifically, in some embodiments, nanosatellite complexes are described herein that have unique surface roughness, epitope organization, and epitope density compared to traditional nanoparticle systems. In work conducted during the development of the embodiments herein, it was found that in the case of B cell immunity and lymph node delivery, these nanoparticle features led to an 18.5-fold improvement in antigen-specific IgG antibody production in a mouse model (see Example 1). From a mechanistic point of view, it was shown that this significant improvement in antibody production was the result of a 3-fold improvement in lymph node delivery and a 2- to 3-fold increase in the retention of related immune cell populations, which promoted an increase in B cell activation and germinal center formation, respectively.

[0048] In certain embodiments, the nanosatellite complex herein uses a mixed Fe@Au core / satellite nanoparticle in which a diblock copolymer containing poly(siloxane) is coated with an iron oxide nanoparticle core (e.g., IONP, 15-20 nm) that anchors a controlled amount of gold nanoparticles (e.g., AuNP, 2-3 nm) to the surface. Through these gold nanoparticles, cysteine-terminal modified peptides are conjugated using Au-S bonds in a defined number and density.

[0049] While the present disclosure is not limited to any particular mechanism, it is believed that the nanosatellite nanoparticle complexes herein combine more biologically relevant surface topologies and spatially confined and locally high-density antigen presentation with virus-like geometric rigidity compared to conventional virus-mimicking nanoparticle systems. The nanosatellite nanoparticles herein can be used in a wide variety of biological applications, including use in B cell immunity. In this context, these unique material properties will manifest as antigen presenting cell uptake and B cell immunity enhancement due to improved B cell receptor cross-linking.

[0050] In work performed during the development of the embodiments herein, results generated (in Example 1) indicate that nanosatellite complexes can be successfully prepared as approximately 60 nm particles with 10-15 AuNPs per IONP core in a hydrodynamic manner, associated with a distance between AuNPs of less than 7.5 nm (ideal for B cell receptor cross-linking). Furthermore, the nanosatellite complexes herein can be prepared with approximately 2,000 peptides per particle and specifically localized to the AuNPs.

[0051] The present disclosure is not limited by the type of antigen used in the nanosatellite complex. In certain embodiments, B cell antigens and / or T cell antigens are used. In certain embodiments, at least a portion of human tumor-associated antigens are used. Examples of human tumor-associated antigens (TAAs) include differentiation antigens (such as melanocyte differentiation antigens), mutant antigens (such as p53), overexpressed cellular antigens (such as HER2), viral antigens (such as human papillomavirus proteins), and cancer / testis (CT) antigens that are expressed in germ cells of the testicles and ovaries but are silent in normal somatic cells (such as MAGE and NY-ESO-1). In other embodiments, antigens from bacteria or viruses are used.

[0052] In certain embodiments, the antigen is provided from the TANTIGEN website, which provides a comprehensive database of tumor T cell antigens (see Olson et al., Cancer Immunol Immunother. March 9, 2017, which is incorporated by reference in its entirety). Table 1 below provides a list of antigens, at least a portion of which can be used with the nanosatellite complexes provided herein. The TANTIGEN website can be used to select portions of specific antigens (see "http: / / projects.met-hilab.org / tadb / index.php"). For example, with respect to the ERBB2 / HER2 antigen, the TANTIGEN website displays the amino acid sequence of this antigen, provides highlighted short antigenic regions that are immunogenic (e.g., Figure 5(as shown, TANTIGEN accession number is "Ag000001"), one or more highlighted regions of this antigen can be used in the complexes described herein. The same procedure can be used with any of the antigens listed in Tables 1 and 4 using the TANTIGEN website or similar resources. In other embodiments, ongoing deep sequencing of cancers provides new tools for additional neoantigen discovery, which can be used with the present disclosure. Nanosatellite complexes and / or serum albumin carrier-antigen-adjuvant complexes are not limited to a specific sequence of the antigenic peptide. Both systems provide methods, compositions and kits for specifically modulating immune responses to additional targeted neoantigens.

[0053] Table 1

[0054]

[0055]

[0056]

[0057]

[0058]

[0059]

[0060] In certain embodiments, the antigens used in the complexes described herein are derived from human oncogenic viruses or tumor viruses. Viruses associated with human malignancies include: HTLV-1 (adult T-cell leukemia (ATL), HPV (cervical cancer, skin cancer in patients with verrucous epidermodysplasia (EV), head and neck cancer, and other anogenital cancers); HHV-8 (Kaposi's sarcoma (KS), primary effusion lymphoma, and Castleman's disease), EBV (Burkitt's Lymphoma (BL), nasopharyngeal carcinoma (NPC), MCPyV (Merkel Cell Carcinoma), post-transplant lymphoma, and Hodgkin's disease. disease), HBV and HCV (hepatocellular carcinoma (HCC)). In addition, viruses that may play a role in human malignancies include: simian vacuolating virus 40 (SV40) (brain cancer, bone cancer and mesothelioma), BK virus (BKV) (prostate cancer), JC virus (JCV) (brain cancer), human endogenous retroviruses (HERV) (germ cell tumors, breast cancer, ovarian cancer and melanoma), human mammary tumor virus (HMTV) (breast cancer) and (vi) Torque teno virus (TTV) (gastrointestinal cancer, lung cancer, breast cancer and myeloma).

[0061] In certain embodiments, antigens from viruses or bacteria are used together with the nanosatellite complexes described herein. Such antigens are well known in the art. Examples of viruses (Table 2) and bacteria (Table 3) as sources of such well-known antigens are provided below.

[0062] Table 2 - Viral diseases

[0063]

[0064]

[0065] Table 3 - Bacterial diseases

[0066] Source of bacterial antigens Disease or condition Bacillus anthracis Anthrax Bordetella pertussis pertussis Clostridium tetani tetanus Corynebacterium diphtheriae diphtheria Coxiella burnetii Q fever Haemophilus influenzae type b (Hib) Epiglottitis, meningitis, pneumonia Mycobacterium tuberculosis Tuberculosis Neisseria meningitidis Meningococcal meningitis Salmonella Typhi Typhoid fever Streptococcus pneumoniae Pneumococcal pneumonia Vibrio cholerae cholera

[0067] Example

[0068] Example 1

[0069] Virus-like nanoparticles for antigen-specific antibody production

[0070] This example describes the production and use of antibody-producing virus-like nanoparticles.

[0071] Materials: All reagents were used as received from commercial sources without further purification. Iron (III) oxide (FeO(OH), hydrated, catalyst grade, 30-50 mesh), oleic acid (technical grade, 90%), 1-octadecene (technical grade, 90%), anhydrous tetrahydrofuran (THF, 99.8%), sodium sulfide, chloroauric acid, iron (II) ammonium sulfate hexahydrate (Fe(NH4)2(SO4)2·6H2O, ACS reagent, 99%), nitric acid (ACS reagent, 70%), and hydrochloric acid (ACS reagent, 37%) were purchased from Sigma-Aldrich. Mouse Uncoated IgG and IgM Total ELISA Ready-SET-Go! The kit, 1-Step Ultra TMB-ELISA substrate solution, HRP-conjugated goat anti-mouse IgG1 secondary antibody, HRP-conjugated goat anti-mouse IgG2a secondary antibody, Nunc Immobilizer amino 96-well ELISA plates, BupH bicarbonate buffer pack (coating buffer), Pierce protein-free PBS tween blocking buffer, 20× PBS-tween wash buffer, Geneticin (G418) selective antibiotic, Invitrogen eBioscience fixable viability dye eFluor 780, and Molecular Probes Streptavidin Alexa Fluor 647 conjugate were all obtained from Thermo Fisher Scientific. HRP-conjugated goat anti-mouse IgG secondary antibody, Zombie UV fixable viability kit, FITC anti-mouse CD19, PE / Dazzle 594 anti-mouse CD38, Brilliant Violet 421 anti-mouse CD138, PE / Dazzle 594 anti-mouse IgD, Alexa Fluor 647 anti-mouse / rat GL7 antigen, BrilliantViolet 421 and PE / Dazzle 594 anti-mouse / human CD45R / B220, FITC anti-mouse CD95, Brilliant Violet421 anti-mouse / human CD11b, FITC anti-mouse CD169, and PE / Dazzle 594 anti-mouse CD11c were all purchased from BioLegend. Custom synthesized HER2 peptides (CDDDPESFDGDPASNTAPLQPEQLQ (SEQ ID NO: 1), biotin-PESFDGDPASNTAPLQPEQLQ (SEQ ID NO: 2), CDDDPESFDGDPASNTAPLQPEQLQGGGK (SEQ ID NO: 3)). 30 nm iron oxide nanoparticle cores stabilized by oleic acid in chloroform were purchased from Ocean Nanotech.DSPE-PEG (2000) and DSPE-PEG (2000) maleimide were obtained from Avanti Polar Lipids. 2'3'-cGAMP was obtained from InvivoGen. Fluorescein was purchased from MP Biomedicals. Sulfo-Cy5.5 NHS ester was obtained from Lumiprobe. Microvette 500Z-Gel serum collection vials with coagulation factors were obtained from Sarstedt. Matrigel basement membrane matrix was purchased from Corning. Gold and iron ICP standards were purchased from Fluka Analytical.

[0072] All animal experiments were performed according to protocols approved by the University of Michigan Committee on Use and Care of Animals (UCUCA). 5- to 7-week-old BALB / c mice were purchased from Charles River Labs.

[0073] Cells. All cells were maintained at 37°C, 5% CO2 / 95% air atmosphere and approximately 85% relative humidity. D2F2 / E2 cells were cultured in complete DMEM high glucose supplemented with 10% NCTC 109 medium, 1% L-glutamine, 1% MEM non-essential amino acids, 0.5% sodium pyruvate, 2.5% sodium bicarbonate, 1% penicillin / streptomycin (pen / strep), 5% cosmic calf serum, 5% fetal bovine serum, 500 μg / mL geneticin and 50 μM 2-mercaptoethanol. RAW264.7 macrophages were cultured in complete RPMI-1640 medium supplemented with 10% fetal bovine serum, 1% L-glutamine, 1% MEM non-essential amino acids, 1% sodium pyruvate and 1% penicillin / streptomycin. DC2.4 dendritic cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 1% L-glutamine, 1% MEM non-essential amino acids, 1% HEPES buffer solution, 1% penicillin / streptomycin, and 50 μM 2-mercaptoethanol.

[0074] Formulation and Characterization of Inorganic Virus-Like Nanoparticles (IVLN) IVLN was formulated as follows.

[0075] Synthesis of IONPs coated with diblock copolymers containing polysiloxane. Spherical IONPs (15 nm in diameter) were synthesized by thermal decomposition in an organic solvent. Cubic IONPs (25 nm in edge length) were also synthesized. Diblock copolymers (PEO-bP) were synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization. γMPS). Preparation of polymer-coated MNPs with single or clustered cores was performed. After digestion with hydrochloric acid (ACS reagent, 37%), the IONP iron concentration was determined using o-phenanthroline (ACS reagent, 99%). The general method for this type of synthesis is described in Chen et al., ACS Appl. Mater. Interfaces 2015, 7, 12814-12823, which is incorporated herein by reference.

[0076] Synthesis of AuNPs. AuNPs were synthesized by using sodium sulfide (Na2S) as a reducing agent. Gold in the form of chloroauric acid (HAuCl4) was prepared into a stock solution with a concentration of 100mM and diluted to 2.0mM before use. Na2S (50mM) was prepared and aged in the dark for 40-48h before use, and diluted to 1.0mM before use. The volume ratio of Na2S to HAuCl4 varied from 2.5 / 1.0 to 3.0 / 1.0. UV / Vis spectra were recorded to monitor the reaction. Where not specified, a reaction with a volume ratio of 3.0 / 1.0 was selected for the following steps. The general method for this type of synthesis is found in Chen et al., ACS Appl. Mater. Interfaces 2015, 7, 12814-12823, which is incorporated herein by reference.

[0077] Synthesis of Inorganic Virus-Like Nanoparticles (IVLNs). IVLNs were prepared by incubating AuNPs with polymer-coated IONPs at 4°C. In a typical experiment, 2 mg of iron-ionized IONPs (0.5 mL) was mixed with AuNP solution (6 mL for spherical IONPs and 4 mL for cubic IONPs, respectively), unless otherwise specified. After overnight incubation, the formed IVLNs were purified by a magnet to remove unbound AuNPs.

[0078] The final Au:Fe ratio of the formulated IVLN was quantified by inductively coupled plasma mass spectrometry using a Perkin-Elmer Nexion 2000 based on established protocols. IVLN formulations were imaged by transmission electron microscopy (TEM) using a JEOL 3011 high-resolution electron microscope. The true particle size of AuNPs, IONPs, and IVLNs was quantified using ImageJ software. The volume-weighted hydrodynamic particle size, polydispersity index, and zeta potential of all formulations were evaluated in milliQ water at 25°C using dynamic light scattering and phase analysis light scattering, respectively, using a Malvern Zetasizer Nano-ZS.

[0079] Lipid-coated iron oxide nanoparticle formulation (lipid-IONP). Lipid-coated iron oxide nanoparticles were prepared based on a previously reported method for film hydration with slight modifications. 10 mg of DSPE-PEG(2000)-maleimide was added to 1 mg of 30 nm iron oxide nanoparticle cores stabilized by oleic acid in chloroform while gently mixing. The resulting solution was subjected to solvent rotary evaporation to remove all chloroform and form a thin film. Simultaneously, this film and 100 mM PBS, pH 7.4, were heated to 75°C in an oven. Once at temperature, hot PBS was quickly added to the film and immediately mixed vigorously to promote film hydration. The resulting nanoparticle solution was stored at 4°C to promote lipid self-assembly. Free phospholipids were removed by magnetic separation overnight at 4°C using a magnetic separator apparatus.

[0080] Lipid-IONP-HER2 and IVLN-HER2 formulations. The HER2 peptide was conjugated to both lipid-IONP and IVLN via a thiol-mediated chemical process. Specifically, lipid-IONP-HER2 was formulated via a maleimide chemistry, and IVLN-HER2 was formulated via a gold-thiol linkage. The HER2 peptide was added to the lipid-IONP at a weight ratio of 1.5x excess in milliQ and incubated overnight at 4°C. The HER2 peptide was added to the IVLN-HER2 at a weight ratio of 5x excess in milliQ and incubated overnight at 4°C. Both materials can be purified by magnetic separation overnight at 4°C using a magnetic separator device or by centrifugation at 10,000xg at 4°C for 30 minutes.

[0081] Immunization and serum collection. On day 0, mice were immunized with the equivalent of 50 μg HER2 peptide plus 10 μg cGAMP, regardless of the type of formulation. Subsequently, on day 14, mice were boosted twice (day 14 and day 28) at two-week intervals with 50% of the original dose of antigen and adjuvant. In order to assess serum antibody titers, blood was collected by submandibular puncture 10 days after each immunization (day 10, 24, and 38). Serum was separated from whole blood by centrifugation at 10,000 x g for 5 minutes at 25°C using a Microvette 500 Ser-Gel collection container with a coagulation activator.

[0082] Enzyme-linked immunosorbent assay (ELISA). Based on the scheme provided by ThermoFisher, the absolute quantification of total IgG and total IgM antibodies was carried out using the uncoated total IgG and total IgM ELISA kits of mice. Based on the indirect ELISA scheme previously established and slightly modified, antigen-specific IgG, IgG1 and IgG2a antibody titers were quantitatively measured. Specifically, by overnight incubation at room temperature under exposure to light, Nunc Immobilizer amino immunoassay plates were used, and HER2 peptide (200 μL, 100 ug / mL in 100 mM carbonate buffer, pH 9.4) was chemically conjugated to ELISA plates by terminal amine groups. After overnight incubation, ELISA plates were washed 3 times with 100 mM PBS with 2% Tween-20 of pH 7.4. Subsequently, ELISA plates were blocked overnight at 4 ° C with 300 μL ELISA blocking agents (PBS blocking buffer without Pierce protein). After blocking, ELISA plates were washed 3 times. Serum samples containing primary antibody were serially diluted (10 1 -10 8 fold) and added to each well in a total amount of 200 μL and incubated at room temperature for 2 hours. After adding the sample, the ELISA plate was washed 3 times. 500-fold diluted anti-IgG-HRP, anti-IgG1-HRP or anti-IgG2a-HRP was added to each well with 100 μL and incubated at room temperature for 1 hour. After 1 hour, the ELISA plate was washed 5 times. Next, 100 μL 1-Step Ultra TMB substrate solution was added to each well and incubated and developed for 15-20 minutes at room temperature with gentle stirring. After 15-20 minutes, color development was stopped by adding 100 μL 100mM sulfuric acid. The color development was quantified by using the absorption spectrum of BioTekCytation 5 at 450 nm. The antibody titer was determined by any absorbance signal that was greater than the PBS control absorbance signal plus 3x standard deviation at a given dilution factor.

[0083] Quantification of nanoparticle delivery to lymph nodes in vivo. Mice were injected subcutaneously in the left elbow joint with 200 μg of total Fe per mouse of lipid-IONP or IVLN-peptide. At designated time intervals, mice were sacrificed and lymph nodes of interest were dissected for ex vivo analysis. The extent of nanoparticle delivery to lymph nodes was quantified using ICP-MS based on a previously reported protocol.

[0084] Quantification of in vivo peptide delivery to lymph nodes. To facilitate quantitative delivery of peptides to lymph nodes, lysine-terminally modified HER2 peptides were chemically conjugated to sulfo-Cy5.5 NHS ester. This conjugation was performed with a 5-fold molar excess of sulfo-Cy5.5 NHS ester to HER2 peptide. After the initial peptide conjugation was complete, IONP-HER2-Cy5.5 and IVLN-HER2-Cy5.5 were functionalized with Cy5.5 to enable easy purification of excess fluorescent dye by magnetic separation. After Cy5.5 functionalization, mice were injected as previously described. After 3 hours, mice were sacrificed and the lymph nodes of interest were dissected for ex vivo analysis by IVIS imaging. Regarding radiation efficiency, IVIS imaging was used for semi-quantification of peptide delivery.

[0085] In vitro cellular uptake. The uptake of IVLN-HER2 and IONP-HER2 cells was evaluated in RAW264.7 macrophages, DC2.4 dendritic cells, and primary B cells isolated from mouse spleens using the EasySep Mouse B Cell Isolation Kit. In blank RPMI medium, the nanoparticle samples were incubated with cells at 50 μg / mL Fe for 18 hours at 37°C, 5% CO2 / 95% air atmosphere, and approximately 85% relative humidity. After 18 hours, the cells were lifted by scraping them off and washed three times with phosphate-buffered saline (PBS). After the washing step, the resulting cell pellet was resuspended in 1 mL of PBS, the cells were counted, and then digested in 1 mL of aqua regia (nitric acid: hydrochloric acid molar ratio of 1:3) for analysis by ICP-MS.

[0086] In vivo cellular uptake. IVLN-HER-Cy5.5 and IONP-HER2-Cy5.5 were injected subcutaneously in the left elbow joint with lipid-IONP or IVLN at 200 μg total Fe per mouse. At 3 hours and 24 hours, mice were sacrificed and the lymph nodes of interest were dissected for in vitro analysis by flow cytometry. Lymph nodes were mechanically dissociated to prepare single cell suspensions. Single cell suspensions of lymph node cells were stained for analysis by flow cytometry using a MoFlo Astrios flow cytometer. The first group was live cells, B cells (B220 + ), subcapsular sinus macrophages (CD169 + CD11b + ), dendritic cells (CD11c + ) and nanoparticle-positive cells (Cy5.5). Flow cytometry data were analyzed by FCS expression.

[0087] Antigen-specific B cells and germinal center flow cytometry. Mice were immunized as previously described. On the 24th and 38th days, mice were sacrificed and spleens and lymph nodes were dissected for in vitro analysis by flow cytometry. Antigen-specific B cell analysis was completed using tetramer staining. In the absence of further purification, HER2 / neu peptide tetramers were prepared by mixing biotin-labeled HER2 peptide with Alexa Fluor 647-labeled streptavidin at a 4:1 molar ratio for 1 hour at room temperature. Using flow cytometry, antigen-specific B cell populations were identified as memory B cells (B220 + CD38 + Tetramer + ) or plasma cells (B220 - CD138 + Tetramer + The germinal center B cell population was identified using the following markers: CD19, IgD, GL7, and CD95.

[0088] Tumor studies. Sixty days after primary immunization, mice were inoculated subcutaneously in the right flank with 500,000 D2F2 / E2 cells. D2F2 / E2 cells were prepared at 5e6 cells / mL in 100 μL and mixed with an equal volume of Matrigel matrix. Tumor size was quantified by caliper measurement every 7 days. Tumor volume was calculated using the following formula:

[0089] Tumor volume = xy 2 / 2

[0090] Endpoints were determined by using a terminal disease scoring system; mice with a terminal disease score greater than 6 were euthanized by CO2 asphyxiation.

[0091] Statistics. Unless otherwise stated, data are expressed as mean ± standard deviation (SD). Comparisons between two groups were performed using an unpaired Student's t-test. The mean values of multiple groups were compared using one-way analysis of variance (ANOVA) followed by post hoc Tukey's paired comparisons. All probability values were two-sided, and values of p < 0.05 were considered statistically significant. Statistical analyses were performed using the GraphPad Prism 7 software package.

[0092] result

[0093] Formulation and viral characterization of inorganic virus-like nanoparticles (IVLN)

[0094] IVLNs were formulated by the self-assembly of two separately prepared nanoparticle systems (iron oxide nanoparticle cores and gold nanoparticle satellites) through the association of hydrolyzed siloxane groups and gold. Figure 1). Iron oxide nanoparticle (IONP) cores were synthesized by thermal decomposition to produce approximately 15 nm spherical cores stabilized by oleic acid in chloroform. To achieve aqueous stability, the IONP cores were coated with a polysiloxane / PEG diblock copolymer (IONP-polymer). Separately, ultrasmall gold nanoparticles (AuNPs) of approximately 3 nm in size were prepared using a modified self-assembly method by reducing chloroauric acid in aged sodium sulfide. After synthesis, AuNPs were added to the polymer-coated IONP cores in solution at a defined weight ratio.

[0095] To quantify the degree of AuNP loading per IONP core after self-assembly, inductively coupled plasma mass spectrometry (ICP-MS) was used. Using ICP-MS, it was possible to determine an average loading efficiency of 73 ± 7% by weight with a linear correlation (R 2 =0.997)( Figure 2 A). It is noteworthy that above an initial loading ratio of 50 wt% Au, the nanoparticles were observed to be unstable in solution and were therefore not the focus of further investigation. To provide visual confirmation of the self-assembly of AuNPs and IONP-polymers to form IVLNs, transmission electron microscopy (TEM) was performed ( Figure 2 B). TEM imaging confirmed that by controlling the initial loading ratio of AuNPs to IONP-polymer core (by weight), IVLNs with variable gold nanoparticle surface density and virus-like features can be obtained ( Figure 2 (B - inset). The diameters of the IONP-polymer core and AuNPs were quantified as 15.9 ± 1.3 nm and 2.3 ± 0.4 nm, respectively. TEM imaging was further used to estimate the AuNP loading per IONP-polymer core. Specifically, at 10%, 20%, and 30% by weight Au, IVLNs with 4 ± 2, 9 ± 3, and 13 ± 5 AuNPs per IONP-polymer core were obtained, respectively.

[0096] After ICP-MS quantification and TEM visualization of IVLN preparations, basic mathematical modeling was performed to determine the structural relevance of IVLN compared to virus-like particles. Given the crystalline nature of AuNPs and IONPs, it was feasible to determine the number of particles of a defined particle size for a given weight of Au or Fe, respectively. Figure 2 ICP-MS measurements performed in A and Figure 2The particle size confirmed by TEM in Figure B allows estimation of the average distance between AuNPs on the IONP surface, as well as the average number of AuNPs per unit area on the IONP surface. From this analysis, it was determined that, depending on the initial weight loading ratio of Au to Fe, IVLNs could be formulated to have a minimum average distance between AuNPs of 6.75 nm, which is the preferred distance for B cell receptor crosslinking ( Figure 2 C). In addition to the spatial distribution of AuNPs, at this same initial loading ratio, the number of AuNPs per unit area was determined to be approximately 12,500-17,000 AuNPs per square micron, a value that compares favorably with the antigen density reported for virus-like particles (e.g., hepatitis B virus) ( Figure 2 D).

[0097] To further evaluate the viral-like potential of IVLNs, we next assessed the capacity and mechanism of peptide loading in this system. The peptide of interest in these studies was a human HER2 / neu-specific peptide that, based on previously published work, contains a B-cell epitope with an overlapping CD4 helper T-cell epitope. In addition to these functional epitopes, a cysteine-containing terminal flank was added to facilitate facile loading into IVLNs via an Au-S linkage (CDDD-PESFDGDPASNTAPLQPEQLQ, SEQ ID NO: 1). A modified fluorochrome peptide assay was used to quantify the capacity of peptide conjugation into IVLNs. Peptide loading was assessed under three separate IVLN formulation conditions: the final weight loading ratio of Au to Fe (w / w Au / Fe) was 0%, 10%, and 30%, respectively. In terms of AuNPs per IONP-polymer core, these values translate to approximately 0, 4, and 10 AuNPs per IONP-polymer core, respectively. Peptide conjugation was performed overnight in water at 4°C, followed by purification by centrifugation. Fluorescent peptide quantification analysis showed that under conditions of maximum peptide loading, 0%, 10% and 30% weight / weight formulations were loaded with 232 ± 73, 888 ± 42 and 1954 ± 157 peptides per IVLN, respectively ( Figure 2 E). When these same values were normalized by total AuNPs, the maximum peptide loading per AuNP was determined to be 227 ± 5. Furthermore, this analysis showed a positive correlation between peptide loading and the number of AuNPs (R = 0.95). Taken together, these results indicate that peptide loading is associated with AuNPs, although there is a low level of nonspecific physical association with the core (approximately 12% under maximum loading conditions). Thus, peptide conjugation to the IVLN surface is AuNP-localized, suggesting that IVLN-peptides are characterized by a heterogeneous and spotty distribution of peptides. This patterned antigen display is inherently viral-like and cannot be reproduced by conventional nanoparticle systems using a uniform distribution of antigens on their surface.

[0098] After evaluating peptide loading, we next evaluated the material properties of the IVLN-peptide to determine whether the material was suitable for in vivo applications and appropriately consistent with virus-like properties ( Figure 2 F). Before incubation with AuNPs, dynamic light scattering (DLS) revealed that the volume-weighted hydrodynamic particle size of the IONP-polymer core of IVLN was 51 ± 2 nm, with a polydispersity index (PDI) of 0.15 ± 0.03. Furthermore, the zeta potential of this material in milliQ water at pH 7 was determined to be -7 ± 4 mV. After formulation at a final weight loading ratio of 30% wt / wt Au / Fe, the IVLN-blank before peptide loading showed a particle size of 55 ± 2 nm, a PDI of 0.20 ± 0.05, and a zeta potential of -16 ± 4 mV. After peptide loading, the IVLN-peptide showed a particle size of 60 ± 4 nm, a PDI of 0.20 ± 0.05, and a zeta potential of -17 ± 1 mV. In summary, the IVLN-peptide was determined to possess optimal material properties for in vivo applications. Furthermore, these properties are considered acceptable within the design criteria for virus-mimicking nanoparticles, which include a particle size between 20-300 nm and an overall negative surface charge.

[0099] Inorganic virus-like nanoparticles (IVLN) for antigen-specific antibody production in mice

[0100] Virus-mimicking nanoparticles have been used for a wide range of in vitro and in vivo applications, but one application for which the properties of virus-like materials are well suited is B cell activation for antigen-specific antibody production. Therefore, a virus-like material based on the properties of IVLN-peptide was established. In this study, BALB / c mice (6-8 weeks old) were immunized with 50 μg of HER2 peptide plus 10 μg of cGAMP as adjuvant on day 0 and boosted once 14 days later. The mice were bled and serum was collected for analysis 10 days after each administration ( Figure 3 A).

[0101] Based on our understanding of the requirement for B cell receptor cross-linking for B cell activation and germinal center formation, we first asked what the role of the number and spatial distribution of AuNPs on the IVLN surface was at saturating peptide loading. To answer this question, we evaluated the titers of antigen-specific IgG antibodies produced in mice following booster immunization with IVLN at final weight loading ratios of 10%, 20%, and 30% weight / weight Au / Fe by indirect ELISA. From this analysis, it was determined that by day 24, the production of antigen-specific IgG in terms of median antibody titers was 7,500, 12,500, and 32,500 for the 10%, 20%, and 30% weight / weight formulations, respectively. Figure 3B). These preliminary results suggest that increasing the number of AuNPs on the IVLN surface can improve antibody production. This improvement is hypothesized to be due to a reduction in the AuNP spacing (10 wt% = approximately 11.25 nm; 20 wt% = approximately 8.05 nm; 30 wt% = 6.25 nm), promoting more efficient B cell receptor crosslinking.

[0102] After evaluating the AuNP surface density, we questioned what the role of peptide conjugation density on the IVLN was for a given Au / Fe weight loading ratio. Peptide density has been shown to positively correlate with increased B cell activation and potentially provide dose savings, as reported previously. Notably, peptide density was negatively correlated with decreased antibody specificity. For a 30 wt% ratio, low-density peptides on the IVLN surface produced a median antigen-specific IgG antibody titer of 6,500, while high-density peptides on the IVLN surface produced a median titer of 32,500 ( Figure 3 B). This trend was also observed at a 20 wt% ratio, producing median titers of 1,300 and 12,500 for low and high densities, respectively. However, this trend did not translate to the 10 wt% ratio. Presumably, this is due to the spacing between AuNPs on the IVLN surface being greater than 10 nm. Based on the above analysis, it was determined that higher peptide densities and a higher number of AuNPs per IVLN surface are generally preferred for antigen-specific antibody production due to the increased viral-like properties of the material. Significantly, peptide density was demonstrated to improve antibody titers without sacrificing antigen specificity. Based on these results, a 30 wt% ratio with high peptide density was used in all future assays and is referred to as IVLN or IVLN-HER2.

[0103] To effectively assess the importance of virus-like features for antigen-specific antibody production applications, we next asked how IVLN performed compared to conventional nanoparticle systems. For this comparison, we used lipid-coated iron oxide nanoparticles (lipid-IONPs) as a control (see Figure 1 ). This nanoparticle has a 30nm iron oxide nanoparticle core and a functionalized DSPE-PEG(2000)-maleimide shell that facilitates easy peptide conjugation. Remarkably, lipid-IONPs have similar material properties in terms of hydrodynamic particle size (69±1nm), PDI (0.20±0.01nm) and maximum number of peptides per particle (2323±394 peptides per IVLN). However, as a traditional nanoparticle system, lipid-IONPs have a smooth PEGylated surface and uniform peptide distribution. In summary, we believe that a side-by-side comparison of IVLNs and lipid-IONPs will provide valuable insights into the role of virus mimics for in vivo function.

[0104] As described above, BALB / c mice were immunized with 50 μg of HER2 peptide plus 10 μg of cGAMP as adjuvant on day 0 and boosted on day 14. On day 24 (10 days after the first boost), sera were analyzed for total IgM, total IgG, antigen-specific IgG, and antigen-specific IgG isotypes IgG1 and IgG2a ( Figure 3 C). At this time point, no statistically significant difference in total IgM antibody production was observed (p = 0.09), but compared to mice treated with PBS and soluble HER2 peptide alone, a significant increase in total IgG was observed for lipid-IONP-HER2 and IVLN-HER2. More specifically, the total IgG of lipid-IONP-HER2 and IVLN-HER2 samples increased by approximately 7 times and 3 times, respectively, compared to PBS alone (p < 0.001; p < 0.001) and soluble HER2 (p < 0.01; p < 0.01). For total IgG, no statistically significant difference (p = 0.98) was observed between lipid-IONP-HER2 and IVLN-HER2. However, when antigen-specific IgG was assessed, significant differences were observed between IVLN-HER2 and lipid-IONP-HER2. Specifically, it was determined that compared to lipid-IONP-HER2, IVLN-HER2 had an 18.5-fold higher antigen-specific IgG titer (39,500 vs. 2,140; p<0.001), a 15-fold higher antigen-specific IgG1 titer (9,600 vs. 640; p<0.001), and a 4.5-fold higher antigen-specific IgG2a titer (5,760 vs. 1,280; p<0.05). Furthermore, compared directly to soluble HER2 peptide, IVLN-HER2 produced a 9-fold higher antigen-specific IgG titer (39,500 vs. 4,300; p<0.001), a 48-fold higher antigen-specific IgG1 titer (9,600 vs. 200; p<0.001), and a 72-fold higher antigen-specific IgG2a titer (5,760 vs. 80; p<0.01).

[0105] Lymph node delivery and nanoparticle distribution

[0106] Having determined that antigen-specific antibodies against IVLN-HER2 were significantly enhanced compared to conventional nanoparticles and soluble controls, we next set out to answer the question. To begin a mechanistic assessment of the effect of the virus-mimetic properties on antibody production, we first evaluated the ability of these materials to be delivered to and retained in lymph nodes. As sites with dense populations of antigen-presenting cells and lymphocytes, lymph nodes are ideal targets for immune activation. Specifically, lymph nodes are the primary sites of B cell activation and germinal center formation, ultimately responsible for initiating antigen-specific IgG antibody production. In the context of virus mimicry, lymph nodes are known to play a crucial role in combating and controlling the spread of viruses throughout the body. This function is the result of unique physiological features developed for viral recognition and virus-specific immune activation. For example, subcapsular sinus macrophages are a highly specialized macrophage phenotype responsible for viral uptake and direct presentation to B cells, promoting targeted viral clearance through the production of antigen-specific antibodies. Therefore, studying lymph node delivery and immune cell interactions within the lymph nodes is essential for evaluating the mechanisms of virus-mimetic nanoparticle function.

[0107] Lymph node delivery of nanoparticles and peptides was quantified following administration via subcutaneous elbow immunization, which was determined to be the most efficient delivery route. Based on a previously established protocol, the kinetics of nanoparticle delivery to lymph nodes (popliteal and inguinal) was determined by quantifying Fe and Au in excised lymph nodes using ICP-MS. Based on this analysis, it was determined that although the tmax for both IVLN-HER2 and lipid-IONP-HER2 was 3 hours after administration, the percentage of the initial nanoparticle dose delivered was 5.1±1.6% and 1.9±0.8% for IVLN-HER2 and lipid-IONP-HER2, respectively (p<0.05) ( Figure 4 A). Over 72 hours, IVLN-HER2 demonstrated a 2.8-fold increase in overall exposure based on AUC compared to lipid-IONP-HER2 (p<0.05). In addition to the observed nearly 3-fold increase in delivery, nanoparticle retention within the lymph nodes was estimated to be 65% for IVLN-HER2 compared to 48% for lipid-IONP-HER2. In addition to direct quantification of nanoparticle delivery by raw elemental analysis, peptide delivery to the lymph nodes at 3 hours was also confirmed by semi-quantitative analysis of fluorescence intensity in excised popliteal and inguinal lymph nodes using IVIS imaging ( Figure 4 B). IVIS imaging showed that IVLN-HER2 resulted in a 4.3-fold increase in peptide delivery compared to lipid-IONP-HER2 and soluble HER2 peptide (p<0.001; p<0.001), which had no statistically significant difference in delivery (p>0.99).

[0108] After quantifying lymph node delivery, we next asked how IVLN and IONPs were distributed within the lymph nodes at the cellular level. To answer this question, fluorescently labeled nanoparticles were delivered to the lymph nodes and flow cytometry was applied to identify the percentage of nanoparticle-positive cells among total cells of different phenotypes. More specifically, lymph node antigen-presenting cells (subcapsular sinusoidal macrophages and dendritic cells) and lymphocytes (B cells and T cells) were identified and nanoparticle uptake was assessed. This analysis showed that after 3 hours and relative to lipid-IONP-HER2, IVLN-HER2 had a 1.7-fold improvement in subcapsular sinus macrophage uptake (86.7±2.4% vs. 52.2±2.8%, p<0.001), a 1.8-fold improvement in dendritic cell uptake (75.2±1.9% vs. 41.1±5.8%, p<0.001), and a 3.4-fold improvement in B cell uptake (63.9±1.1% vs. 19.7±2.2%, p<0.001). Figure 4 C).

[0109] Finally, we asked what the mechanism for improved cellular uptake is. Based on previous studies, nanoparticle surface topology was positively correlated with improved cellular uptake. Given the rough surface topology of IVLN compared to the smooth PEGylated surface of lipid-IONPs, we hypothesized that cellular uptake depends on the degree of AuNP loading on the IVLN surface. To test this hypothesis, in vitro cellular uptake was assessed using RAW264.7 macrophages, DC2.4 dendritic cells, and primary B cells isolated from mouse spleen. From this analysis, it was determined that regardless of the cell type, cellular uptake was determined by the degree of AuNP loading on the IVLN surface and was matched with an exponential function (RAW264.7, R 2 =0.902; DC2.4, R 2 =0.893; B cells, R 2More specifically, compared to treatment with the IONP-polymer core alone, IVLN-HER2 at a 30% weight / weight loading ratio improved cellular uptake by 6-fold (30.3±1.1 pg Fe / cell vs. 4.7±0.9 pg Fe / cell, p<0.001), 4.5-fold (12.5±1.4 pg Fe / cell vs. 2.9±0.9 pg Fe / cell, p<0.001), and 4-fold (2.0±0.4 pg Fe / cell vs. 0.5±0.2 pg Fe / cell, p<0.001) for RAW264.7 macrophages, 4.7±0.9 pg Fe / cell vs. DC2.4 dendritic cells, and 4-fold (2.0±0.4 pg Fe / cell vs. 0.5±0.2 pg Fe / cell, p<0.001) for primary B cells, respectively. Notably, compared directly with the lipid-IONP-HER2 control group, IVLN-HER2 improved cellular uptake by 3-fold (30.3±1.1 pg Fe / cell vs. 11.8±1.3 pg Fe / cell, p<0.001) in RAW264.7 macrophages, 3-fold (12.5±1.4 pg Fe / cell vs. 3.9±1.9 pg Fe / cell, p<0.01) in DC2.4 dendritic cells, and 2-fold (2.0±0.4 pg Fe / cell vs. 0.8±0.4 pg Fe / cell, p<0.01) in primary B cells.

[0110] Virus-like characteristics are ideal material properties for delivery vehicles, immunostimulants, and cellular uptake carriers that are urgently needed for the advancement of rationally designed and engineered nanotechnology in biotechnology and medical applications. Therefore, here we report the development and evaluation of virus-like particle alternatives (inorganic virus-like nanoparticles (IVLNs) and IVLN-peptides) based on a more holistic virus-mimicking material design approach. In certain embodiments, IVLNs are composed of a mixed Au@Fe core-satellite nanoparticle system that uses a 16nm iron oxide nanoparticle core (IONP-polymer) coated with a diblock polymer containing polysiloxane and a 2.5nm gold nanoparticle satellite (AuNP). Based on the formulation conditions, IVLNs can be produced with variable surface topology, antigen density, and antigen spatial resolution. In addition, IVLNs have optimal particle size, shape, and surface charge for efficient lymph node delivery and retention. Therefore, these properties inform the virus-like characteristics and functional potential of IVLNs.

[0111] Our results demonstrate that the virus-like features are indeed significant for B cell activation and antigen-specific antibody production. Specifically, by manipulating the spatial distribution of AuNPs on the IVLN surface and the peptide density regions confined to those AuNPs, the median antigen-specific IgG antibody titer could be increased from 6,300 to 32,500 (a 5-fold increase). This result was further confirmed by directly comparing IVLN with lipid-IONPs. Remarkably, antigen-specific IgG, IgG1, and IgG2a titers were determined to be 18.5-fold, 15-fold, and 4.5-fold higher for IVLN-HER2 compared to lipid-IONP-HER2, respectively. Given the highly comparable hydrodynamic particle size, surface charge, core shape, and peptide loading per unit nanoparticle, any quantifiable differences in antibody production by these two structures can be attributed to the increased virus-like features. Mechanistically, this virus-like feature leads to enhanced antigen-specific antibody production as a result of improved delivery and retention in lymph nodes due to improved immune cell uptake, thereby promoting a significant increase in overall B cell activation and germinal center formation.

[0112] Example 2

[0113] Virus-like nanoparticles for antigen-specific antibody production

[0114] This example describes the production and use of antibody-producing virus-like nanoparticles. To achieve the virus-like structure, we engineered IVLNs using a controllable and robust self-assembly process to resemble the viral spike protein, with a spike antigen cluster topology, defined distances between clusters, and high localized antigen density on the spike.

[0115] To test viral functional mimicry of IVLN, we assessed IVLN activation of antigen-specific B cells and persistent antigen-specific antibody responses. We selected well-known HER2 B cell epitopes with overlapping CD4 T cell epitopes because an in vivo model for assessing persistent antibody responses is readily available by monitoring HER2 tumor growth without the need for a biosafety level 4 laboratory. Three important viral-like functions of IVLN were assessed: (1) antigen delivery efficiency and B cell compartment uptake in secondary lymph nodes; (2) antigen-specific B cell activation by the varying density and spatial arrangement of antigens on the IVLN surface; and (3) follicular helper T cell activation in germinal centers for B cell activation and persistent antibody responses. The persistent function of antigen-specific antibodies was assessed in vivo to inhibit HER2 cancer growth.

[0116] Materials and methods

[0117] Material

[0118] All reagents were used as received from commercial sources without further purification, except for γ-methacryloxypropyltrimethoxysilane (98%) which was purified by distillation under reduced pressure, and 2,2-azobis(isobutyronitrile) (98%) which was purified by recrystallization from ethanol. Iron (III) oxide (FeO(OH), hydrated, catalyst grade, 30-50 mesh), oleic acid (technical grade, 90%), iron (II) ammonium sulfate hexahydrate (ACS reagent, 99%), 1-octadecene (technical grade, 90%), anhydrous tetrahydrofuran (THF, 99.8%), carbon disulfide (99.9%), magnesium turnings (>99.5%), 2-chloro-2-phenylacetyl chloride (CPAC, 90%), poly(ethylene oxide) monomethyl ether (PEO), anhydrous dioxane (99.8%), dimethylformamide (DMF, 99.9%), dimethyl sulfoxide (DMSO, 99.9%), o-phenanthroline monohydrate (ACS reagent, 99%), hydroquinone (ACS reagent, 99%), sodium sulfide, chloroauric acid, nitric acid (ACS reagent, 70%), and hydrochloric acid (ACS reagent, 37%) were purchased from Sigma-Aldrich. Mouse uncoated IgG and IgM total ELISA kit, 1-Step Ultra TMB-ELISA substrate solution, HRP-conjugated goat anti-mouse IgG1 secondary antibody, HRP-conjugated goat anti-mouse IgG2a secondary antibody, Nunc Immobilizer amino 96-well ELISA plates, BupH bicarbonate buffer pack (coating buffer), Pierce protein-free PBS tween blocking buffer, 20× PBS-tween wash buffer, Geneticin (G418) selective antibiotic, Invitrogen eBioscience fixable viability dye eFluor 780, and Molecular Probes Streptavidin Alexa Fluor 647 conjugate were obtained from Thermo Fisher Scientific. HRP-conjugated goat anti-mouse IgG secondary antibody, Zombie UV fixable viability kit, FITC anti-mouse CD19, PE / Dazzle 594 anti-mouse IgD, Alexa Fluor 647 anti-mouse / rat GL7 antigen, BrilliantViolet 421 and PE / Dazzle 594 anti-mouse / human CD45R / B220, FITC anti-mouse CD95, Brilliant Violet421 anti-mouse / human CD11b, FITC anti-mouse CD169, and PE goat anti-mouse IgG secondary antibodies were purchased from BioLegend.HER2 peptides (CDDDPESFDGDPASNTAPLQPEQLQ (SEQ ID NO: 1), biotin-PESFDGDPASNTAPLQPEQLQ (SEQ ID NO: 2), and CDDDPESFDGDPASNTAPLQPEQLQGGGK (SEQ ID NO: 3) were custom synthesized by LifeTein. Iron oxide nanoparticles (30 nm) stabilized by oleic acid in chloroform were purchased from Ocean Nanotech. DSPE-PEG(2000) and DSPE-PEG(2000)-maleimide were obtained from Avanti Polar Lipids. 2'3'-cGAMP was obtained from InvivoGen. Fluorescein was purchased from MP Biomedicals. Sulfo-Cy5.5 NHS ester was obtained from Lumiprobe. Microvette 500 Z-Gel serum collection vials with coagulation factors were obtained from Sarstedt. Matrigel basement membrane matrix was purchased from Corning. Gold and iron ICP standards were purchased from Fluka Analytical.

[0119] mice

[0120] All animal experiments were performed according to protocols approved by the University of Michigan Committee on Use and Care of Animals (UCUCA). BALB / c mice, 5-7 weeks old, were purchased from Charles River Labs.

[0121] cell

[0122] All cells were maintained at 37°C, 5% CO2 / 95% air atmosphere and approximately 85% relative humidity. D2F2 / E2 cells ((83)) were cultured in complete DMEM high glucose supplemented with 10% NCTC 109 medium, 1% L-glutamine, 1% MEM non-essential amino acids, 0.5% sodium pyruvate, 2.5% sodium bicarbonate, 1% penicillin / strep, 5% cosmic calf serum, 5% fetal bovine serum, 500 μg / mL geneticin and 50 μM 2-mercaptoethanol. RAW264.7 macrophages were cultured in complete RPMI-1640 medium supplemented with 10% fetal bovine serum, 1% L-glutamine, 1% MEM non-essential amino acids, 1% sodium pyruvate and 1% penicillin / strep. Primary B cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum.

[0123] Formulation and Characterization of Inorganic Virus-Like Nanoparticles (IVLN)

[0124] IVLNs were formulated generally as in Example 1. The final Au to Fe ratio of the formulated IVLNs was quantified by inductively coupled plasma mass spectrometry (ICP-MS) using a Perkin-Elmer Nexion 2000 based on a previously reported protocol modified for ICP-OES analysis (78). IVLN formulations were imaged by s-scanning transmission electron microscopy (STEM) using a JEOL 2100F with a CEOS probe calibrator. The true particle size of AuNPs, IONPs, and IVLNs was quantified using ImageJ software. The volume-weighted hydrodynamic particle size, polydispersity index, and zeta potential of all formulations were evaluated in milliQ water at 25°C using dynamic light scattering and phase analysis light scattering, respectively, using a Malvern Zetasizer Nano-ZS.

[0125] Lipid-coated iron oxide nanoparticle formulations (IONPs)

[0126] Lipid-coated iron oxide nanoparticles were prepared as follows. DSPE-PEG(2000)-maleimide (10 mg) was added to 1 mg of 30 nm iron oxide nanoparticles stabilized by oleic acid in chloroform while gently mixing. The resulting solution was subjected to solvent rotary evaporation to remove all chloroform and form a film. Simultaneously, this film and 100 mM PBS at pH 7.4 were heated to 75°C in an oven. Once the temperature was reached, hot PBS was quickly added to the film and immediately mixed vigorously to promote film hydration. The resulting nanoparticle solution was stored at 4°C to promote lipid self-assembly. Free phospholipids were removed by magnetic separation overnight at 4°C using an EasySep magnetic separator device (StemCell).

[0127] IONP-HER2 and IVLN-HER2 formulations

[0128] HER2 peptide was conjugated to both IONP and IVLN via thiol-mediated chemistry. Specifically, IONP-HER2 was formulated via maleimide chemistry and IVLN-HER2 was formulated via gold-thiol linkage. HER2 peptide was added to IONP at a weight ratio of 1.5x excess in milliQ and incubated overnight at 4°C. HER2 peptide was added to IVLN-HER2 at a weight ratio of 5x excess in milliQ and incubated overnight at 4°C. Both materials were purified by magnetic separation at 4°C overnight or by centrifugation at 10,000xg for 30 minutes at 4°C. In the presence of nanoparticles, peptide loading was determined using a modified fluorescein peptide quantification assay (Ex / Em: 390 / 465nm, Biotek Cytation 5) (86). Quantification was performed using a standard curve to illustrate the quenching effect, where the peptide concentration was increased with the normalized concentration of nanoparticles (IONP or IVLN).

[0129] Immunization and serum collection

[0130] At day 0, regardless of the type of formulation, mice were immunized with the equivalent of 50 μg or 5 μg of HER2 peptide plus 10 μg of cGAMP. Subsequently, on day 14, mice were boosted twice (day 14 and day 28) at two-week intervals with 50% of the original dose of antigen and adjuvant. To assess serum antibody titers, blood was collected by submandibular puncture 10 days after each immunization (days 10, 24, and 38). Serum was separated from whole blood by centrifugation at 10,000 x g for 5 minutes at 25°C using a Microvette 500 Ser-Gel collection container with a coagulation activator.

[0131] Enzyme-linked immunosorbent assay (ELISA)

[0132] Based on the scheme (Thermo Fisher) recommended by the manufacturer, the total IgG and total IgM ELISA kits uncoated by mice were used to carry out the absolute quantification of total IgG and total IgM antibodies. Based on the indirect ELISA scheme previously established and slightly modified, antigen-specific IgG, IgG1 and IgG2a antibody titers were quantitatively measured (87). Specifically, by overnight incubation at room temperature under exposure to light, Nunc Immobilizer amino immunoassay plates were used, and HER2 peptide (200 μL, 100 μg / mL in 100mM carbonate buffer, pH 9.4) was chemically conjugated to ELISA plates by terminal amine groups. After overnight incubation, ELISA plates were washed 3 times with 100mM PBS with 2% Tween-20 of pH 7.4. Subsequently, ELISA plates were blocked overnight at 4°C with 300 μL ELISA blocking agents (PBS blocking buffer without Pierce protein). After blocking, ELISA plates were washed 3 times. Serum samples containing primary antibody were serially diluted (10 1 -10 8 fold) and added to each well in a total amount of 200 μL and incubated at room temperature for 2 hours. After adding the sample, the ELISA plate was washed 3 times. 500-fold diluted anti-IgG-HRP, anti-IgG1-HRP or anti-IgG2a-HRP was added to each well in 100 μL and incubated at room temperature for 1 hour. After 1 hour, the ELISA plate was washed 5 times. Next, 100 μL of 1-Step UltraTMB substrate solution was added to each well and incubated and developed for 15-20 minutes at room temperature with gentle stirring. After 15-20 minutes, the color development was stopped by adding 100 μL of 100 mM sulfuric acid. The color development was quantified by using the absorption spectrum at 450 nm using a BioTek Cytation 5 plate reader. Antibody titer (88) was determined by any absorbance signal that was greater than the PBS control absorbance signal plus 3 standard deviations at a given dilution factor.

[0133] Quantification of nanoparticle delivery to lymph nodes in vivo

[0134] Mice were injected subcutaneously in the left elbow joint with IONP or IVLN at a dose of 200 μg Fe per mouse. At the indicated time intervals, mice were sacrificed and the lymph nodes of interest were dissected for ex vivo analysis. The extent of nanoparticle delivery to lymph nodes was quantified using ICP-MS based on a previously reported protocol (77).

[0135] Quantification of peptide delivery to lymph nodes in vivo

[0136] To facilitate quantitative delivery of peptides to lymph nodes, lysine-terminally modified HER2 peptides were chemically conjugated to sulfo-Cy5.5 NHS ester. This conjugation was performed with a 5-fold molar excess of sulfo-Cy5.5 NHS ester relative to the HER2 peptide. After the initial peptide conjugation was complete, IONP-HER2-Cy5.5 and IVLN-HER2-Cy5.5 were functionalized with Cy5.5 to enable easy purification of excess fluorescent dye by magnetic separation. After Cy5.5 functionalization, mice were injected as previously described. After 3 hours, the mice were sacrificed and the lymph nodes of interest were dissected for in vitro analysis by IVIS imaging. Regarding radiation efficiency, IVIS imaging was used for semi-quantification of peptide delivery.

[0137] In vivo cellular uptake

[0138] IVLN-HER-Cy5.5 and IONP-HER2-Cy5.5 were injected subcutaneously in the left elbow joint with lipid-IONP or IVLN at 200 μg total Fe per mouse. At 3 hours and 24 hours, mice were sacrificed and the lymph nodes of interest were dissected for ex vivo analysis by flow cytometry. Lymph nodes were mechanically dissociated to prepare single cell suspensions. Single cell suspensions of lymph node cells were stained for analysis by flow cytometry using a MoFlo Astrios flow cytometer. Live cells (Zombie UV) were identified as B cells (B220 + ) or subcapsular sinus macrophages (CD169 高 CD11b + ), and positive nanoparticle interactions (Cy5.5) were assessed. Flow cytometry data were analyzed by FCS expression.

[0139] In vitro cellular uptake

[0140] The uptake of IVLN-HER2 and IONP-HER2 cells was assessed in RAW264.7 macrophages, dendritic cells (DC 2.4) and primary B cells isolated from mouse spleen using the EasySep mouse B cell isolation kit. In blank RPMI culture medium, at 37°C, 5% CO2 / 95% air atmosphere and about 85% relative humidity, the nanoparticle samples were incubated with cells for 18 hours with 50 μg / mL Fe. After 18 hours, the cells were lifted by scraping the cells and washed three times with PBS. After the washing step, the resulting cell pellet was resuspended in 1 mL of PBS, the cells were counted, and then digested in 1 mL of aqua regia (nitric acid: hydrochloric acid molar ratio of 1:3) for analysis by ICP-MS.

[0141] Mass cytometry (CyTOF) for analyzing all immune cells

[0142] The fixed and frozen cell suspension was thawed on ice. As previously described (89,90), the samples were stained and prepared for CyTOF analysis using an optimized mixture of 40 metal-conjugated antibodies designed to identify the primary and secondary immune cell subsets in the lymph nodes. After collection on CyTOF II (Fluidigm, San Francisco, CA), the samples were normalized to internal bead standards. Cell subsets were identified by gating using FlowJo software. Based on the expression of marker genes in different subsets of immune cells, SPADE was used to perform an overall analysis for unsupervised cluster analysis.

[0143] Antigen-specific B cells and germinal center flow cytometry

[0144] Mice were immunized as previously described. On the 10th day, mice were sacrificed and lymph nodes were dissected for in vitro analysis by flow cytometry. Based on the previously established protocol and with slight modifications, antigen-specific B cell analysis (63) was completed using tetramer staining. In the absence of further purification, HER2 / neu peptide tetramers were prepared by mixing biotinylated HER2 peptide with AlexaFluor647 labeled streptavidin at a 4:1 molar ratio for 1 hour at room temperature. Antigen-specific B cell populations were identified using CD19, and HER2-peptide tetramers were identified using flow cytometry. The following markers B220, IgD, GL7, and CD95 (B220 + IgD 低 GL7 + CD95 + ) Identification of germinal center B cell populations.

[0145] Cancer Research

[0146] Forty-nine days after the first immunization, 2.5 × 10 5 Mice were subcutaneously inoculated with 2.5 × 10 D2F2 / E2 cells in 100 μL. 6 D2F2 / E2 cells were prepared at 10 cells / mL and mixed with an equal volume of Matrigel matrix. Tumor size was quantified by caliper measurement every 7 days. Volume = (width) 2 Tumor volume was calculated by multiplying length / 2. The endpoint was determined using a terminal disease scoring system; mice with a terminal disease score greater than 6 were euthanized by CO2 asphyxiation.

[0147] statistics

[0148] Unless otherwise stated, data are expressed as mean ± standard deviation (SD). Comparisons between the two groups were performed using an unpaired Student's t-test. The mean values of the multiple groups were compared using one-way analysis of variance (ANOVA) followed by post hoc Tukey's paired comparisons. All probability values were two-sided, and values of p < 0.05 were considered statistically significant. Statistical analysis was performed using the GraphPad Prism 7 software package.

[0149] result

[0150] Design of Inorganic Virus-Like Nanoparticles (IVLN) that Mimic Virus-Like Spike Structures

[0151] We have developed a controllable and robust method to fabricate IVLNs using a self-assembly process. To obtain a virus-like spike topology, AuNPs (2 nm) were attached to the surface of IONPs (15 nm) to produce IVLNs ( Figure 7 A) Attachment of AuNPs and IONPs is achieved through self-assembly via interactions between the reactive AuNP surface and the free siloxane moieties present in the polymer used to coat the IONPs. This approach is controllable and robust for large-scale manufacturing.

[0152] IONPs were synthesized by thermal decomposition to produce 15 nm spherical cores stabilized by oleic acid in chloroform. To achieve aqueous stabilization, IONPs were coated with a diblock copolymer containing poly(siloxane) and poly(ethylene glycol) based on a previously reported procedure (51). Ultrasmall gold nanoparticles or satellites (AuNPs) of approximately 2–3 nm in size were prepared by reducing chloroauric acid in aged sodium sulfide using a modified precipitation method (53). The AuNP solution was added to the IONP solution at a defined weight ratio and incubated overnight at 4°C to allow self-assembly of IVLNs. In order to control the number of virus-like spike structures on IVLNs to 4–14 ( Figure 7 B, Figure 7 C), the ratio of AuNPs to IONPs was adjusted to 10%, 20%, and 30% AuNPs / IONP, resulting in IVLNs with 4±2, 9±3, and 13±5 AuNPs per IVLN as measured by ICP-MS (54,55). Figure 7 B). Scanning transmission electron microscopy (STEM) confirmed the virus-like structure of IVLN ( Figure 7 C). High-angle annular dark-field (HAADF) image of a single IVLN (with 14 AuNPs) shows a close resemblance to a virus-like structure ( Figure 8 C).

[0153] IVLN conjugated to a non-capsid antigenic peptide resembles the spike protein structure of the virus with three virus-like features

[0154] To simulate an antigenic structure similar to the viral S protein, three features were used: the topology of the spike antigen clusters, the optimal distance between antigen clusters (5nm), and the local high antigen density on the spike.

[0155] The spike antigen cluster topology was achieved by conjugating only the antigenic peptide to the spike AuNP of IVLN, but not to the polymer of the IONP core. We used a non-viral capsid as well as a well-known HER2 B-cell epitope (56-58) with an overlapping CD4 T-cell epitope (CDDD-PESFDGDPASNTAPLQPE QLQ-(GGK). We chose the HER2 B-cell epitope as a proof-of-concept study to investigate viral-like structure and functional mimicry of IVLN because the in vivo model is easy to test antibody function by monitoring tumor growth without the need for a biosafety level 4 laboratory. Conjugation of the HER2 peptide (with a cysteine at the N-terminus) to the spike AuNP was achieved only by S-Au reaction on IVLN (and not on the adjacent polymer coating on IONP). Figure 7 F, Figure 1 IONPs coated with polymer alone without AuNPs were used as controls. High levels of peptide conjugation were observed for IVLNs with AuNPs, but not on IONP cores alone ( Figure 7 F, black symbols). In addition, a positive correlation was observed between peptide loading and the number of AuNPs (R = 0.95). These results indicate that, although there is a low level of nonspecific association of HER2 peptide with the INOP core (approximately 12%), peptide conjugation is associated with AuNPs, which enables the antigen spike cluster topology on IVLNs; these virus-mimicking properties cannot be achieved by conventional nanoparticles with only a uniform distribution of antigens on the surface ( Figure 7 I).

[0156] To control the distance between the two spike antigen clusters to 5–10 nm, which is the ideal distance for B cell receptor (BCR) cross-linking and activation (41, 59), we used different AuNP / IONP ratios to adjust the number of AuNPs on the IVLN surface to 14, resulting in distances between 5.1 and 6.3 nm ( Figure 7 D).

[0157] To control the antigen density on the spikes to highly localize, we conjugated different amounts of HER2 peptides on IVLNs at AuNP / IONP ratios of 0%, 10%, and 30% (which correspond to 0, 4, and 13 AuNPs on the IVLN surface, respectively). Figure 7F). Peptide loadings were 232 ± 73, 888 ± 42, and 1954 ± 157 peptides per IVLN (227 ± 5 peptides per AuNP, Figure 7 F). Therefore, IVLN reaches every μ 2 The density of 20,000–25,000 antigen clusters is comparable to the antigen density reported for VLPs (9) (e.g., hepatitis B virus, approximately 20,000 antigens) ( Figure 7 E).

[0158] To mimic the optimal virus size and surface charge (32,34), IVLN particles were 50–60 nm in size, with a PDI of 0.2 and a zeta potential of −16 mV ( Figure 7 H). Furthermore, under in vivo relevant serum conditions, IVLN-HER2 was shown to be stable between 12 and 24 hours.

[0159] To effectively evaluate these three features of the virus-mimicking structure of IVLN, a conventional lipid-coated IONP (IONP-HER2) with similar size, charge, and peptide density on the surface (2323 ± 394 peptides per IONP, but uniform antigen distribution) was generated as a control ( Figure 7 G, Figure 7 I). IONP-HER2 has a 30 nm IONP core and a functionalized DSPE-PEG(2000)-maleimide shell that facilitates easy peptide conjugation. IONP-HER2 has similar material properties in terms of volume-weighted hydrodynamic particle size (68 ± 5 nm), PDI (0.22 ± 0.02 nm), and maximum number of peptides per particle (2323 ± 394 peptides per IONP). Figure 7 G). Therefore, a side-by-side comparison of IVLN-HER2 and IONP-HER2 will provide valuable insights into the role of viral mimetic functions.

[0160] IVLN-HER2 enhanced HER2-specific antibody production 7- to 18-fold compared to INOP-HER2

[0161] Virus-mimetic nanoparticles have been widely used for in vitro and in vivo applications, but the most important application of virus-like structures and functions is the activation of B cells to produce antigen-specific antibodies (2,6,13,57,60-62). Therefore, we first tested whether IVLN-HER2 induced the production of antigen-specific IgG in vivo against a non-capsid oncogenic human HER2-specific peptide (HER2).

[0162] Compared with conventional lipid-coated IONP-HER2 with similar HER2 peptide density (about 2000 peptides / INOP, uniform antigen distribution), the optimized IVLN (about 14 spike antigen clusters, 5-6 nm distance between two antigen clusters, about 2000 peptides / IVLN, 150 peptides / AuNP) was used to immunize BALB / c mice to produce HER2-specific antibodies. IVLN-HER2 and IONP-HER2 have similar sizes under TEM (30 nm) and DLS (65 nm). On day 0, the same dose of HER2 peptide in all groups was used for immunization of BALB / c mice (5ug or 50ug peptide, 10μg cGAMP as adjuvant) and boosted twice at 14-day intervals ( Figure 8 A). The HER2 of two dosages (5 μg and 50 μg) is used for carrying out immunization.After two booster immunizations (the 38th day), complete serum analysis is carried out, because this time point is determined to react the strongest, and therefore the most relevant.After two booster immunizations (the 38th day), ELISA is used to analyze the antibody response of total IgM, total IgG, HER2-specific IgG and HER2-specific IgG isotype (IgG1 and IgG2a).

[0163] At low doses, IVLN-HER2 (5 μg) produced 8-fold higher HER2-specific IgG titers, 18-fold higher HER2-specific IgG1 titers, and 13-fold higher HER2-specific IgG2a titers compared to IONP-HER2. In addition, IVLN-HER2 produced 14-fold higher Ag-specific IgG titers, 7-fold higher Ag-specific IgG1 titers, and 14-fold higher Ag-specific IgG2a titers compared to soluble HER2 peptide ( Figure 8 B).

[0164] Similarly, at high doses (50 μg), IVLN-HER2 (50 μg) enhanced antigen-specific IgG titers by 12-fold, antigen-specific IgG1 titers by 8-fold, and antigen-specific IgG2a titers by 14-fold compared to soluble HER2 peptide. Compared to INOP-HER2, IVLN-HER2 (50 μg) enhanced antigen-specific IgG titers by 4-5-fold, antigen-specific IgG1 titers by 3-fold, and antigen-specific IgG2a titers by 5-fold ( Figure 8 C). No statistically significant differences in total IgM and total IgG antibody production were observed between any treatment groups. Overall, these data suggest that the virus-like properties of IVLN are more efficient for the production of antigen-specific antibodies.

[0165] The number of spike antigen clusters, the distance between two antigen clusters, and the local antigen density on the IVLN affect its ability to produce antigen-specific antibodies.

[0166] Virus-like features, including different numbers of spike antigen clusters, different distances between clusters, and different local antigen densities, are important for B cell activation via multivalent B cell receptor cross-linking (9,40,41). Therefore, we tested whether different IVLN-HER2 virus-mimicking features affect anti-HER2 antibody production in BALB / c mice. We immunized mice on day 0 and boosted them once on day 14 with IVLN-HER2 with different numbers and distances of spike clusters but with the same amount of HER2 peptide (50 μg HER2 peptide, 10 μg cGAMP as adjuvant). Figure 9 A).

[0167] We first evaluated the effect of the number of spike antigen clusters and the distance between the antigen clusters on the production of HER2-specific IgG antibodies using two different AuNP / IONP ratios: 10% and 30%, which correspond to the ratio of the number of AuNPs on IONPs to 4 and 14, and the distance between the AuNPs to be approximately 15nm and approximately 5nm. Although the same dose of HER2 antigen was used in the immunization, the HER2-specific IgG titer (64,500) generated by IVLN (with 14 antigen clusters and an optimal distance between clusters of 5nm) was 6 times higher than that of IVLN (with 4 antigen clusters and an optimal distance between clusters of 15nm) which generated a HER2-specific IgG titer of 10,540. Figure 9 B).

[0168] We then evaluated the effect of local antigen density on IVLN on HER2-specific antibodies. Although the same HER2 antigen dose was used in the immunization, the titer of antigen-specific antibodies generated by IVLN (14 antigen clusters with a distance between clusters of approximately 5 nm and a high density of 150 peptides / cluster) was 4 times higher than that of antigen-specific antibodies generated by IVLN (with 4 antigen clusters with a distance between clusters of 15 nm and a low antigen density of 30 peptides / cluster) ( Figure 9 B). The data indicate that the number of antigen clusters (14 clusters in this example), the distance between antigen clusters (5-6 nm in this example), and the local antigen density (2000 peptides / IVLN, approximately 150 peptides / AuNP in this example) result in highly HER2-specific antibodies.

[0169] IVLN-HER2 increased antigen-specific B cell activation and GC formation 6-fold compared with IONP-HER2

[0170] To generate a high antibody response, antigen-specific B cell activation in the germinal center is required (9,40,41). Therefore, we tested the effect of IVLN-HER2 on GC formation and antigen-specific B cells in the draining lymph nodes of immunized BALB / c mice. HER2-specific B cells in the lymph node immune response were measured using fluorescently labeled streptavidin HER2 peptide tetramer staining (63) ( Figure 10 A). Antigen-specific B cells were identified as double positive for CD19 and HER2 peptide tetramers.

[0171] The data showed that IVLN-HER2 generated 6-fold more HER2-specific B cells in the lymph nodes compared to the IONP-HER2 immunization group (3%) ( Figure 10 B). No differences in HER-specific B cells were detected in mice immunized with IONP-HER2 versus HER2 peptide. In addition, we evaluated GC formation at day 10 (the peak of the GC response) after primary immunization with IVLN-HER2, IONP-HER2, and soluble HER2 peptide by flow cytometric analysis. GC B cells were identified as B220 + IgD 低 cells, which are double positive for CD95 and GL-7 markers (64,65) ( Figure 10 C). IVLN-HER2 resulted in a 2.6-fold and 8-fold increase in GC formation compared to the IONP-HER2 and HER2 peptide immunization groups ( Figure 10 D) Importantly, the 3% activation of antigen-specific B cells and the greater than 17% germinal center formation observed with any other delivery system are rare. These data suggest that the viral mimetic properties of IVLN-HER2 uniquely enhance antigen-specific B cells compared to traditional nanodelivery systems.

[0172] CyTOF analysis of immune cells reveals that IVLN-HER2 enhances Tfh-dependent B cell activation in lymph nodes

[0173] In the germinal center (GC), B cell activation requires interaction with follicular T cells to generate long-lived plasma cells (PCs), which in turn generate long-lasting antibody responses. Therefore, CyTOF analysis was used to assess T cell-dependent B cell activation by evaluating immune cells in the lymph nodes, including macrophages, dendritic cells, B cells, CD4+ and CD8+ T cells, and NK cells in the lymph nodes and spleen, following immunization with heavy metal-labeled antibodies from 40 manufacturers. (66-68)

[0174] Global analysis using SPADE showed that IVLN-HER increased GCs, plasma B cells, and follicular T cells in lymph nodes ( Figure 11 A, Figure 11 B), but no other significant changes in immune cells in lymph nodes and spleen were shown. Detailed analysis showed that IVLN-HER2 stimulated more germinal center B cells (CD19+ / GL7+ or B220+ / GL7+) compared with the IONP-HER2 immune group ( Figure 11 C), follicular helper T cells (Tfh) (CD4+ / CXCR5+ / PD-1+) ( Figure 11 D) and plasma cells (PC) ( Figure 11 E), which is essential for antibody secretion. These data provide strong evidence that IVLN-HER2 induces Tfh-dependent B cell activation in lymph node GCs, explaining why IVLN-HER2 produces higher titers of antigen-specific antibodies.

[0175] IVLN-HER2 improves lymph node delivery efficiency and B cell compartment uptake compared to IONP-HER2

[0176] Efficient antigen delivery to lymph nodes is a prerequisite for effective B cell activation and antibody responses. We first assessed the delivery efficiency and retention of IVLN-HER2 in lymph nodes (69,70). Second, within the lymph nodes, we determined whether IVLN-HER2 could specifically target the B cell compartment, as lymph nodes are the primary site of B cell activation and germinal center formation that are ultimately responsible for initiating antigen-specific IgG antibody production (71-73). Third, we also tested whether IVLNs have a virus-like cell distribution pattern within the lymph nodes. As sites with dense populations of antigen-presenting cells and lymphocytes, lymph nodes are known to be crucial in virus sequestration and directed immune activation (74,75). This function is a result of unique physiological characteristics developed for virus recognition and virus-specific immune activation. For example, subcapsular sinus macrophages are a highly specialized macrophage phenotype that is responsible for virus uptake and direct presentation to B cells to promote directed viral clearance via the production of antigen-specific antibodies (75,76).

[0177] The delivery efficiency and retention of IVLN-HER2 in lymph nodes compared to IONP-HER2 were assessed using two different approaches (69,70): ICP-MS quantification of Fe in excised lymph nodes (77,78) and IVIS imaging of fluorescently labeled IVLN-HER2 peptide (79). max The total exposure of IVLN-HER2 was 3.5-fold higher than that of IONP-HER2 over 48 hours, based on the area under the curve (AUC). Figure 12A). In addition, fluorescently labeled peptides were used to monitor lymph node delivery of IVLN-HER2, IONP-HER2, and soluble HER2 peptides using IVIS imaging of excised popliteal and inguinal lymph nodes at 3 hours ( Figure 12 B) IVIS imaging showed that IVLN-HER2 resulted in a 4.3-fold improvement in lymph node delivery compared to IONP-HER2 and soluble HER2 peptide, with no statistically significant difference in delivery between IONP-HER2 and HER2.

[0178] We then tested whether IVLN-HER2 has a viral-like distribution within the lymph nodes, specifically in the subcapsular sinusoidal macrophage and B cell populations, compared to IONP-HER2 (72,73,76). Fluorescently labeled IVLN-HER2 was injected subcutaneously into the elbow joint, and flow cytometry was performed 3 hours after administration to identify cells positive for IVLN-HER2 or IONP-HER2 of different phenotypes. Subcapsular sinusoidal macrophages were identified as CD11b + CD169 高 Double positive and B cells were identified as B220 + (76,80). Compared with INOP-HER2, IVLN-HER2 improved uptake by subcapsular sinus macrophages by 1.7-fold and by B cells by 3.4-fold ( Figure 12 C).

[0179] In vitro, the intracellular uptake of IVLN-HER2 and IONP-HER2 was confirmed in RAW 264.7 macrophages and primary B cells isolated from mouse spleens. IVLN-HER2 improved cellular uptake in macrophages by 3-fold and in B cells by 2-fold compared to the IONP-HER2 control group ( Figure 12 D) Taken together, these data suggest that viral structural mimicry of IVLN improves lymph node delivery efficiency and distribution to the preferred B cell compartment in lymph nodes.

[0180] IVLN-HER2 induces antigen-specific antibodies with long-lasting functionality

[0181] We used a well-known B cell epitope of the HER2 antigen, so we could easily test the functionality of the induced antibodies in the established model by monitoring tumor growth after IVLN-HER2 immunization in vivo. The HER2 peptide on IVLN is the one that generates pertuzumab. (56) is a B cell epitope of pertuzumab, currently used to treat HER2+ breast cancer in humans (81,82). Therefore, we used IVLN-HER2 as a vaccine to prevent tumor growth in a HER2 breast cancer xenograft model (D2F2 / E2 murine breast cancer with high human HER2 expression) because of its in vivo prophylactic efficacy (83). Prophylactic tumor suppression was achieved by administering 2.5 × 10 per mouse on day 49 after primary immunization. 5 The initial subcutaneous flank inoculation of 10 cells was followed by three additional booster administrations at 14-day intervals. Figure 13 A). At 50μg (125±239mm 2 1843±661mm 2 , p<0.001)( Figure 13 B) and 5 μg (583 ± 392 mm 2 1843±661mm 2 , p<0.001)( Figure 13 C), IVLN-HER2 vaccination significantly inhibited tumor growth over 6 weeks, outperforming the INOP-HER2 and HER2 peptide groups alone. Furthermore, the prophylactic anticancer efficacy appeared to be directly correlated not only with the specificity of these endogenous antibodies for the D2F2 / E2 cell line, but also with the potency of the antigen-specific antibodies. These data suggest that antigen-specific antibodies against IVLN-HER2 have persistent function in vivo.

[0182] In order to prevent infection, B cell immunity against viral capsid protein antigens on the surface of the virus is very much needed. In such cases, the capsid antigens on the inactivated / attenuated live virus and the virus-like structure of virus-like particles (VLPs) using viral capsid proteins are highly effective for the active B cell immunity against viral infection (1-4). However, in the other three cases, B cell immunity against non-capsid protein antigens is also needed to resist bacterial toxins of deadly bacterial infections, oncogenic proteins of cancer, and peptide antigens for antibody production (20,21). However, it is very difficult to prepare virus-like particles using these non-capsid antigens and thus activate B cell immunity. B cell vaccines against bacterial toxins are very much needed to prevent deadly bacterial infections, such as anthrax (C.Anthracis) (anthrax) and clostridium botulinum (C.Botulinum) (20,21). These bacterial toxin B cell vaccines usually use toxinoids as antigens to strengthen neutralizing antibodies (20,21). Successful bacterial toxin vaccines are currently used to resist tetanus and diphtheria. However, the safety and efficacy of bacterial toxin-like B cell vaccines against Bacillus anthracis (anthrax) and Clostridium botulinum are two major concerns (20,21). Due to the highly toxic nature of these two toxins, the use of peptide antigens of these toxins as vaccines is preferred (22). However, the efficiency of peptide antigens in enhancing B cell immunity using nanoparticle delivery systems without viral structure mimicry is very low. In addition, B cell immunity against oncogenic antigens may have potential benefits in the prevention / treatment of cancer. For example, although the benefits / risks of B cell activation in cancer are still under debate, several HER2 B cell vaccines are currently in clinical trials (23,24). In addition, efficient antibody production against various peptide antigens is highly desirable in disease detection / treatment (25). However, these peptide antigens have low efficiency in generating antibodies and they can only produce low-titer antibodies in the short term.

[0183] The current strategy to enhance B cell immunity against non-capsid antigens is to use nano-delivery systems to mimic virus-like structures. However, most nano-delivery systems do not have true virus-like structures and are insufficient to activate B cell immunity. Although nano-delivery systems without virus-like structures are superior to soluble peptides in terms of B cell immunity, they can only activate low levels of antigen-specific B cells (less than 1-3%) and the antibody response lifespan is short (35-38). In contrast, the inorganic virus-like nanoparticles (IVLN) with HER2 peptides herein produced more than 17% antigen-specific B cells in a follicular helper T cell-dependent manner. These characteristics induced a long-lasting antibody response to inhibit HER2 tumor growth in vivo. We chose HER2 B cell epitopes to study the virus-like structure and functional mimicry of IVLN because the in vivo model is easy to test antibody function by monitoring tumor growth without the need for a biosafety level 4 laboratory. However, the same principles can be applied to activate B cell immunity to achieve other applications, such as antibody production against peptides, or B cell immunity against bacterial toxins of Bacillus anthracis (anthrax) and Clostridium botulinum.

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[0275] All publications and patents mentioned in this application are incorporated herein by reference. Various modifications and variations of the described methods and compositions of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in conjunction with specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. In fact, various modifications of the described modes for carrying out the present invention that are apparent to those skilled in the relevant art are intended to be within the scope of the following claims.

[0276] Table 4

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[0280]

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[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309]

[0310]

[0311]

[0312]

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[0323] Sequence Listing <110> The Regents of the University of Michigan <120> Nanosatellite complex <130> UM-37046.601 <150> US 62 / 746,755 <151> 2018-10-17 <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> synthetic peptides <400> 1 Cys Asp Asp Asp Pro Glu Ser Phe Asp Gly Asp Pro Ala Ser Asn Thr 1 5 10 15 Ala Pro Leu Gln Pro Glu Gln Leu Gln 20 25 <210> 2 <211> twenty one <212> PRT <213> Artificial Sequence <220> <223> synthetic peptides <400> 2 Pro Glu Ser Phe Asp Gly Asp Pro Ala Ser Asn Thr Ala Pro Leu Gln 1 5 10 15 Pro Glu Gln Leu Gln 20 <210> 3 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> synthetic <400> 3 Cys Asp Asp Asp Pro Glu Ser Phe Asp Gly Asp Pro Ala Ser Asn Thr 1 5 10 15 Ala Pro Leu Gln Pro Glu Gln Leu Gln Gly Gly Gly Lys 20 25 <210> 4 <211> 1255 <212> PRT <213> Homo sapiens <400> 4 Met Glu Leu Ala Ala Leu Cys Arg Trp Gly Leu Leu Leu Ala Leu Leu 1 5 10 15 Pro Pro Gly Ala Ala Ser Thr Gln Val Cys Thr Gly Thr Asp Met Lys 20 25 30 Leu Arg Leu Pro Ala Ser Pro Glu Thr His Leu Asp Met Leu Arg His 35 40 45 Leu Tyr Gln Gly Cys Gln Val Val Gln Gly Asn Leu Glu Leu Thr Tyr 50 55 60 Leu Pro Thr Asn Ala Ser Leu Ser Phe Leu Gln Asp Ile Gln Glu Val 65 70 75 80 Gln Gly Tyr Val Leu Ile Ala His Asn Gln Val Arg Gln Val Pro Leu 85 90 95 Gln Arg Leu Arg Ile Val Arg Gly Thr Gln Leu Phe Glu Asp Asn Tyr 100 105 110 Ala Leu Ala Val Leu Asp Asn Gly Asp Pro Leu Asn Asn Thr Thr Pro 115 120 125 Val Thr Gly Ala Ser Pro Gly Gly Leu Arg Glu Leu Gln Leu Arg Ser 130 135 140 Leu Thr Glu Ile Leu Lys Gly Gly Val Leu Ile Gln Arg Asn Pro Gln 145 150 155 160 Leu Cys Tyr Gln Asp Thr Ile Leu Trp Lys Asp Ile Phe His Lys Asn 165 170 175 Asn Gln Leu Ala Leu Thr Leu Ile Asp Thr Asn Arg Ser Arg Ala Cys 180 185 190 His Pro Cys Ser Pro Met Cys Lys Gly Ser Arg Cys Trp Gly Glu Ser 195 200 205 Ser Glu Asp Cys Gln Ser Leu Thr Arg Thr Val Cys Ala Gly Gly Cys 210 215 220 Ala Arg Cys Lys Gly Pro Leu Pro Thr Asp Cys Cys His Glu Gln Cys 225 230 235 240 Ala Ala Gly Cys Thr Gly Pro Lys His Ser Asp Cys Leu Ala Cys Leu 245 250 255 His Phe Asn His Ser Gly Ile Cys Glu Leu His Cys Pro Ala Leu Val 260 265 270 Thr Tyr Asn Thr Asp Thr Phe Glu Ser Met Pro Asn Pro Glu Gly Arg 275 280 285 Tyr Thr Phe Gly Ala Ser Cys Val Thr Ala Cys Pro Tyr Asn Tyr Leu 290 295 300 Ser Thr Asp Val Gly Ser Cys Thr Leu Val Cys Pro Leu His Asn Gln 305 310 315 320 Glu Val Thr Ala Glu Asp Gly Thr Gln Arg Cys Glu Lys Cys Ser Lys 325 330 335 Pro Cys Ala Arg Val Cys Tyr Gly Leu Gly Met Glu His Leu Arg Glu 340 345 350 Val Arg Ala Val Thr Ser Ala Asn Ile Gln Glu Phe Ala Gly Cys Lys 355 360 365 Lys Ile Phe Gly Ser Leu Ala Phe Leu Pro Glu Ser Phe Asp Gly Asp 370 375 380 Pro Ala Ser Asn Thr Ala Pro Leu Gln Pro Glu Gln Leu Gln Val Phe 385 390 395 400 Glu Thr Leu Glu Glu Ile Thr Gly Tyr Leu Tyr Ile Ser Ala Trp Pro 405 410 415 Asp Ser Leu Pro Asp Leu Ser Val Phe Gln Asn Leu Gln Val Ile Arg 420 425 430 Gly Arg Ile Leu His Asn Gly Ala Tyr Ser Leu Thr Leu Gln Gly Leu 435 440 445 Gly Ile Ser Trp Leu Gly Leu Arg Ser Leu Arg Glu Leu Gly Ser Gly 450 455 460 Leu Ala Leu Ile His His Asn Thr His Leu Cys Phe Val His Thr Val 465 470 475 480 Pro Trp Asp Gln Leu Phe Arg Asn Pro His Gln Ala Leu Leu His Thr 485 490 495 Ala Asn Arg Pro Glu Asp Glu Cys Val Gly Glu Gly Leu Ala Cys His 500 505 510 Gln Leu Cys Ala Arg Gly His Cys Trp Gly Pro Gly Pro Thr Gln Cys 515 520 525 Val Asn Cys Ser Gln Phe Leu Arg Gly Gln Glu Cys Val Glu Glu Cys 530 535 540 Arg Val Leu Gln Gly Leu Pro Arg Glu Tyr Val Asn Ala Arg His Cys 545 550 555 560 Leu Pro Cys His Pro Glu Cys Gln Pro Gln Asn Gly Ser Val Thr Cys 565 570 575 Phe Gly Pro Glu Ala Asp Gln Cys Val Ala Cys Ala His Tyr Lys Asp 580 585 590 Pro Pro Phe Cys Val Ala Arg Cys Pro Ser Gly Val Lys Pro Asp Leu 595 600 605 Ser Tyr Met Pro Ile Trp Lys Phe Pro Asp Glu Glu Gly Ala Cys Gln 610 615 620 Pro Cys Pro Ile Asn Cys Thr His Ser Cys Val Asp Leu Asp Asp Lys 625 630 635 640 Gly Cys Pro Ala Glu Gln Arg Ala Ser Pro Leu Thr Ser Ile Ile Ser 645 650 655 Ala Val Val Gly Ile Leu Leu Val Val Val Leu Gly Val Val Phe Gly 660 665 670 Ile Leu Ile Lys Arg Arg Gln Gln Lys Ile Arg Lys Tyr Thr Met Arg 675 680 685 Arg Leu Leu Gln Glu Thr Glu Leu Val Glu Pro Leu Thr Pro Ser Gly 690 695 700 Ala Met Pro Asn Gln Ala Gln Met Arg Ile Leu Lys Glu Thr Glu Leu 705 710 715 720 Arg Lys Val Lys Val Leu Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys 725 730 735 Gly Ile Trp Ile Pro Asp Gly Glu Asn Val Lys Ile Pro Val Ala Ile 740 745 750 Lys Val Leu Arg Glu Asn Thr Ser Pro Lys Ala Asn Lys Glu Ile Leu 755 760 765 Asp Glu Ala Tyr Val Met Ala Gly Val Gly Ser Pro Tyr Val Ser Arg 770 775 780 Leu Leu Gly Ile Cys Leu Thr Ser Thr Val Gln Leu Val Thr Gln Leu 785 790 795 800 Met Pro Tyr Gly Cys Leu Leu Asp His Val Arg Glu Asn Arg Gly Arg 805 810 815 Leu Gly Ser Gln Asp Leu Leu Asn Trp Cys Met Gln Ile Ala Lys Gly 820 825 830 Met Ser Tyr Leu Glu Asp Val Arg Leu Val His Arg Asp Leu Ala Ala 835 840 845 Arg Asn Val Leu Val Lys Ser Pro Asn His Val Lys Ile Thr Asp Phe 850 855 860 Gly Leu Ala Arg Leu Leu Asp Ile Asp Glu Thr Glu Tyr His Ala Asp 865 870 875 880 Gly Gly Lys Val Pro Ile Lys Trp Met Ala Leu Glu Ser Ile Leu Arg 885 890 895 Arg Arg Phe Thr His Gln Ser Asp Val Trp Ser Tyr Gly Val Thr Val 900 905 910 Trp Glu Leu Met Thr Phe Gly Ala Lys Pro Tyr Asp Gly Ile Pro Ala 915 920 925 Arg Glu Ile Pro Asp Leu Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro 930 935 940 Pro Ile Cys Thr Ile Asp Val Tyr Met Ile Met Val Lys Cys Trp Met 945 950 955 960 Ile Asp Ser Glu Cys Arg Pro Arg Phe Arg Glu Leu Val Ser Glu Phe 965 970 975 Ser Arg Met Ala Arg Asp Pro Gln Arg Phe Val Val Ile Gln Asn Glu 980 985 990 Asp Leu Gly Pro Ala Ser Pro Leu Asp Ser Thr Phe Tyr Arg Ser Leu 995 1000 1005 Leu Glu Asp Asp Asp Met Gly Asp Leu Val Asp Ala Glu Glu Tyr 1010 1015 1020 Leu Val Pro Gln Gln Gly Phe Phe Cys Pro Asp Pro Ala Pro Gly 1025 1030 1035 Ala Gly Gly Met Val His His Arg His Arg Ser Ser Ser Thr Arg 1040 1045 1050 Ser Gly Gly Gly Asp Leu Thr Leu Gly Leu Glu Pro Ser Glu Glu 1055 1060 1065 Glu Ala Pro Arg Ser Pro Leu Ala Pro Ser Glu Gly Ala Gly Ser 1070 1075 1080 Asp Val Phe Asp Gly Asp Leu Gly Met Gly Ala Ala Lys Gly Leu 1085 1090 1095 Gln Ser Leu Pro Thr His Asp Pro Ser Pro Leu Gln Arg Tyr Ser 1100 1105 1110 Glu Asp Pro Thr Val Pro Leu Pro Ser Glu Thr Asp Gly Tyr Val 1115 1120 1125 Ala Pro Leu Thr Cys Ser Pro Gln Pro Glu Tyr Val Asn Gln Pro 1130 1135 1140 Asp Val Arg Pro Gln Pro Pro Ser Pro Arg Glu Gly Pro Leu Pro 1145 1150 1155 Ala Ala Arg Pro Ala Gly Ala Thr Leu Glu Arg Pro Lys Thr Leu 1160 1165 1170 Ser Pro Gly Lys Asn Gly Val Val Lys Asp Val Phe Ala Phe Gly 1175 1180 1185 Gly Ala Val Glu Asn Pro Glu Tyr Leu Thr Pro Gln Gly Gly Ala 1190 1195 1200 Ala Pro Gln Pro His Pro Pro Pro Ala Phe Ser Pro Ala Phe Asp 1205 1210 1215 Asn Leu Tyr Tyr Trp Asp Gln Asp Pro Pro Glu Arg Gly Ala Pro 1220 1225 1230 Pro Ser Thr Phe Lys Gly Thr Pro Thr Ala Glu Asn Pro Glu Tyr 1235 1240 1245 Leu Gly Leu Asp Val Pro Val 1250 1255

Claims

1. A vaccine composition comprising a nanosatellite complex, wherein the surface of the nanosatellite complex has a negative charge, and wherein the nanosatellite complex comprises: a) a core nanoparticle composite comprising a biocompatible coating of a diblock copolymer of polysiloxane and polyethylene glycol surrounding a Fe3O4 nanoparticle core; b) 10-20 gold satellite particles attached to or absorbed into the biocompatible coating; c) a plurality of antigenic peptides conjugated to or absorbed into the satellite particle, wherein the antigenic peptides comprise B cell epitopes; and d) wherein the nanosatellite complex comprises all of the following characteristics: i) the weight ratio of all the satellite particles to the nanoparticle core is 10-40%; ii) each of the satellite particles has a diameter of 1-5 nm, and the diameter of the nanosatellite complex is 20-70 nm; iii) the satellite particles are present at a density of 15,000 to 30,000 per square micron; iv) the plurality of antigenic peptides is 1500-3000 antigenic peptides; v) 100-400 of said plurality of said antigenic peptides are present on each said satellite particle; and vi) The average distance between each of the satellite particles is 4-7 nm.

2. The composition of claim 1, wherein the 10-20 satellite particles are 10-15 satellite particles.

3. The composition of claim 1, wherein the weight ratio of all of the satellite particles to the nanoparticle core is 25-35%.

4. The composition of claim 1, wherein the weight ratio of all of the satellite particles to the nanoparticle core is 29-31%.

5. The composition of claim 1, wherein 225-275 of said plurality of antigenic peptides are present on each of said satellite particles.

6. The composition of claim 1, wherein the average distance between each of the satellite particles is 5-7 nm.

7. The composition of claim 1, wherein the antigenic peptide comprises: i) a neoantigenic determinant; ii) at least one epitope from a tumor antigen; iii) at least one epitope from a viral oncoprotein; iv) at least one epitope from an infectious virus; v) at least one epitope from a parasite; or vi) at least one epitope from an infectious bacterium.

8. The composition of claim 1, further comprising a physiologically compatible aqueous solution.

9. The composition of claim 1, further comprising cancer cells or antigen-presenting cells.

10. The composition of claim 1, wherein the plurality of antigenic peptides are unevenly distributed on the satellite particles.

11. The composition of claim 1, wherein the composition further comprises a type I interferon agonist.

12. The composition of claim 11, wherein the Type I interferon agonist is electrostatically attracted or absorbed to i) the antigenic peptide, ii) the plurality of satellite particles, and / or iii) the core nanoparticle.

13. The composition of claim 1, wherein the composition is adjuvant-free.

14. The composition of claim 1, further comprising an immune checkpoint inhibitor.

15. Use of the composition according to any one of claims 1 to 14 in the preparation of a vaccine for eliciting an immune response in a subject, wherein the vaccine is capable of producing antibodies against the antigenic peptide.

16. The use of claim 15, wherein the subject is a human.

17. The use of claim 15, wherein the subject is an animal.

18. The use of claim 15, further comprising obtaining a sample from the subject and purifying at least some of the antibody from the sample.

19. The use of claim 15, wherein no adjuvant is administered as part of the composition or otherwise.

20. The use of claim 15, wherein the subject is administered a type I interferon agonist in the form of the composition or alone.

21. The method of claim 15, wherein the subject is administered an immune checkpoint inhibitor in the form of the composition or alone.

22. The use of claim 15, wherein the nanosatellite complex does not generate detectable non-specific antibodies against the nanosatellite complex.

23. The use of claim 15, wherein the nanosatellite complex homes to the subject's lymph nodes.

24. The use of claim 15, wherein the nanosatellite complex homes to the B cell region or T cell region of the subject's lymph nodes.

25. The use of claim 15, wherein the nanosatellite complex is taken up by the subject's subcapsular sinus macrophages at a rate equivalent to that of the virus.

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