Compositions for increasing immune response and methods of use thereof

Abstract

Nanoparticles are provided having a calcium core, such as calcium hydroxide (Ca (OH) 2) and calcium carbonate (CaCO3) particles. The nanoparticles may further include a shell, such as a shell formed from silica or oleic acid. The nanoparticles may further include a coating, such as a coating formed from polyethylene glycol, and optionally further include a coating of lipids. The nanoparticles may further include a targeting agent, such as a targeting agent that targets dendritic cells, T cells, or other immune cells. The nanoparticles may further include or otherwise be used in combination with an active agent, optionally selected from the group consisting of an antigen, a chemotherapeutic drug, an immune system modulator or an immune checkpoint modulator. Also provided are pharmaceutical compositions comprising the nanoparticles and methods of use thereof for increasing an immune response against, for example, cancer and infection.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of and priority to USSN 63 / 384,922, filed November 23, 2022, and USSN 63 / 498,238, filed April 25, 2023, which are specifically incorporated herein by reference in their entireties.

[0003] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0004] This invention was made with government support under Grants CA257851 and CA247769 from the National Institutes of Health. The government has certain rights in this invention.

[0005] References to sequence listings

[0006] The Sequence Listing, submitted as a text file named "UGA2023-053-03-PCT.xml" created on November 22, 2023 and having a size of 9,937 bytes, is hereby incorporated by reference pursuant to 37 CFR §1.52(e)(5). Technical Field

[0007] The present invention generally relates to enhancing immune responses, for example against cancer. Background Art

[0008] The role of the immune system in combating cancer is now recognized. For example, dendritic cells (DCs) are the most effective types of antigen presenting cells (APCs), and play an important role in resisting malignant tumors (Palucka et al., "Nature Reviews Cancer (Nature Reviews Cancer)" 2012, 12 (4), 265-277.). DC continuously samples and processes the antigens in its environment, and migrates to secondary lymphoid tissues, where they activate naive T cells. During this process, DC undergoes maturation, and its hallmark is the increase in antigen presenting molecules (major histocompatibility complex MHC-I and MHC-II) and costimulatory molecules (e.g., CD80, CD86 and CD40) (Wculek et al., "Nature Reviews Immunology (Nature Reviews Immunology)" 2020, 20 (1), 7-24). DCs also secrete cytokines, including interleukin 12 (IL-12) and type I interferons, which shape T cell responses (Vignali et al., Nature Immunology 2012, 13(8), 722-728, Parker et al., Nature Reviews Cancer 2016, 16(3), 131-144). Therefore, DCs are an important bridge between the innate and adaptive immune responses.

[0009] However, the tumor microenvironment (TME) is often rich in immunosuppressive factors, which have a negative impact on DC infiltration and antigen cross-presentation, thereby suppressing immunity or inducing tolerance. This effect is widespread because many cancer treatments rely on or benefit from DC-mediated immunity. For example, radiotherapy (RT) and chemotherapy can induce immunogenic cell death (ICD), but as monotherapy, they often cannot effectively stimulate strong immunity. Immune checkpoint inhibitors may cause long-term remission, but many tumors are identified as immune "cold" and do not respond to immunotherapy (Binnewies et al., Nature Medicine 2018, 24 (5), 541-550). Some people have explored immunomodulators, such as toll-like receptor (TLR) agonists, which can stimulate DC cells and overcome immunosuppressive TME (Pham et al., Experimental & Molecular Medicine 2010, 42 (6), 407-419). However, issues including rapid clearance, off-target toxicity, and suboptimal efficiency limit their clinical application.

[0010] Additionally, cytotoxic T cells also play an important role. Cancer cells have tumor-associated antigens (TAAs), which, like viruses and bacteria, can be recognized by the immune system and killed by cytotoxic T cells (CTLs) in an antigen-specific manner. However, solid tumors are often characterized by an immunosuppressive environment that inhibits T cell activation and proliferation, or inactivates them. Various strategies, including immune checkpoint inhibitors (ICIs), are being developed to directly or indirectly enhance the function of endogenous T cells. However, a considerable number of patients do not respond to ICIs. Alternatively, antigen-specific T cells can be expanded or engineered outside the patient's body and reintroduced into the host. These include adoptive T cell transfer and CAR-T therapy, which have made significant progress and are entering the clinic. However, the efficacy of these therapies may still be limited by problems such as toxicity and a harsh tumor microenvironment. New immunotherapy options that can be used as a single treatment or in combination are needed to enhance existing immunotherapies.

[0011] Thus, there remains a need for compositions and methods for enhancing immune responses, such as immune cell maturation and infiltration, for immune response therapies, such as cancer therapies.

[0012] It is an object of the present invention to provide compositions and methods for enhancing immune responses, including but not limited to immune cell maturation and infiltration, for immune response therapies, such as cancer therapy. Summary of the Invention

[0013] Disclosed are compositions and methods for enhancing responses such as immune cell maturation and infiltration in cancer therapy.

[0014] The composition includes nanoparticles having a core comprising calcium. Examples include calcium hydroxide cores (herein CHNP), calcium carbonate (herein CCNP), calcium citrate (CaCit), calcium phosphate (Ca3(PO4)2), CaCL2, calcium sulfate (CaSO4), CaC2O4, Ca(NO3)2, calcium silicate (Ca2SiO4), calcium fluoride (CaF2), CaBr2, and CaI2.

[0015] The nanoparticles may include a shell, such as a silica or oleic acid shell.

[0016] In some forms, the shape of the particles is hexagonal. In the experiments provided below, the average diameter of CHNP (the long diagonal of the hexagon) is about 219.9 ± 17.8 nm, and the average diameter of CCNP is 150 nm to 160 nm, but other diameters are also considered. In some forms, the thickness of the silica shell is about ~ 20 nm. In certain embodiments, the nanoparticles further include a coating or other portion, such as polyethylene glycol (PEG) or lipid-PEG coating, which is on, above, or in combination with the shell.

[0017] In certain embodiments, the nanoparticles include one or more targeting agents, such as dendritic cells or T cell targeting agents. Such targeting particles can be referred to as AnCHNP, CCNP-Ab, etc. The targeting agent can be covalently bound to the nanoparticles directly or indirectly through a linker. In certain embodiments, the targeting agent targets one or more immune cells, such as dendritic cells, T cells (effector T cells (e.g., cytotoxic T cells, helper T cells, regulatory T cells, or a combination thereof), memory T cells, gamma-delta T cells (gamma delta T cells), follicular helper T cells (Tfh), natural killer T cells (NKT cells)), macrophages, natural killer cells, and / or neutrophils.

[0018] In some embodiments, the nanoparticles and / or the preparations comprising the nanoparticles may further include tumor antigens and / or immunomodulators, such as immune system modulators or immune cell modulators. In still another embodiment, the nanoparticles may include additional cancer therapeutics, such as immune checkpoint inhibitors or chemotherapeutic agents. Some embodiments further include adjuvants and / or antigens (e.g., antigenic peptides).

[0019] Also provided are pharmaceutical compositions comprising an effective amount of the disclosed calcium particle compositions, and / or cells treated in vitro or ex vivo with the disclosed calcium particles.

[0020] Methods of using the nanoparticle formulations, treated cells, and pharmaceutical compositions are also provided. The methods generally include increasing an immune response, such as the activity of dendritic cells or T cells. The immune response can be induced by increasing calcium signaling mediated by the calcium core particles.

[0021] The method can be performed in a subject in need (i.e., in vivo), in vitro, or ex vivo. The subject may be suffering from a benign or malignant tumor or infection. In some embodiments, the subject has cancer and is optionally undergoing cancer therapy, e.g., vaccination, radiotherapy, chemotherapy, or immunotherapy. Exemplary cancers include, but are not limited to, vascular cancer, bone cancer, muscle cancer, bladder cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, uterine cancer, or germ cell cancer.

[0022] For example, a method of treating a subject in need thereof may comprise administering to the subject an effective amount of the disclosed nanoparticle formulations or ex vivo treated cells, preferably in a pharmaceutical composition, optionally further comprising one or more of the following: an antigen, an immunomodulator (e.g., an immune system modulator, an immune cell modulator, etc.), an immune checkpoint inhibitor, and a chemotherapeutic agent. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figures 1A-1I The synthesis and properties of AnCHNPs are shown. Figure 1A Schematic diagram showing the nanoparticle synthesis, surface coating, and antibody conjugation steps. FIG1B shows SEM images of CHNPs and SCHNPs. Scale bar, 200 nm. FIG1C shows TEM images of CHNPs (left) and SCHNPs (right). Scale bars indicate lengths of 200 nm (black) and 100 nm (white), respectively. Figure 1D Shown is an EDS elemental distribution map illustrating the core / shell structure of SCHNPs. Scale bar, 250 nm. Figure 1E XRD spectra of SCHNPs, CHNPs, and a Ca(OH)2 reference (Ca(OH)2Ref) are shown. Figure 1F Shown is the EDS spectrum of SCHNPs. Figure 1G Shown are the DLS spectra of CHNPs, PCHNPs, and AnCHNPs tested in water. Figure 1H is a bar graph showing the zeta potential of CHNPs, SCHNPs, PCHNPs, and AnCHNPs measured in PBS (solution). Figure 1I Figure 2 is a schematic diagram illustrating the use of AnCHNPs to enhance anti-cancer immunity. AnCHNPs are taken up by DCs and promote their maturation and migration to secondary lymphoid organs such as tumor-draining lymph nodes (TDLNs), where they activate naive T cells. Activated DCs also secrete cytokines such as IL-12 that enhance the efficacy of effector T cells. The immunomodulatory effects of AnCHNPs are most effective when used after radiotherapy or chemotherapy, which triggers the release of tumor antigens and possible DAMPs.

[0024] Figures 2A-2H The stability and intracellular degradation of AnCHNPs are shown. Figure 2A Shown are the time-dependent Ca2+ release from PCHNPs tested in ammonium acetate buffer at pH 7.4 and 5.5. Figure 2B TEM images showing the degradation of the calcium core of PCHNPs in water are shown. Scale bar: 100 nm. Figure 2C : is a bar graph showing the uptake of AnCHNP (Cy5-labeled, 5 μg / mL) by DCs. Compared with PCHNP, AnCHNP showed significantly increased cellular uptake. *, p<0.05. Figure 2D is a bar graph showing the inhibition of AnCHNP (Cy5-labeled, 5 μg / mL) uptake by DCs by endocytosis inhibitors including sodium azide (50 mM), dynasore (80 μM), nystatin (25 μM), and chlorpromazine (100 μM). *, p<0.05. Figure 2E is a bar graph showing changes in DC lysosomal pH after incubation with AnCHNPs (5 μg / mL or 10 μg / mL). Figure 2F is a bar graph showing changes in DC [Ca 2+ ]int after cells were treated with AnCHNP or CaCl 2 (5 μg / mL or 10 μg / mL). Figure 2G is a bar graph showing changes in DC [Na + ]int after incubation with AnCHNPs or CaCl 2 (5 μg / mL or 10 μg / mL). Figure 2H is a bar graph showing changes in DC [K + ]int after incubation with AnCHNPs or CaCl 2 (5 μg / mL or 10 μg / mL).

[0025] Figures 3A-3E Shown are the effects of AnCHNPs on DC maturation and migration tested with BMDCs after incubation with AnCHNPs or CaCl2 (5 μg / mL or 10 μg / mL). Figures 3A-3C The population and mean fluorescence intensity (MFI) of MHC-II+ and CD205+ in DCs are shown. Figure 3A Quadrants showing population variation are shown. Figure 3B and 3C are histograms showing fluorescence intensity (MFI) and population variation. Figure 3D is a bar graph showing the effect of silica nanoparticles on DC maturation. Figure 3EFigure 2 shows a transwell assay using CFSE-labeled DCs. B16F10-OVA cells pre-irradiated with (+) or without (-) 100 Gy were seeded onto the bottom chamber. CFSE+ cells in the bottom chamber were quantified by flow cytometry at 24 hours. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

[0026] Figures 4A-4C Shown are the effects of AnCHNPs on DC maturation tested with BMDC / B16F10-OVA (pre-irradiated, 100 Gy) co-cultures in the presence of AnCHNPs or CaCl2 (5 μg / mL or 10 μg / mL). Figure 4A Figure 2 shows DCs (CD11c + )CD80 + CD86 + and MHC-II + SIINFEKL-H-2Kb + Quadrant plot of population changes. In the control group, live B16F10-OVA was used in the co-culture and PBS was added to the incubation medium. Figure 4B Including CD80 in DC + CD86 + 、CD40 + , MHC-II + and MHC-II + SIINFEKL-H-2Kb + Histogram of cell frequencies. Figure 4C Bar graph showing pro-inflammatory cytokines (IL-6, IL-12, and TNF-α) and anti-inflammatory cytokines (IL-10) in the supernatants of co-cultures analyzed by ELISA. *, p<0.05; **, p<0.01; ***, p<0.001.

[0027] Figures 5A-5F Shown are studies evaluating AnCHNP activation of DCs at the molecular level. Figure 5A This is a cartoon showing that endocytosis of AnCHNP leads to its degradation in lysosomes and release of Ca2+ in the cytoplasm. The increase in intracellular [Ca2+] activates the transcription factor pathways of NF-κB and NFAT, which can lead to gene expression of activation markers and cytokine release. Figure 5B is a heat map of the top 10 most upregulated genes in AnCHNP-treated BMDCs (relative to controls). Figure 5C GO enrichment analysis of the top 10 GO terms resulting from upregulated DGE in AnCHNP-treated BMDCs (vs. control) is shown. Figure 5D GSEA analysis of the enrichment plots for the a priori gene sets of the top four most upregulated pathways in AnCHNP-treated BMDCs (relative to controls) is shown. Figure 5E Western blot analysis of target proteins is shown. BMDCs were treated with OVA (10 μg / mL) (control) or OVA (10 μg / mL) + AnCHNPs (5 μg / mL) (AnCHNPs) for 24 hours and then lysed for Western blot analysis. Equal amounts of cell lysates were used for immunoblotting. NF-κB, phosphorylated-NF-κB, IκBα phosphorylated-IκBα, NFAT1, and calcineurin were examined. GAPDH was used as a loading control for cytoplasmic proteins. Figure 5F : is a bar graph showing the expression of selected genes for cytokines and chemokines measured by RT-qPCR. *, p < 0.05; **, p < 0.01; ***, p < 0.001. GSEA, gene set enrichment analysis; NES, normalized enrichment score.

[0028] Figures 6A-6D Shown are the effects of AnCHNPs on immune responses tested in B16F10-OVA tumor-bearing C57BL / 6 mice. Figure 6A Figure 2 is a diagram showing the experimental design. On day 0, animals received tumor irradiation (10 Gy) followed by intratumoral injection of AnCHNP (200 μg / kg) (n=10). In the control group, CaCl2 plus RT and PBS plus RT were tested (n=10). Half of the animals in each group were euthanized on day 3, while the remaining animals were euthanized on day 7. Tumor, TDLN, spleen, and serum samples were collected for flow cytometry or ELISA analysis. Figure 6B Total DC populations in day 3 and day 7 tumors are shown. Figure 6C CD86 in both tumors and TDLNs at days 3 and 7 is shown. + CD80 + 、CD40 + , MHC-II + and MHC-II + SIINFEKL-H-2Kb + DC group. Figure 6D T lymphocyte populations, including CTLs (CD45 + CD3 + CD8 + ), effector CTL (IFN-γ + CD45 + CD3 + CD8 + ) and Treg (CD45+ CD3 + CD4 + Foxp3 + ). The CTL / Treg ratio was also calculated. Figure 6E is a bar graph showing serum levels of cytokines including IL-12, IFN-γ, IL-10, IL-1β, IL-6, and TNF-α on day 3 and day 7. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.

[0029] Figures 7A-7F The therapeutic benefit of AnCHNPs when used in combination with RT was shown, tested in both B16F10-OVA and MB49 tumor-bearing C57BL / 6 mice. Figures 7A-7D Shown are the results of a therapy study using the B16F10-OVA model. Figure 7A The protocol for the B16F10-OVA study is shown. On days 0 and 2, animals were irradiated with 10 Gy of radiation applied to the tumor, followed by intratumoral injection of 200 μg / kg AnCHNPs (RT+AnCHNPs, n=5). PBS alone (PBS), PBS plus RT (RT+PBS), and AnCHNPs alone (AnCHNPs) (n=5) were tested. For T cell depletion, in addition to RT and AnCHNP combinations (RT+AnCHNP+αCD4 and RT+AnCHNP+αCD8, respectively; n=5), anti-CD4 or anti-CD8 antibodies were also administered (intraperitoneal injection, 10 mg / kg, day 0 and day 4). Figure 7B Mean tumor growth, animal survival and body weight curves are shown. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Figure 7C Individual tumor growth curves are shown. Figures 7D-7F Shown are therapeutic studies performed with the MB49 model. Figure 7D The study protocol is shown. On days 0 and 2, animals received radiation applied to the tumor (10 Gy) followed by intratumoral administration of AnCHNPs (200 μg / kg) (n=5). PBS alone (PBS) and PBS plus RT (RT+PBS) were tested (n=5). Figure 7E Mean tumor growth, animal survival and body weight curves are shown. *, p<0.05. Figure 7F Individual tumor growth curves are shown.

[0030] Figures 8A-8E Shown are results evaluating the benefit of AnCHNPs when used in combination with chemotherapy or immunotherapy. Figures 8A-8CShown is the efficacy of dual therapy with AnCHNPs and carboplatin tested in the B16F10-OVA model. Figure 8A The protocol for the therapy study was as follows. On days 0 and 2, animals received carboplatin (intraperitoneal injection, 40 mg / kg on day 0) followed by intratumoral administration of 200 μg / kg AnCHNPs (Carboplatin + AnCHNPs, n=5). PBS alone (PBS) and carboplatin alone (Carboplatin) were tested for comparison (n=5). Figure 8B Mean tumor growth, animal survival and body weight curves are shown. *, p<0.05; **, p<0.01. Figure 8C Individual tumor growth curves are shown. Figures 8D-8E Shown is the efficacy of dual therapy with AnCHNPs and anti-PD-L1 antibody tested in the B16F10 model. Figure 8D Figure 2 is a diagram showing the experimental design. Animals received anti-PD-L1 antibody (intraperitoneal injection, 10 mg / kg) on ​​days -2, 0, 2, and 4, followed by intratumoral injection of 200 μg / kg AnCHNP (αPD-L1+AnCHNP, n=5). PBS alone (PBS) and anti-PD-L1 alone (αPD-L1) were tested for comparison (n=5). Figure 8E Mean tumor growth, animal survival and body weight curves are shown. **, p<0.01.

[0031] Figures 9A-9C Additional physicochemical characterization of calcium nanoparticles, including calcium hydroxide nanoparticles (CHNPs), silica-coated calcium hydroxide nanoparticles (SCHNPs), and PEGylated calcium hydroxide nanoparticles (PCHNPs), is shown. Figure 9A FT-IR spectra of CHNPs, SCHNPs, and PCHNPs are shown. APTES (3-aminopropyl)triethoxysilane) for silica coating and PEG-diacid for surface PEGylation were also analyzed. Figure 9B EDS analysis of CHNPs is shown. The molar ratio of Ca to O is about 1:2. Figure 9C Shown are the zeta potentials of CHNPs, SCHNPs, PCHNPs, and AnCHNPs tested in PBS.

[0032] Figures 10A-10D Calcium release in solution and in vitro is shown. Calcium levels in solution were quantified using an ion-selective electrode. In vitro quantification is based on the formation of a chromogenic complex between calcium ions and 0-cresolphthalein, with this chromophore measured at OD = 575 nm. Figure 10A is a standard calibration curve prepared for potentiometric measurements using known concentrations of calcium salts (CaCl2, 150 ppm and 2000 ppm). Figure 10B Shown are the time-dependent Ca from CHNPs tested in ammonium acetate buffer at pH 7.4 and 5.5 2+ release. Figure 10C is a bar graph showing the cytotoxicity of AnCHNPs, CaCl2, and PEGylated silica nanoparticles tested with BMDCs using the ATPlite-1-step luminescence assay. Figure 10D Contains instructions for using LysoSensor TM Yellow / blue DND-160 (PDMPO) is a bar graph showing changes in lysosomal pH after cells were treated with AnCHNPs (5 μg / mL and 10 μg / mL) using BMDC. The PDMPO fluoresces primarily yellow (440 nm) in acidic organelles and blue (540 nm) in less acidic organelles. Dual emission measurements allow ratiometric imaging of pH in acidic organelles.

[0033] Figures 11A-11B Shown are the effects of AnCHNPs on the immune responses of DCs and T cells tested in B16F10-OVA tumor-bearing C57BL / 6 mice. Figure 11A Populations of CD86+CD80+, CD40+, MHC-II+, and MHC-II+SIINFEKL-H-2Kb+ DCs in spleens on days 3 and 7 are shown. Figure 11B Figure 3 shows T lymphocyte populations in TDLN on days 3 and 7, including CTLs (CD45+CD3+CD8+), effector CTLs (IFN-γ+CD45+CD3+CD8+), and Tregs (CD45+CD3+CD4+Foxp3+). The CTL / Treg ratio was also calculated. *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001.

[0034] Figure 12 The effect of AnCHNP on antigen-specific cellular immunity was shown. Splenocytes from the AnCHNP-treated group were co-incubated with B16F10-OVA cells for 6 hours in vitro, and IFN-γ was measured by flow cytometry. + CTL frequencies. Splenocytes from the PBS and CaCl2-treated groups were studied.

[0035] Figure 13 Shown is the flow cytometry gating strategy used for DC migration studies.

[0036] Figure 14 Shown is the flow cytometric gating strategy used for in vivo immune profiling studies detecting populations of DCs in tumors and TDLNs.

[0037] Figure 15 Shown is the flow cytometry gating strategy used for in vivo immunoprofiling studies detecting lymphocyte populations in tumors and spleens.

[0038] Figures 16A-16N Results of nanoparticle synthesis and characterization are shown. Figure 16A is a TEM image of CaCO3 nanoparticles. Figure 16B is an enlarged TEM image of CaCO3 nanoparticles; scale bar: 100 nm. Figure 16C is the SEM image of CaCO3 nanoparticles. Figure 16D is a graph showing the size distribution of CaCO 3 nanoparticles based on TEM results. Figure 16E is the TEM image of CaCO3@OA nanoparticles. Figure 16F is an enlarged TEM image of CaCO3@OA nanoparticles; scale bar, 100 nm. Figure 16G is the SEM image of CaCO3@OA nanoparticles. Figure 16H is a graph showing the size distribution of CaCO3@OA nanoparticles based on TEM results. Figure 16I This is the TEM energy dispersive X-ray spectroscopy (EDX) image of CaCO3 nanoparticles. Figure 16J X-ray diffraction (XRD) of CaCO3 nanoparticles (top) and bulk CaCO3 (bottom). Figure 16K is a comparison of the Fourier transform infrared spectra (FT-IR) of OA, CaCO3@OA and CaCO3 nanoparticles. Figure 16L It is a graph showing the zeta potential of CCNP and CCNP-Ab measured in HEPES buffer (pH=7.4). Figure 16M is a graph showing the DLS measurement results of CaCO 3 nanoparticles (in ethanol), CaCO 3 @OA (in hexane), CCNP (in HEPES), and CCNP-Ab (in HEPES). Figure 16N Graphs showing calcium release from CCNP-Ab tested at pH = 7.4 and 5.0 at room temperature.

[0039] Figures 17A-17J Results of in vitro studies are shown. Figure 17A is a graph showing the cytotoxicity of PMA@CCNP-Ab and CaCl2 measured in EL4 cells. Nanoparticle doses are based on equivalent calcium concentrations. Figure 17B Is based on Figure 17A IC50 activity curve of activity data. Figure 17CGraph showing cellular uptake data. PMA@CCNP and PMA@CCNP-Ab were labeled with Cy5. After co-culture with EL4 for 24 hours, the mean fluorescence intensity (MFI) of Cy5 was measured. Figure 17D is a graph showing changes in intracellular calcium levels. Fluo-3AM was used as a calcium indicator. Figure 17E and 17F The effect of PMA@CCNP-Ab on NF-κB ( Figure 17E ) and NFAT( Figure 17F ) pathways were analyzed by Western blot. Figure 17G and 17H Each is a series showing that cells were treated with PMA@CCNP-Ab for 48 hours ( Figure 17G ) and 72 hours ( Figure 17H ) is a diagram showing immune profile analysis of OT-1 CTLs after RT-PCR. Figure 17I is a graph showing IFN-γ secretion from OT-1 cells (pre-activated with irradiated B16-OVA) after treatment with PMA@CCNP-Ab and a control group. Figure 17J is a graph showing IL-2 secretion from OT-1 cells (pre-activated with irradiated B16-OVA) after treatment with PMA@CCNP-Ab and a control group.

[0040] Figures 18A-18D Figure 2 shows the evaluation of the in vivo immunostimulatory effect of PMA@CCNP-Ab. C57BL / 6 mice bearing B16-OVA tumors were irradiated (15 Gy) and then injected intratumorally with PMA@CCNP-Ab on days 2, 5, and 8. Figure 18A ),spleen( Figure 18B ) and lymph nodes ( Figure 18C ) samples were analyzed by flow cytometry. Figure 18D Figure 2 shows the results of co-culture of splenocytes and B16-OVA cancer cells and the evaluation of activated CTLs (CD8 + IFN-γ + ) dot plot.

[0041] Figures 19A-19C Figure 2 shows an in vivo evaluation of the therapeutic benefit of PMA@CCNP-Ab in C57BL / 6 mice bearing B16 tumors. PMA@CCNP-Ab nanoparticles were injected intratumorally on days 0, 1, and 3. PBS or CaCl2 salt was injected for comparison. In addition to PMA@CCNP-Ab, anti-CD8 antibody was also injected to assess the impact of CTLs on the therapeutic effect. Figure 19A is the animal survival curve. Figure 19B It is the tumor growth curve. Figure 19Cis a series of graphs showing individual tumor growth curves. DETAILED DESCRIPTION

[0042] The disclosed compositions are based at least on the discovery that safe and effective calcium modulators can promote immune cell activation, such as DC-mediated and / or T cell-mediated anti-cancer immunity.

[0043] Ca 2+ As a second messenger, it plays an important role in DC maturation and migration. In the resting state, immature DCs maintain low levels of cytoplasmic calcium ions or [Ca 2+ ] int Cytokines, pathogen-associated molecular patterns, or damage-associated molecular patterns can bind to DC receptors and trigger [Ca 2+ ] int Increased, in turn, activates a signaling cascade that ultimately induces co-stimulatory and antigen-presenting molecules (Shumilina et al., American Journal of Physiology-Cell Physiology 2011, 300(6), C1205-C1214). [Ca 2+ ] int It is tightly regulated by calcium-selective ion channels and transporters on the plasma membrane, endoplasmic reticulum, and inner mitochondrial membrane. Previously, in laboratory settings, calcium ionophores (such as ionomycin) were shown to be able to increase [Ca 2+ ] int and activate DCs (Liu et al., Journal of Biological Chemistry 1978, 253(17), 5892-5894). However, these ion carriers lack specificity for DCs and may cause toxicity when administered systemically (Jiang et al., Nature 1995, 375(6527), 151-155). In addition, DC maturation and activation require [Ca 2+ ] int The sustained increase in the amount of leukocytes required for administration of ionophores (Santegoets et al., Journal of leukocyte biology 2008, 84(6), 1364-1373) is challenging or impossible to achieve with small molecule ionophores that are rapidly cleared after injection.

[0044] Calcium also plays a central role as a second messenger in T cell activation. Calcium signaling begins with stimulation of the TCR pathway and ultimately leads to activation of the transcription factor NFAT through activation of the calcium-sensitive phosphatase calcineurin.

[0045] There is an unmet need for safe and effective calcium modulators that can enhance DC-mediated and T cell-mediated anti-cancer immunity.

[0046] Herein, the examples show the use of calcium nanoparticles as DC-targeted immunomodulators. In short, Ca(OH)2 nanoparticles were synthesized by coprecipitation and coupled with antibodies specific for CD205 (also known as DEC205), a type I integral membrane protein primarily expressed on DCs (Jiang et al., Nature 1995, 375(6527), 151-155). The results showed that antibody-conjugated calcium hydroxide nanoparticles (AnCHNPs) were selectively taken up by DCs and released calcium therein, so that [Ca 2+ ] int Can continue to increase. [Ca 2+ ] int The increase in DCs promotes DC maturation, migration, and cross-presentation, thereby enhancing T cell immunity ( Figure 1I These results were validated in vitro using bone marrow-derived dendritic cells (BMDCs) and in vivo using AnCHNPs as adjuvants in combination with RT, immunotherapy, or chemotherapy. The examples demonstrate that CHNPs comprising a DC-targeting moiety can be used as adjuvants in combination with RT, immunotherapy, or chemotherapy.

[0047] Additional examples show that T cells effectively internalize calcium nanoparticles, such as PMA@CCNP-Ab, resulting in elevated intracellular calcium levels. Delivery of calcium and PMA to T cells promotes their activation, as evidenced by increased expression or secretion of CD69, IFN-γ, and TNF-α. This was observed in both the EL4 cell line and primary T cells from OT1 mice. In vivo testing in C57 / BL6 mice bearing B16-OVA tumors showed that PMA@CCNP-Ab enhanced tumor infiltration by cytotoxic T cells and increased the CTL / Treg ratio. The observed therapeutic benefit correlated with the ability of PMA@CCNP-Ab to enhance T cell activation. Furthermore, PMA@CCNP-Ab can also be used to enhance cell-based therapies, including adoptive T cell transfer and CAR-T therapy.

[0048] I. Definition

[0049] The term "nanoparticle" refers to any particle having a diameter greater than 1 nm and less than 1000 nm.

[0050] The terms "targeting agent" and "targeting moiety" refer to a chemical compound that can direct a nanoparticle to a site on a selected cell or tissue type, can act as an attachment molecule, or be used to couple or link another molecule. The term "directing," as used with respect to a chemical compound, means causing the nanoparticle to preferentially attach to a selected cell or tissue type. Such targeting agents typically bind to their target with high affinity and specificity.

[0051] As used herein, the terms "treatment" and "treating" refer to the medical management of a subject with the intention of curing, improving, stabilizing or preventing a disease, pathological condition or disorder. This term includes active treatment, that is, treatment specifically directed to improving a disease, pathological condition or disorder, and also includes etiological treatment, that is, treatment directed to eliminating the cause of the disease, pathological condition or disorder in question. In addition, this term includes palliative treatment, i.e., treatment designed to relieve symptoms rather than cure a disease, pathological condition or disorder; preventive treatment, i.e., treatment intended to minimize or partially or completely inhibit the development of a disease, pathological condition or disorder in question; and supportive treatment, i.e., treatment that is used to supplement another specific therapy for the improvement of a disease, pathological condition or disorder in question. It should be understood that while treatment is intended to cure, improve, stabilize or prevent a disease, pathological condition or disorder, it does not necessarily actually cure, improve, stabilize or prevent. The effect of treatment can be measured or evaluated as described herein and as known in the art to be suitable for the disease, pathological condition or disorder in question. Such measurements and evaluations can be performed qualitatively and / or quantitatively. Thus, for example, characteristics or features of a disease, pathological condition or disorder and / or symptoms of a disease, pathological condition or disorder can be reduced to any effect or to any amount.

[0052] The term "tumor" or "neoplasm" refers to an abnormal mass of tissue containing neoplastic cells. Neoplasms and tumors may be benign, precancerous, or malignant.

[0053] The term "cancer" or "malignant neoplasm" refers to cells that exhibit uncontrolled growth, invade neighboring tissues, and often metastasize to other locations in the body.

[0054] The terms "individual," "subject," and "patient" are used interchangeably to refer to any individual who is the target of administration or treatment. A subject can be a vertebrate, such as a mammal. Thus, a subject can be a human or a veterinary patient.

[0055] The term "therapeutically effective" means that the amount of the composition used is an amount sufficient to ameliorate one or more causes or symptoms of the disease or condition. Such amelioration need only reduce or alter, not eliminate. A therapeutically effective amount of a composition for treating cancer is preferably an amount sufficient to cause tumor regression or sensitize the tumor to radiation or chemotherapy.

[0056] The term "treatment" refers to the medical management of a patient with the goal of curing, ameliorating, stabilizing, or preventing a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment specifically directed toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward eliminating the cause of the relevant disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed to relieve symptoms rather than cure the disease, pathological condition, or disorder; preventive treatment, that is, treatment intended to minimize or partially or completely inhibit the development of the relevant disease, pathological condition, or disorder; and supportive treatment, that is, treatment used to supplement another specific therapy directed toward the improvement of the relevant disease, pathological condition, or disorder.

[0057] The use of the terms "a," "an," "the," and similar referents in the context of describing the presently claimed invention (especially in the context of the claims) are to be construed to cover both the singular and the plural unless otherwise indicated herein or clearly contradicted by context.

[0058] Recitation of ranges of values ​​herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated, and each separate value is incorporated into the specification as if it were individually recited herein.

[0059] The use of the term "about" is intended to describe a value that is within a range of approximately + / -10% above or below the stated value; in other embodiments, the range of the value may be within a range of approximately + / -5% above or below the stated value; in other embodiments, the range of the value may be within a range of approximately + / -2% above or below the stated value; in other embodiments, the range of the value may be within a range of approximately + / -1% above or below the stated value. The foregoing ranges are intended to be determined in accordance with the context and no further limitations are implied. Unless otherwise indicated herein or otherwise clearly contradicted by the context, all methods described herein can be performed in any suitable order. Unless otherwise required, the use of any and all examples or exemplary language (e.g., "such as") provided herein is intended solely to better illustrate the invention and does not limit the scope of the invention. No language in this specification should be construed as indicating that any unclaimed element is necessary to practice the invention.

[0060] Disclosed are materials, compositions, and components that can be used in, in conjunction with, or to prepare the disclosed methods and compositions, or as products of the disclosed methods and compositions. These and other materials are disclosed herein, and it should be understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed, while specific reference to each different individual and collective combination and arrangement of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a ligand is disclosed and discussed, and a number of modifications that can be made to multiple molecules comprising the ligand are discussed, then unless specifically indicated to the contrary, each combination and arrangement of the ligand and possible modifications are specifically contemplated. Thus, if a class of molecules A, B, and C and a class of molecules D, E, and F are disclosed, and an example of a combination molecule AD is disclosed, then each is individually and collectively contemplated, even if each is not individually listed. Thus, in this example, each of the combinations AE, AF, BD, BE, BF, CD, CE, and CF is specifically contemplated and should be considered disclosed by the disclosure of A, B, and C; D, E, and F; and the example combination AD. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the subgroups of AE, BF, and CE are specifically contemplated and should be considered disclosed by the disclosure of A, B, and C; D, E, and F; and the example combination AD. Further, each of the materials, compositions, components, etc. contemplated and disclosed above may also be specifically and independently included or excluded from any group, subgroup, list, set, etc. of such materials.

[0061] These concepts apply to all aspects of this application, including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed, it should be understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods, and each such combination is specifically contemplated and should be considered disclosed.

[0062] Unless otherwise specified or clearly contradicted by the context, all methods described herein can be performed in any suitable order. Unless otherwise required, the use of any and all examples or exemplary language (e.g., "such as") provided herein is intended only to better illustrate the embodiments and does not constitute a limitation on the scope of the embodiments. No language in this specification should be construed as indicating that any unclaimed element is essential to practicing the invention.

[0063] II. Composition

[0064] A. Nuclear Composition

[0065] The composition includes particles, typically nanoparticles, having a core comprising calcium. The particles are designed to enter cells and release calcium ions (e.g., Ca 2+ ). Particles typically include one or more of the following characteristics:

[0066] Controlled calcium release. T cell activation utilizes intracellular calcium concentration ([Ca 2+ ] int ) continues to increase, and [Ca2+] int Increased [Ca] activates a signaling cascade that ultimately induces co-stimulatory and antigen-presenting molecules in dendritic cells. This is difficult to achieve using calcium salts (due to the ion-impermeable plasma membrane) or bare calcium nanoparticles (due to the rapid dissolution of the particles in the tumor microenvironment). 2+ ] int To address this problem, a shell and / or coating can be used that prevents the nanoparticles from rapid degradation, thereby allowing the nanoparticles to enter the cell via endocytosis and gradually release calcium ions within the cell.

[0067] Low toxicity: Unlike cytokine- or interferon-based immunomodulators, calcium nanoparticles can have low toxicity and can be administered repeatedly without causing systemic toxicity. After treatment, the nanoparticles can be degraded into Ca 2+ and other synergistic components, such as CO3 2- , these components can be safely excreted, metabolized or absorbed by the host.

[0068] Targeted delivery: Nanoparticles can be conjugated with targeting ligands to facilitate targeted delivery of calcium, and / or loaded with active agents, e.g., antigens and / or PKC antagonists.

[0069] Unique mechanism of action: Cell activation can be inhibited or blocked at multiple stages, thereby impairing cellular immunity. In the disclosed method, calcium delivery can bypass upstream signaling, which is believed to enable cell activation even in an immunosuppressive environment.

[0070] In some embodiments, the particles have a calcium hydroxide core. The experiments below show that such particles, also known as Ca(OH)2 nanoparticles and CHNPs, can be synthesized by a co-precipitation method using CaCl2 and NaOH as precursors.

[0071] In some embodiments, the particles have a calcium carbonate core. Referring also to CaCO3 and CCNP, the experiments below show that such particles can be synthesized using calcium chloride and ammonium bicarbonate precursors by a co-precipitation method.

[0072] Other calcium core particles may also be considered and include, but are not limited to, calcium citrate (CaCit), calcium phosphate (Ca3(PO4)2), CaCL2, calcium sulfate (CaSO4), CaC2O4, Ca(NO3)2, calcium silicate (Ca2SiO4), calcium fluoride (CaF2), CaBr2 and CaI2, each of which may also be specifically excluded.

[0073] The following experiments discuss an exemplary method for preparing calcium core particles.

[0074] See also, e.g., Rimsueb et al., ACSOmega, 5, 13, 7418-7423 (2020) doi.org / 10.1021 / acsomega.0c00032; Khalifehzadeh and Arami, Advances in Colloid and Interface Science, 2010. Science, Vol. 279, May 2020, 102157, doi.org / 10.1016 / j.cis.2020.102157; Leukel et al., Langmuir, 34, 24, 7096-7105 (2018) doi.org / 10.1021 / acs.langmuir.8b00927; Li et al., Theranostics. 2016 Oct 7; 6(13): 2380-2393. doi:10.7150 / thno.15914; Putnis et al., Crystallization via Nonclassical Pathways Volume 2: Accumulation, Biomineralization, Imaging, and Applications. 2: Aggregation, Biomineralization, Imaging & Application), Chapter 1, pp. 1-35, ACS Symposium Series, Vol. 1383 (2021). DOI: 10.1021 / bk-2021-1383.ch001; Jiang et al., ACS Appl. Nano Mater. 2022, 5, 9, 13069-13077, doi.org / 10.1021 / acsanm.2c02852, WO 2020 / 150623 and WO 2023 / 039415.

[0075] The size of the disclosed particles is typically nanometer-scale, for example, having a diameter of 10 nm up to (but not including) about 1 micron. However, it should be understood that in some embodiments and for some uses, the particles can be smaller or larger (e.g., microparticles, etc.). Although many compositions disclosed herein are referred to as nanoparticle compositions, it should be understood that in some embodiments and for some uses, the particles can be slightly larger than nanoparticles. For example, the composition can also include particles with a diameter between about 1 micron and about 1000 microns. Such compositions can be referred to as microparticle compositions. Therefore, all particle compositions provided herein can be microparticles, but are generally more preferably nanoparticles of nanometer size.

[0076] Nanoparticle is generally used for application and cell infiltration in tissue.Therefore, in certain embodiments, particle is diameter from 10nm until about 1,000nm, or the nanoparticle of any subrange or specific integer therebetween.For example, the diameter of nanoparticle can be 10nm to 900nm, 10nm to 800nm, 10nm to 700nm, 10nm to 600nm, 10nm to 500nm, 20nm to 500nm, 30nm to 500nm, 40nm to 500nm, 50nm to 500nm, 50nm to 400nm, 50nm to 350nm, 50nm to 300nm or 50nm to 200nm, 10nm to 100nm.For example, in certain embodiments, particle is any other integer value or the value range between about 15nm, 25nm, 60nm, 100nm, 150nm, 200nm, 250nm, 300nm or 1nm to 1000nm (comprising endpoints). In some embodiments, the diameter of the nanoparticles can be less than 400 nm, less than 300 nm, or less than 200 nm. For example, the diameter of the nanoparticles can be 50 nm to 300 nm.

[0077] The sizes disclosed can be the size of the particles with or without a shell and / or coating. Thus, in some embodiments, the size is the average diameter of the particle core.

[0078] In one embodiment, the average diameter of the core of the nanoparticle is from about 15 nm to about 800 nm, or from about 20 nm to about 500 nm, or from about 50 nm to about 350 nm, or any sub-range or specific integer therebetween. In certain embodiments, the average diameter of the nanoparticle is from about 100 nm or 150 nm or 200 nm to about 200 nm or 250 nm or 300 nm.

[0079] For example, particle size can be measured or determined by dynamic light scattering, electron microscopy, such as scanning electron microscopy (SEM) and transmission electron microscopy (TEM).

[0080] In some embodiments, the particles in the particulate composition are monodisperse. In some embodiments, the particles in the particulate composition are of varying sizes (ie, polydisperse).

[0081] B. Shell

[0082] In some embodiments, the calcium core is surrounded by a shell. The shell can be or include a metal organic framework, a protein shell (e.g., ferritin, albumin, and virus-like particles), a noble metal (Au, Ag, Pt, etc.), carbon, etc. The shell can be formed of, for example, silica, mesoporous silica, carbon; sulfides such as ZnS, CoS, CuS, Cu2S, FeS, MoS, Al2S3, Y2S3 and MnS; oxides such as Fe3O4, Fe2O3, Gd2O3, TiO2, Al2O3, MnO2, etc.; fluorides such as NaYF4, YF3, LaF3, CeF3, PrF3 and GdFe3; fatty acids such as oleic acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, arachidic acid, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA); alkylamines such as octylamine, nonylamine, decylamine, undecylamine, laurylamine, tridecylamine, tetradecylamine, pentadecylamine, hexadecylamine, heptadecylamine, octadecylamine, oleylamine; MgO, CuO and ZnO.

[0083] In a preferred embodiment, the shell is formed from silica. In some of the experiments below, a silica shell was added to the core particles using a mixture of tetraethylorthosilicate (TEOS) and (3-aminopropyl)triethoxysilane (APTES) as the silane precursor, such that amine groups were present on the surface of the resulting nanoparticles.

[0084] In some embodiments, the shell is formed from oleic acid. In some of the experiments below, an oleic acid shell was added to the core particles by dispersing the particles in a mixture of ethanol and oleic acid.

[0085] In some embodiments, a protective shell is added to reduce, prevent, or otherwise delay degradation of the particles. Preferably, the shell is composed of a material that is low in toxicity, stable at neutral pH, and / or biodegradable. In some embodiments, the shell is hydrophobic.

[0086] C. Coating

[0087] In order to further enhance the nanoparticles, a coating can be added. In certain embodiments, the coating can improve dispersion in aqueous solution and / or delay core release and / or improve half-life. Such coatings are preferably applied on the shell or combined with the shell, but it is also envisioned that they can be applied directly on the core (e.g., in the absence of a shell).

[0088] In some of the following experiments, a PEG-diacid coating was added to silica-shelled CHNPs by dispersing the particles in a mixture of dimethyl sulfoxide (DMSO) and PEG-diacid.

[0089] In some of the following experiments, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (DSPE-PEG-COOH) was added to oleic acid-shelled CaCO3 (CaCO3@OA) particles for hydrophobic interaction by dispersing the particles in a mixture of hexane and DSPE-PEG-COOH.

[0090] Therefore, the particles optionally but preferably include a coating. The coating / layer (also referred to herein as a layer or outer layer) is generally above the core of the particle and optionally but preferably on or combined with the shell of the particle. In certain embodiments, the coating enhances the compatibility of the particle with an aqueous solution. Additionally or alternatively, a coating can be added to extend the half-life of the nanoparticles in an aqueous environment and / or to improve the uptake of the nanoparticles by cells.

[0091] 1. Composition of coating

[0092] The coating can be composed of, for example, polar or non-polar polymers and copolymers, peptides, proteins, lipids, silica, metal oxides, or combinations thereof. In some embodiments, the coating is composed of a conjugate or fusion of two or more of the foregoing substances, alone or in combination with one or more active agents and / or targeting molecules.

[0093] For example, in some embodiments, the thickness of the coating (including or excluding the shell) is in the range of 1 nm to 200 nm, or 10 nm to 100 nm, or 25 nm to 75 nm (inclusive), or any subrange or specific integer therebetween, such as 50 nm.

[0094] While PEG is the preferred polymeric substrate for forming coatings, optionally with additional moieties such as charge-modifying moieties (e.g., carboxyl groups) and / or targeting moieties (e.g., antibodies) or other moieties mentioned herein or elsewhere, other coatings are also contemplated, and examples are discussed below.

[0095] a.Polymer

[0096] In some embodiments, the layer or coating surrounding the particle is formed from one or more polymers. The polymer can be polar, non-polar, or amphiphilic, and can be a single polymer or a copolymer. A polymer refers to a molecular structure comprising one or more repeating units (monomers) linked by covalent bonds. A biocompatible polymer is a polymer that generally does not cause an adverse reaction when inserted or injected into a living subject. A copolymer is a polymer formed from two or more different monomers. The different units can be arranged in a random order, in an alternating order, or as a "block" copolymer, i.e., one or more regions, each of which includes a first repeating unit (e.g., a first monomer or block of a monomer) and one or more regions, each of which includes a second repeating unit (e.g., a second block), etc. Block copolymers can have two (diblock copolymers), three (triblock copolymers), or more different blocks.

[0097] In a preferred embodiment, the coating is formed of amphiphilic molecules, especially when the surface in contact with the coating (e.g., core or shell, etc.) is hydrophobic. The term "amphiphilic" refers to a molecule having both a polar portion and a non-polar portion. In some embodiments, the polar portion (e.g., a hydrophilic portion such as a hydrophilic polymer) is soluble in water, while the non-polar portion (e.g., a hydrophobic portion such as a hydrophobic polymer) is insoluble in water. The polar portion can have a formal positive charge or a formal negative charge. Alternatively, the polar portion can have both a formal positive charge and a negative charge and be a zwitterion or an inner salt.

[0098] The hydrophilic portion of the amphiphilic material can form a corona around the particle, which increases the solubility of the particle in aqueous solution. In a specific embodiment, the amphiphilic material is a hydrophobic biodegradable polymer terminated by a hydrophilic block.

[0099] The hydrophilic portion and the hydrophobic portion can be a biocompatible hydrophilic polymer and a hydrophobic polymer, respectively. Exemplary biocompatible polymers include, but are not limited to, polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terephthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinyl pyrrolidone, polylactic acid, polyglycolides, polysiloxanes, polyurethanes and copolymers thereof, celluloses including alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitrocelluloses, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxyethyl cellulose, cellulose triacetate, and cellulose sulfate sodium salt; polyacrylic acid polymers such as propylene glycol; Polymers of acrylates and methacrylates, such as poly(methyl methacrylate), poly(ethyl methacrylate), poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexyl methacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), polyalkylenes, such as polyethylene, polypropylene poly(ethylene glycol), poly(ethylene oxide) and poly(ethylene terephthalate), poly(vinyl alcohol), poly(vinyl acetate), polyvinyl chloride, polystyrene and polyvinyl pyrrolidone, derivatives thereof, linear and branched copolymers and block copolymers thereof and blends thereof.

[0100] Other exemplary biodegradable polymers include, but are not limited to, polyesters, polydopamine, poly(orthoesters), poly(ethyleneimine), poly(caprolactone), poly(hydroxybutyrate), poly(hydroxyvalerate), polyanhydrides, poly(acrylic acid), polyglycolide, poly(urethane), polycarbonate, polyphosphate, polyphosphazene, derivatives thereof, linear and branched copolymers thereof, and block copolymers thereof, and blends thereof. In particularly preferred embodiments, the copolymer comprises one or more biodegradable hydrophobic polyesters, such as poly(lactic acid), poly(glycolic acid), and poly(lactic-co-glycolic acid), and / or with polyalkylene oxides (such as polyethylene glycol) or block copolymers (such as polyoxypropylene-polyoxyethylene). ) conjugated to these polymers.

[0101] The molecular weight of the biodegradable oligomer or polymer segments or polymers can be varied to tailor the properties of the polymer.

[0102] In some embodiments, the hydrophilic polymers or segments or blocks include, but are not limited to, homopolymers or copolymers of polyolefin glycols, such as poly(ethylene glycol), poly(propylene glycol), poly(butylene glycol), and acrylates and acrylamides, such as hydroxyethyl methacrylate and hydroxypropyl-methacrylamide.

[0103] The hydrophobic portion of the amphiphilic material can provide a nonpolar polymer matrix coating for loading nonpolar drugs.

[0104] b. Lipids

[0105] The coating can be or include one or more lipids. The lipids and other components useful in preparing the disclosed nanoparticle compositions with lipid-based coatings are known in the art. Suitable neutral, cationic and anionic lipids include, but are not limited to, sterols and lipids such as cholesterol, phospholipids, lysolipids, lysophospholipids and sphingolipids. Neutral and anionic lipids include, but are not limited to, phosphatidylcholines (PC) (such as egg PC, soybean PC), including but not limited to 1,2-diacyl-glycero-3-phosphocholine; phosphatidylserine (PS), phosphatidylglycerol, phosphatidylinositol (PI); glycolipids; phospho-sphingolipids such as sphingomyelin and glycosphingolipids (also known as 1-ceramidoglycosides), such as ceramide galactoside, gangliosides and cerebrosides; fatty acids, sterols containing carboxylic acid groups, such as cholesterol; phosphoethanolamines, such as 1,2- Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-diacyl-sn-glycero-3-phosphoethanolamine, including but not limited to 1,2-dioleoylphosphoethanolamine (DOPE), 1,2-hexadecylphosphoethanolamine (DHPE); and phosphatidylcholine, such as 1,2-distearoylphosphatidylcholine (DSPC), 1,2-dipalmitoylphosphatidylcholine (DPPC) and 1,2-dimyristoylphosphatidylcholine (DMPC). Lipids can also include various natural (e.g., tissue-derived L-α-phosphatidyl: egg yolk, heart, brain, liver, soybean) and / or synthetic (e.g., saturated and unsaturated 1,2-diacyl-sn-glycero-3-phosphoethanolamine) lipids. - Glycerol-3-phosphocholine, 1-acyl-2-acyl-sn - Glycerol-3-phosphocholine, 1,2-diheptanoyl-SN-glycerol-3-phosphocholine) lipid derivatives.

[0106] The lipid may be a sphingomyelin metabolite, such as, but not limited to, ceramide, sphingosine, or sphingosine-1-phosphate (S1P).

[0107] Exemplary cationic lipids include, but are not limited to, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium salts, also known as TAP lipids, such as methyl sulfate. Suitable TAP lipids include, but are not limited to, DOTAP (dioleoyl-), DMTAP (dimyristoyl-), DPTAP (dipalmitoyl-), and DSTAP (distearoyl-). Suitable cationic lipids in liposomes include, but are not limited to, dimethyldioctadecyl ammonium bromide (DDAB), 1,2-diacyloxy-3-trimethylammonium propane, N-[1-(2,3-dioleoyloxy)propyl]-N,N-dimethylamine (DODAP), 1,2-diacyloxy-3-dimethylammonium propane, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1,2-dioctadecyl ammonium bromide (DDAB), 1,2-diacyloxy-3-trimethylammonium propane, Alkyloxy-3-dimethylammonium propane, dioctadecylamido glycyl spermine (DOGS), 3-[N-(N',N'-dimethylamino-ethane)carbamoyl] cholesterol (DC-Chol); 2,3-dioleoyloxy-N-(2-(sperminecarboxamido)-ethyl)-N,N-dimethyl-1-propanium trifluoroacetate (DOSPA), β-alanyl cholesterol, cetyltrimethylammonium bromide (CTAB), di-C 14-amidine, N-tert-butyl-N'-tetradecyl-3-tetradecylamino-propionamidine, N-(α-trimethylaminoacetyl)docosyl-D-glutamic acid chloride (TMAG), tetracosanoyl-N-(trimethylammonio-acetyl)diethanolamine chloride, 1,3-dioleoyloxy-2-(6-carboxy-sperminyl)-propionamide (DOSPER), and N,N,N',N'-tetramethyl-, N'-bis(2-hydroxyethyl)-2,3-dioleoyloxy-1,4-butanediammonium iodide. In one embodiment, the cationic lipid can be a 1-[2-(acyloxy)ethyl]2-alkyl(enyl)-3-(2-hydroxyethyl)-imidazolinium chloride derivative, for example, 1-[2-(9(Z)-octadecenoyloxy))ethyl]-2-(8(Z)-heptadecenyl-3-(2-hydroxyethyl)-imidazolinium chloride (DOTIM) and 1-[2-(hexadecanoyloxy)ethyl]-2-pentadecyl-3-(2-hydroxyethyl)imidazolinium chloride (DPTIM). In one embodiment, the cationic lipid can be a 2,3-dialkoxypropyl quaternary ammonium compound derivative containing a hydroxyalkyl moiety on the quaternary amine, for example, 1,2-dioleoyl-3-dimethyl-hydroxyethylammonium bromide (DORI), 1,2-dioleoyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DORIE), 1,2-dioleoyloxypropyl The lipids include, but are not limited to, 1,2-dioleoyloxypropyl-3-dimethyl-hydroxypropyl ammonium bromide (DORIE-HP), 1,2-dioleoyl-oxy-propyl-3-dimethyl-hydroxybutyl ammonium bromide (DORIE-HB), 1,2-dioleoyloxypropyl-3-dimethyl-hydroxypentyl ammonium bromide (DORIE-Hpe), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DMRIE), 1,2-dipalmitoyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DPRIE) and 1,2-distearoyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DSRIE). Lipids can be formed by a combination of more than one lipid, for example, a charged lipid can be combined with a nonionic or uncharged lipid at physiological pH. Nonionic lipids include, but are not limited to, cholesterol and DOPE (1,2-dioleoylglycerylphosphatidylethanolamine).

[0108] A sterol component may be included to impart physicochemical and biological behavior. Such a sterol component may be selected from cholesterol or a derivative thereof, for example, ergosterol or cholesterol hemisuccinate.

[0109] The coating may include a single type of lipid, or a combination of two or more lipids, or a combination of one or more lipids with other materials.

[0110] c. Polyether and polyquaternium salt

[0111] The coating can be or include a polyether. Exemplary polyethers include, but are not limited to, oligomers and polymers of ethylene oxide. In a preferred embodiment, the polyether is polyethylene glycol (PEG). PEG is prepared by the polymerization reaction of ethylene oxide and is commercially available in a wide molecular weight range of 300 g / mol to 10,000,000 g / mol and can have a branched, star-shaped or comb-shaped geometry. The number typically included in the PEG name indicates its average molecular weight (e.g., the average molecular weight of a PEG with n=9 is approximately 400 daltons and is labeled PEG 400.) Most PEGs include molecules with a molecular weight distribution (i.e., they are polydisperse). The size distribution can be statistically characterized by its weight average molecular weight (Mw) and its number average molecular weight (Mn), and the ratio of weight average molecular weight to number average molecular weight is called the polydispersity index (Mw / Mn). Mw and Mn can be measured by mass spectrometry. In some embodiments, PEG is amino (polyethylene glycol) (also known as PEG amine).

[0112] In some embodiments, the PEG or PEG amine is up to about 25,000 or more. In some embodiments, the PEG or PEG amine is about PEG 350 to about PEG 25,000, or about PEG 350 to about PEG 20,000. In some embodiments, the PEG or PEG amine is about PEG 350 to about PEG 5000, or about PEG 750 to about PEG 5000, or about PEG 1000 to PEG 3000. In a specific embodiment, the PEG is PEG 2000.

[0113] In certain embodiments, the coating is a polyether-lipid (eg, phospholipid) conjugate coating.

[0114] In certain embodiments, coating comprises one or more polyquaternium salts or is formed by them.Polyquaternium salt is that the International Nomenclature for Cosmetic Ingredients (International Nomenclature for Cosmetic Ingredients) names several polycationic polymers used in the personal care industry.Polyquaternium salt is a new word, and it is used to emphasize that there is quaternary ammonium center in the polymer.INCI has approved at least 40 kinds of different polymers to use the polyquaternium salt name.Different polymers are distinguished with the numerical value behind the word "polyquaternium salt", and for example, comprise, polyquaternium-1 to polyquaternium-20, polyquaternium-22, polyquaternium-24, polyquaternium-27 to polyquaternium-37, polyquaternium-39 and polyquaternium-42 to polyquaternium-47.In a particular embodiment, polyquaternium salt is polyquaternium-7, polyquaternium-10 or polyquaternium-30.

[0115] d. Charge modification part

[0116] The coating and / or shell can include a charge modification portion, for example, at the end of some or all of the molecules forming it. For example, it can be formed by a material having a structure AX, wherein A is a hydrophobic molecule or a hydrophobic polymer, and X is a terminal portion that imparts a charge (e.g., a negative charge) to the particle. The material can have an ABX structure, wherein A is a hydrophobic molecule or a hydrophobic polymer, B is a hydrophilic molecule or a hydrophilic polymer, and X is a terminal portion that imparts a charge (e.g., a negative charge). In some embodiments, the shell includes an anionic lipid; a negatively charged portion connected to a cation, a neutral lipid, an anionic lipid, and / or a linker such as PEG; or a combination thereof. In a particular embodiment, the terminal portion is an acidic group or an anionic group hanging on a hydrophilic group (PEG). Acidic groups include, for example, carboxylic acids, protonated sulfates, protonated sulfonates, protonated phosphates, monoprotonated or diprotonated phosphonates, and monoprotonated or diprotonated hydroxamic acids. Anionic groups include, for example, carboxylates, sulfates, sulfonates, mono- or di-deprotonated phosphates, mono- or di-deprotonated phosphonates, and hydroxamic acids. Positively charged moieties include, but are not limited to, primary, secondary, and tertiary amines, guanidine groups, imine groups, and imidazolyl groups.

[0117] In some embodiments, the coating is formed partially or entirely of a material comprising a lipid (eg, a phospholipid such as DSPE conjugated to PEG conjugated to a negatively charged terminal moiety such as COOH).

[0118] D. Targeting agents and other functional molecules

[0119] Functional molecules can be bound, linked, conjugated or otherwise attached directly or indirectly to the disclosed particles.One type of functional element is a targeting molecule.

[0120] For example, the disclosed particles can also be injected systemically and rely on the passive or active targeting of the NP to the target tissue. Therefore, in some embodiments, the particle includes a targeting agent, most typically conjugated to one or more components of the coating. The targeting moiety can specifically identify or bind to a target molecule that is specific to a cell type, tissue type, or organ. The target molecule can be or target a cell surface polypeptide, lipid, glycolipid, or its ligand. The targeting agent can be covalently bound to the nanoparticle directly or indirectly through a linker.

[0121] 1. Exemplary Forms of Targeting Agents

[0122] The targeting molecule can be a protein, peptide, nucleic acid molecule, sugar or polysaccharide that binds to a receptor or other molecule on the surface of the target cell. The degree of specificity and affinity of binding to the transplant can be adjusted by selecting the targeting molecule. For example, antibodies are very specific. These can be polyclonal, monoclonal fragments, recombinant antibodies or single-chain antibodies, many of which are commercially available or easily obtained using standard techniques.

[0123] In some embodiments, the targeting agent is an antibody. The term "antibody" refers to a natural or synthetic antibody that selectively binds to a target antigen. The term includes polyclonal antibodies and monoclonal antibodies. The antibody can be any type of immunoglobulin known in the art. For example, the antibody can be any isotype, for example, IgA, IgD, IgE, IgG, IgM, etc. The antibody can be monoclonal or polyclonal. The antibody can be a naturally occurring antibody, for example, an antibody isolated and / or purified from a mammal (e.g., mouse, rabbit, goat, horse, chicken, hamster, human, etc.). Alternatively, the antibody can be a genetically engineered antibody, for example, a humanized antibody or a chimeric antibody or a fragment, variant or fusion protein thereof. The antibody can be in monomeric or polymeric form.

[0124] In addition to intact immunoglobulin molecules, the term "antibody" also includes fragments or polymers or fusions of those immunoglobulin molecules and human or humanized immunoglobulin molecule versions that selectively bind to the target antigen. Exemplary fragments and fusions include, but are not limited to, single-chain antibodies, single-chain variable fragments (scFv), double scFv, triple scFv, bifunctional antibody fragments, trifunctional antibodies, tetrafunctional antibody fragments, disulfide-linked Fv (sdFv), Fab', F(ab')2, Fv, and single domain antibody fragments (sdAb).

[0125] In some embodiments, the targeting moiety can be or include one, two or more scFvs. For example, the targeting moiety can be an scFv or a dual scFv.

[0126] 2. Exemplary Methods for Linking Targeting Agents

[0127] Targeting moieties, therapeutic molecules and other functional moieties can be coupled to the particles using standard techniques. For example, the moieties and molecules can be coupled directly or indirectly to the shell or coating.

[0128] Functionality refers to the conjugation of the ligand to the particle surface through functional chemical groups (carboxylic acids, aldehydes, amines, sulfhydryls, and hydroxyls) present on the particle surface and the ligand to be attached. Functionality can be introduced into the particle in at least two ways. The first is during the preparation of the particle, for example, during the introduction of the shell and / or chemical group coating. The second is post-particle preparation, by directly cross-linking the particle and ligand with a homobifunctional or heterobifunctional cross-linking agent. This second procedure can use suitable chemistry and a class of cross-linking agents (CDI, EDAC, glutaraldehyde, etc., as discussed in detail below) or any other cross-linking agent, which couples the ligand to the particle surface after preparation by chemical modification of the particle surface.

[0129] One useful method involves "activating" hydroxyl groups on polymer chains with the agent carbonyldiimidazole (CDI) in an aprotic solvent such as DMSO, acetone, or THF. CDI forms an imidazolylcarbamate complex with the hydroxyl groups, which can be displaced by binding to free amino groups of ligands such as proteins. This reaction is an N-nucleophilic substitution and results in the formation of a stable N-alkylcarbamate linkage between the ligand and the polymer. Typically, "coupling" of the ligand to the "activated" polymer is maximized in the pH range of 9-10 and typically requires at least 24 hours. The resulting ligand-polymer complex is stable and resists hydrolysis for extended periods of time.

[0130] Another coupling method involves combining 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) or "water-soluble CDI" with N-hydroxysulfosuccinimide (sulfo-NHS) to couple the exposed carboxyl groups of the polymer to the free amino groups of the ligand in an all-aqueous environment at a physiological pH of 7.0. In short, EDAC and sulfo-NHS form activated esters with the carboxylic acid groups of the polymer, which react with the amine end of the ligand to form a peptide bond. The resulting peptide bond is resistant to hydrolysis. Using sulfo-NHS in the reaction increases the efficiency of EDAC coupling tenfold and provides particularly mild conditions, ensuring the vitality of the ligand-polymer complex.

[0131] By using either of these methods, virtually any polymer containing hydroxyl or carboxyl groups can be "activated" in a suitable solvent system that does not dissolve the polymer matrix (ie, shell and / or coating).

[0132] A useful coupling procedure for connecting ligands with free hydroxyl and carboxyl groups to polymers involves the use of the cross-linking agent divinyl sulfone. This method is suitable for connecting sugars or other hydroxy compounds with bioadhesive properties to hydroxyl matrices. In short, activation involves reacting divinyl sulfone with the hydroxyl groups of the polymer to form a vinylsulfonyl ethyl ether of the polymer. The vinyl group will couple with alcohols, phenols, and even amines. Activation and coupling occur at a pH of 11. This connection is stable in the pH range of 1-8 and is suitable for transport through the intestine.

[0133] Any suitable coupling method known to those skilled in the art for coupling ligands to polymers via double bonds, including the use of UV cross-linking, can be used to attach the molecule to the polymer.

[0134] The coupling is preferably performed by covalent bonding, but may also be indirect, for example, via a linker attached to a polymer, or by an interaction between two molecules such as streptavidin and biotin. It may also be performed by electrostatic attraction via dip coating.

[0135] In the following experiments, antibodies were coupled to the coated particles using EDC / NHS chemistry.

[0136] 3. Exemplary Target Molecules

[0137] a. Targeting antigen-presenting cells

[0138] In a preferred embodiment, the targeting agent helps to target the nanoparticles to antigen presenting cells such as dendritic cells. Among the main types of antigen presenting cells (B cells, macrophages and DC cells), the effect of DC cells is the strongest and is responsible for starting all antigen-specific immune responses. A biological characteristic of DC is that it can sense the conditions of encountering antigens, thereby starting the process of DC maturation. DC uses the receptors of various microorganisms and inflammatory products, and according to the nature of the pathogens (viruses, bacteria, protozoa) encountered, reacts to antigen exposure in different ways. When presenting antigens in the lymph nodes, the change of cytokine release pattern can pass this information to T cells, thereby changing the type of T cell response caused. Therefore, targeting DC not only generally provides the opportunity of quantitatively enhancing antigen delivery and antigen response, but also qualitatively controls the nature of immune response according to the expected vaccination results.

[0139] Dendritic cells express a variety of cell surface receptors that can mediate endocytosis of bound antigens. Targeting exogenous antigens to internalizing surface molecules on systemically distributed antigen-presenting cells can facilitate antigen uptake and overcome a major limiting step in immunization, and therefore vaccination.

[0140] Dendritic cell targeting molecules include monoclonal or polyclonal antibodies or fragments thereof that recognize and bind to epitopes displayed on the dendritic cell surface. Dendritic cell targeting molecules also include ligands that bind to cell surface receptors on dendritic cells. One such receptor, lectin DEC-205, has been used in vitro and in mice to enhance both humoral (antibody-based) and cellular (CD8 T cell) responses by 2-4 orders of magnitude (Hawiger et al., Journal of Experimental Medicine (J. Exp. Med.), 194 (6): 769-79 (2001); Bonifaz et al., Journal of Experimental Medicine, 196 (12): 1627-38 (2002); Bonifaz et al., Journal of Experimental Medicine, 199 (6): 815-24 (2004)). In these experiments, antigens were fused to anti-DEC205 heavy chains and immunized using recombinant antibody molecules.

[0141] A variety of other endocytic receptors, including mannose-specific lectin (mannose receptor) and IgG Fc receptor, have also been targeted in this manner with similar enhancement of antigen presentation efficiency. Other suitable receptors that may be targeted include, but are not limited to, DC-SIGN, 33D1, SIGLEC-H, DCIR, CD11c, heat shock protein receptors, and scavenger receptors.

[0142] Other receptors that may be targeted include toll-like receptors (TLRs). TLRs recognize and bind pathogen-associated molecular patterns (PAMPs). PAMPs target TLRs on the surface of dendritic cells and send signals internally, thereby potentially increasing DC antigen uptake, maturation, and T cell stimulation capabilities. PAMPs conjugated or co-encapsulated with the particle surface include: unmethylated CpG DNA (bacteria), double-stranded RNA (viruses), lipopolysaccharides (bacteria), peptidoglycans (bacteria), lipoarabinomannan (bacteria), zymosan (yeast), mycoplasma lipoproteins such as MALP-2 (bacteria), flagellin (bacteria), poly (inosinic-cytidylic) acid (bacteria), lipoteichoic acid (bacteria), or imidazoquinoline (synthetic). Therefore, in some embodiments, the disclosed nanoparticles are preferably conjugated with a targeting moiety to enhance DC uptake of the nanoparticles. In one embodiment, the nanoparticles are conjugated with antibodies that specifically bind to molecules on the surface of the DC. Antigens present on the surface of DCs include, but are not limited to, DEC-205 (CD-205), DC-SIGN, mannose receptor (MR), Fc receptor, and CD40. For example, anti-DEC-205 antibodies can be conjugated to carbon nanoparticles to enhance the uptake of nanoparticles by DCs. Antibodies against CD-205, DC-SIGN, MR, and CD40 are commercially available (Bio-Rad, Catalog No. MCA4755 (anti-CD205); R&D systems; Catalog No. MAB161 (anti-DC-SIGN); Abcam, an8918 (anti-MR antibody). CP-870,893 (Pfizer) is a fully humanized CD40 agonist IgG2 mAb that has both immune-mediated and non-immune-mediated effects on tumor cell death (Gladue et al., J Clin Oncol. Oncol. 2006; 24(18S): 103s). SGN-40 is a humanized IgG1 immunoglobulin and a partial agonist of CD40 that induces apoptosis and antibody-dependent cellular cytotoxicity against a panel of malignant B cell lines in vitro and causes tumor regression in human multiple myeloma and lymphoma xenograft models in vivo (Tai et al., Cancer Res. 2004; 64(8): 2846-52; Law et al., Cancer Res. 2005; 65(18): 8331-8 and Kelley et al., Br J Pharmacol. 2006; 148(8): 1116-23). ​​In another embodiment, the nanoparticles can be conjugated to a receptor ligand, wherein the corresponding receptor is expressed on the surface of DCs. For example, DC receptors can include, but are not limited to, ICAM-2 and PDI.

[0143] In some of the following embodiments, the targeting agent is anti-CD205 targeting dendritic cells.

[0144] b. Targeting T cells and other immune cells

[0145] In some embodiments, the targeting moiety targets a T cell. The T cell can be an effector cell (e.g., a cytotoxic cell, a helper cell, a regulatory cell, or a combination thereof), a memory T cell, a gamma-delta T cell (gamma delta T cell), a follicular helper T cell (Tfh), a natural killer T cell (NKT cell), or a combination thereof.

[0146] Targets include, but are not limited to, CD3, CD4, CD8, CD103, CXC motif chemokine receptor 6 (CXCR6), CD69, PD-1, CD90, TIGIT, CCR7, CD45RA, CD45RO, CD62L, CD95, 4-1BB, LAG-3, TIM-3, and CTLA4.

[0147] In certain embodiments, the target T cells are or include CD8 T cells.

[0148] Exemplary T cell targeting molecules are discussed in U.S. Published Application No. 20210386782.

[0149] Exemplary antibodies are discussed in more detail below. It should be understood that not only the antibodies themselves can be used in the disclosed compositions and methods, but also the complementary determining regions (CDRs), preferably the complementary determining regions in the heavy and light chain variable region frameworks, and in some examples the entire heavy and light chain variable regions, can be used to form other antibody formats discussed herein, including but not limited to humanized and / or chimeric antibodies, fusion proteins (such as scFv), etc. Therefore, this document clearly provides such antibodies and antibody fragments for each of the exemplary antibodies, including CDRs, preferably in their natural orientation, preferably in suitable heavy and light chain variable regions.

[0150] CD3

[0151] All T cells express CD3. GenBank accession numbers for exemplary sequences of human CD3 proteins include, for example, T cell surface glycoprotein CD3 delta chain isoform A P_000723.1 precursor; T cell surface glycoprotein CD3 delta chain isoform BNP_001035741.1; precursor T cell surface glycoprotein CD3 epsilon chain P07766.2 precursor; T cell surface glycoprotein CD3 gamma chain NP_000064.1 precursor; T cell surface glycoprotein CD3 zeta chain isoform 1 NP_932170.1 precursor and T cell surface glycoprotein CD3 zeta chain isoform 2 NP_000725.1 precursor.

[0152] Exemplary anti-CD3 antibodies include, but are not limited to, those disclosed in US20150166661, US20170204194, U.S. Pat. Nos. 7,728,114, 8,551,478, US20140193399, US20030216551, US20060275292, WO 2017136659, and WO 2016179003. Anti-CD3 antibodies specific for human CD3 that are available from commercial suppliers include, but are not limited to, clone 289-13801 (Product No. ABIN234581, Antibodies-Online), clone 4AOKT3 (Product No. ABIN2145039, Antibodies-Online), clone 4D10A6 (Product No. ABIN969472), clone B477 (Product No. ABIN965782, Antibodies-Online), clone B-B11 (Product No. ABIN1383795, Antibodies-Online), clone hCD3 (Product No. ABIN2136389, Antibodies-Online), clone HIT3a (Product No. ABIN2136387, Antibodies-Online), clone Okt 03 (Product No. ABIN457398, Antibodies-Online), clone UCHT1 (Product No. ABIN135720, Antibodies-Online), clone BC3 (Product No. 830301, BioLegend), clone Hu113 (Product No. MAB9929-100, R&D Systems), clone B-B11 (Product No. AM31215PU-N, Origene), clone N26-R (Product No. NBP1-79054, Novus Biologicals Canada), clone 1A7E5G5 (Product No. 10977-MM03, Sino Biological Inc), clone UCHT-1 (Product No. T-1363, BMA Biomedicals).

[0153] CD4

[0154] CD4 is expressed by helper T cells. In diseases where CD4 T cells preferentially contribute to pathology, targeting this antigen can be used to selectively deplete CD4 T cells. For example, the malignant T cells in cutaneous T-cell lymphoma are often CD4+, and targeting these cells can be used to selectively deplete malignant T cells from the skin while sparing the CD8+ T cell population.

[0155] The sequence of the human CD4 protein is available in GenBank at accession number NP_000607.1. Anti-CD4 antibodies include, but are not limited to, those disclosed in U.S. Pat. Nos. 7,452,534, 5,871,732, 8,877,913, 8,399,621, 7,947,272, 7,452,981, 8,440,806, 8,586,715, 8,673,304, and 8,685,651. Anti-CD4 antibodies specific for human CD4 that are available from commercial suppliers include, but are not limited to, clone 8 (product number 10400-MM08, Sino Biological Technology Co., Ltd.), clone 22 (product number 10400-MM22, Sino Biological Technology Co., Ltd.), clone 6F7B4C5 (product number 10400-MM03, Sino Biological Technology Co., Ltd.), clone CE9.1 (product number A1091-200, Biovision Inc.), clone CL0395 (product number AMAb90754, Atlas Antibodies), clone 34915 (product number MAB3791, R&D Systems), clone 34930 (product number MAB379-100, R&D Systems), clone 10B5 (product number GTX84720, GeneTex), clone 13B8.2 (product number GTX44212, GeneTex), clone MEM-241 (product number GTX21089, GeneTex), clone 4A11 (product number ABIN2136522, Antibodies Online), clone 4B12 (product number ABIN180655, Antibodies Online), and clone 6Eli) (product number ABIN2136524, Antibodies Online).

[0156] CD8

[0157] CD8 is expressed by cytotoxic T cells. In some inflammatory diseases, such as allogeneic transplant rejection, CD8+ T cells are considered to be the main cause of tissue damage (Harper, SJ et al., (2015). Proceedings of the National Academy of Sciences of the United States of America (Proc Natl Acad Sci USA) 112 (41): 12788-12793). Therefore, according to the biology of the inflammatory process, it may be desirable to exhaust CD8+ T cells without damaging other T cell subsets. The sequence of human CD8 protein can be obtained in the accession number NP_001759.3 of GenBank. Anti-CD8 antibodies include but are not limited to those disclosed in: U.S. Patent No. 9,518,131, WO9015152 and US20090304659. Anti-CD8 antibodies specific for human CD8 that are available from commercial suppliers include, but are not limited to, clone C8 / 144B (Product No. 925-MSM2-P1, Enquire Bioreagents), clone C8 / 468 (Product No. 925-MSM1-P1, Enquire Bioreagents), clone 37006 (Product No. MAB1509, R&D Systems), clone 2ST8.5H7 (Product No. GTX75282, GeneTex), clone LT8 (Product No. LT8, GeneTex), clone OKT-8 (Product No. GTX14198, GeneTex), clone Bu88 (Product No. AM05583PU-N, Origene Technologies), clone B-Z31 (Product No. AM31251PU-N, Origene Technologies), clone MCD8 (Product No. No. AM39011PU-N, Aurigene Technologies), clone RAVB3 (Product No. AM06078PU-N, Aurigene Technologies), clone RFT-8 (Product No. AM08158PU-N, Aurigene Technologies), clone 14 (Product No. NBP2-50467, Novus Biologics, Canada), clone X107 (Product No. NBP2-50469, Novus Biologics, Canada) and clone UCH-T4 (Product No. NBP2-50468, Novus Biologics, Canada).

[0158] CD103

[0159] CD103 is expressed by resident memory T cells (TRMs) in peripheral tissues in both humans and mice and is enriched on TRMs that populate mucosal and epithelial cells (Sathaliyawala, T. et al., (2013). Immunity (1) 38: 187-197). CD103 is also known as integrin αE subunit (ITGAE). The sequence of human CD103 protein is available in GenBank under accession number NP_002199.3. Anti-CD103 antibodies include, but are not limited to, those disclosed in the following documents: US20110142861, US20110142860, and US20050266001. Anti-CD103 antibodies specific for human CD103 available from commercial suppliers include, but are not limited to, clone B-Ly7 (Product No. NBP1-43370H, Novus Biologics, Canada), clone BP6 (Product No. NBP2-50446H, Novus Biologics, Canada), clone LF61 (Product No. NB100-65272H, Novus Biologics, Canada), clone AX.14 (Product No. AM05205PU-N, OriGene Technologies), clone B-ly7 (Product No. AM39027PU-N, OriGene Technologies), clone 3H1798 (Product No. C2445-63A, United States Biotechnology Corporation), and clone LF61 (Product No. NB100-65272H, OriGene Technologies, Inc.). Biological)), clone 3H1797 (product number C2445-63, American Biological), clone 3H1797 (product number C2445-63J1, American Biological), and clone 3H1797 (product number C2445-63K, American Biological).

[0160] CXCR6

[0161] CXCR6 is expressed by TRMs in tissues and is required for their optimal development (Zaid, A., (2017). J Immunol 199(7):2451-2459). The sequence of human CXCR6 protein is available in GenBank under accession number NP_006555.1. Anti-CXCR6 antibodies include, but are not limited to, those disclosed in U.S. Patent No. 9,872,905 and WO 2004019046. Anti-CXCR6 antibodies specific for human CXCR6 available from commercial suppliers include, but are not limited to, clone 56811 (Product No. MAB699-100, R&D Systems), clone MM0226-2B44 (Product No. NBP2-12243, R&D Systems), clone 14L333 (Product No. 216429, R&D Systems), clone K041E5 (Product No. 356001, Biogen), clone K041E5 (Product No. 356002, Biogen), and polyclonal antibodies specific for human CXCR6 (e.g., Product No. GTX77935, GeneTex; Product No. SP1286P, OriGene Technologies; Product No. NLS1102, Novus Biologics, Canada; Product No. abx148716, Abbexa; Product No. 170358, American Biotech).

[0162] CD69

[0163] CD69 is a surface molecule that is consistently expressed at high levels by T cells in all tissues tested to date, regardless of their activation state, and is the most widely expressed T cell marker in human skin (Watanabe, R. et al. (2015). Science Translational Medicine 7(279):279ra239). CD69 is also expressed by activated T cells in tissues, for example, at sites of inflammation, and is upregulated within 12 hours of in vitro stimulation. At least in human skin, CD69 is not expressed by circulating T cells or FOXP3 regulatory T cells (Clark, RA et al. (2007). Blood 109(1):194-202). The sequence of the human CD69 protein is available in GenBank under accession number NP_001772.1. Anti-CD69 antibodies known in the art and useful for the methods of the present invention include, but are not limited to, those disclosed in US20150118237, U.S. Patent No. 8,440,195, US20130224111, U.S. Patent Nos. 7,867,475, 8,182,816, WO 2018074610, and WO 2018150066.Anti-CD69 antibodies specific for human CD19 are available from commercial suppliers, including but not limited to clone 4AF50 (product number ABIN2145225, Antibodies-Online), clone FN50 (product number ABIN302090, Antibodies-Online), clone 298633 (product number MAB2359-SP, R&D Systems), clone 298614 (product number MAB23591, R&D Systems), monoclonal anti-CD69 antibody (product number AM03132PU-N, OriGene Technologies), clone 15B5G2 (product number NBP2-25242SS, Novus Biologics, Canada), clone 7H192 (product number C2424-01E, U.S. Biological Life Sciences, Inc.), and monoclonal anti-CD69 antibody (product number AM03132PU-N, OriGene Technologies, Inc.). Sciences), clone 4H3 (Product No. 124672, American Biosciences), clone 7H192 (Product No. C2424-01, American Biosciences), clone HP-4B3 (Product No. LS-C134543-100, LifeSpan BioSciences), or select a polyclonal antibody specific for human CD69 (for example, Product No. ABIN2136942, Antibodies-Online; Product No. AF2359, R&D Systems; Product No. GTX37447, GeneTex; Product No. AP21168PU-N, OriGene Technologies; Product No. 124671, American Biosciences).

[0164] PD-1 and CTLA4

[0165] PD-1 and CTLA4 are proteins found on T cells that help control the body's immune response. When PD-1 binds to another protein called PD-L1, it helps prevent T cells from killing other cells, including cancer cells. Similarly, when CTLA-4 binds to another protein called B7, it also helps prevent T cells from killing other cells. Some anti-cancer drugs called immune checkpoint inhibitors are used to block PD-1 and CTLA4. When these proteins are blocked, the "brake" on the immune system is released, and the ability of T cells to kill cancer cells is enhanced.

[0166] Anti-PD-1 and anti-CTLA antibodies are known in the art and are discussed in more detail elsewhere herein. Any of these antibodies can be used as an active agent and / or targeting moiety.

[0167] CD90

[0168] Thy-1 or CD90 (cluster of differentiation 90) is a conserved cell surface protein with a molecular weight of 25-37 kDa, heavily N-glycosylated, glycophosphatidylinositol (GPI)-anchored, and single V-like immunoglobulin domain. It was originally discovered as a thymocyte antigen. Antibodies to human CD90 are known in the art, see, for example, F15-42-1 (e.g., Thermo Fisher product number MA5-16671), eBio5E10 (5E10) (e.g., Thermo Fisher product number 11-0909-42), 2V9S6 (Thermo Fisher product number MA5-42657), SU35-07 (e.g., Thermo Fisher product number MA5-32124), HL1766 (Thermo Fisher product number MA5-47174), and the like.

[0169] TIGIT

[0170] TIGIT is expressed in humans by activated CD8+T and CD4+T cells, natural killer (NK) cells, regulatory T cells (Treg) and follicular T helper cells. In sharp contrast to DNAM-1 / CD226, TIGIT expression in naive T cells is very weak. Antibodies to human TIGIT are known in the art, see, for example, MBSA43 (e.g., Thermo Fisher product number 12-9500-42), BLR047F (e.g., Thermo Fisher product number A700-047), OTI3B6 (e.g., Thermo Fisher product number CF812550), OTI5G1 (e.g., Thermo Fisher product number CF812567), OTI3A10 (e.g., Thermo Fisher product number CF813029), etc.

[0171] CD45RA and CD45RO

[0172] The tyrosine phosphatase CD45 generates isoforms of varying molecular weight (180-220 kDa) through alternative splicing that are differentially expressed on hematopoietic cells (LaSalle and Haflter et al., Cell Immunol. 1991 Nov;138(1):197-206. doi:10.1016 / 0008-8749(91)90144-z.). Monoclonal antibodies reactive with either the 180-kDa (UCHL-1, CD45RO) or the 200-kDa to 220-kDa (2H4, CD45RA) isoforms have been used to subdivide T cell populations based on the expression of one or the other of these two epitopes. CD45RA T cells have a “naive” character, that is, they are unresponsive to recall antigens and are prominent in cord blood, whereas CD45RO T cells are considered “memory” T cells because they proliferate in response to recall antigens and increase after PHA activation in cord blood.

[0173] Antibodies to human CD45RA are known in the art, see, for example, HI100 (e.g., Thermo Fisher Scientific Product No. 11-0458-42), MEM-56 (e.g., Thermo Fisher Scientific Product No. MHCD45RA01), 4KB5 (e.g., Thermo Fisher Scientific Product No. MA5-12490), JS-83 (e.g., Thermo Fisher Scientific Product No. 11-9979-42), etc.

[0174] Antibodies to human CD45RO are known in the art, see for example, UCHL1 (e.g., Thermo Fisher Scientific Product No. MA5-11532), IL-A116 (e.g., Thermo Fisher Scientific Product No. MA5-28402), T200, 797 (e.g., Thermo Fisher Scientific Product No. 5788-MSM7-P1), etc.

[0175] CD62L

[0176] L-selectin, also known as CD62L, is a cell adhesion molecule found on the cell surface of leukocytes and blastocysts. L-selectin is expressed on naive T cells and is rapidly shed after T cell priming. Once cytotoxic T cells leave the lymph nodes, L-selectin expression is reactivated. Mature central memory T cells express L-selectin, while effector memory cells do not. L-selectin is also expressed by naive B cells, and its absence distinguishes activated B cells destined to differentiate into antibody-secreting cells. L-selectin is expressed on circulating neutrophils and is shed after neutrophil priming. L-selectin expression in neutrophils decreases with neutrophil aging. Monocytes typically express high levels of L-selectin while circulating. L-selectin shedding from monocytes occurs during transendothelial migration.

[0177] Antibodies to human CD62L are known in the art, see for example, LT-TD180 (e.g., Thermo Fisher Scientific Product No. MA1-19715), DREG56 (e.g., Thermo Fisher Scientific Product No. 17-0629-42), IVA94 (e.g., Thermo Fisher Scientific Product No. MA5-44129), etc.

[0178] CD95

[0179] The Fas receptor, also known as Fas, FasR, apoptosis antigen 1 (APO-1 or APT), cluster of differentiation 95 (CD95), or tumor necrosis factor receptor superfamily member 6 (TNFRSF6), is a protein in humans encoded by the FAS gene. CD95 (Fas / APO-1) and its ligand, CD95L, have long been considered a death receptor / death ligand system that mediates apoptosis induction to maintain immune homeostasis. Furthermore, these molecules are important for the immune clearance of virus-infected cells and cancer cells.

[0180] Antibodies to human CD95 are known in the art, see, for example, JJ0942 (e.g., Thermo Fisher Scientific Product No. MA5-32489), DX2 (e.g., Thermo Fisher Scientific Product No. 11-0959-42), H.831.6 (e.g., Thermo Fisher Scientific Product No. MA5-14882), SM1 / 23 (e.g., Thermo Fisher Scientific Product No. 17-0959-42), etc.

[0181] 4-1BB

[0182] 4-1BB (CD137; TNFRS9), an activation-induced co-stimulatory molecule, is an important regulator of the immune response. 4-1BB was originally discovered in activated cells and was therefore initially referred to as induced lymphocyte activation (ILA) in humans, but it is also constitutively expressed in a variety of cells, albeit at lower levels, including Foxp3+ Tregs and DCs (Vinay and Kwon BMB Rep. 2014 March; 47(3): 122-129).

[0183] Antibodies to human 4-1BB are known in the art, see, e.g., 4B4 (e.g., Thermo Fisher Scientific Product No. 11-1379-42), ARC1963 (e.g., Thermo Fisher Scientific Product No. MA5-38063), BBK-2 (e.g., Thermo Fisher Scientific Product No. MA5-13739), 4H3 (e.g., Thermo Fisher Scientific Product No. 25-5906-42), 2G1 (e.g., Thermo Fisher Scientific Product No. MA5-42580), 819 (e.g., Thermo Fisher Scientific Product No. MA5-46628), etc.

[0184] LAG-3

[0185] LAG-3 (CD223) is a cell surface molecule expressed on activated T cells (Huard et al. Immunogenetics 39:213-217, 1994), NK cells (Triebel et al. J Exp Med 171:1393-1405, 1990), B cells (Kisielow et al. Eur J Immunol 35:2081-2088, 2005), and plasmacytoid dendritic cells (Workman et al. J Immunol 182:1885-1891, 2009), where it plays an important role in the function of these lymphocyte subsets. Furthermore, the interaction between LAG-3 and its primary ligand, MHC class II, is thought to play a role in regulating dendritic cell function (Andreae et al. J Immunol 168:3874-3880, 2002), and recent preclinical studies have documented a role for LAG-3 in CD8 T cell exhaustion (Blackburn et al. Nat Immunol 10:29-37, 2009).

[0186] Antibodies to human LAG-3 are known in the art, see, for example, 3DS223H (e.g., Thermo Fisher Scientific Product No. 17-2239-42), BLR028F (e.g., Thermo Fisher Scientific Product No. A700-028), 1F14 (e.g., Thermo Fisher Scientific Product No. 80867-1-RR100UL), OTI8F6 (e.g., Thermo Fisher Scientific Product No. A700-027), etc.

[0187] TIM-3

[0188] Tim-3 is a co-inhibitory receptor expressed on IFN-γ-producing T cells, FoxP3+ Treg cells, and innate immune cells (macrophages and dendritic cells) that has been shown to inhibit the responses of these cells upon interaction with its ligand (Das et al., Immunol Rev. 2017 Mar;276(1):97-111).

[0189] Antibodies to human TIM-3 are known in the art, see, for example, F38-2E2 (e.g., Thermo Fisher Scientific Product No. 78-3109-42), 4C4G3 (e.g., Thermo Fisher Scientific Product No. 60355-1-IG), 1E5 (e.g., Thermo Fisher Scientific Product No. MA5-32841), 1E6 (e.g., Thermo Fisher Scientific Product No. MA5-32839), 1E3 (e.g., Thermo Fisher Scientific Product No. 368-3109-42), etc.

[0190] E. Active agent

[0191] The disclosed particles can have molecular and even therapeutic effects without any additional active agents, and thus in some embodiments, the particles alone are the active material, and the particles do not include (i.e., do not contain) additional active agents. Alternatively, the particles can optionally include one or more active agents. For example, in some embodiments, the outer layer or coating is an active agent or includes an active agent. In some embodiments, one or more active agents are conjugated to a component of the hydrophilic layer, or otherwise connected to the surface of the hydrophilic layer, or bound to, loaded into, or encapsulated into the hydrophilic layer itself. In some such embodiments, the core of the particle still does not contain additional active agents. Additionally or alternatively, active agents, including but not limited to those discussed herein, can be separated from the particles and administered in different formulations (i.e., different mixtures) or the same formulation (i.e., the same mixture). Therefore, it is envisioned that the particles contain or do not contain active agents, the pharmaceutical compositions include separate particles containing or not containing active agents or particles further combined with active agents, and methods including administering the pharmaceutical compositions alone or in combination with one or more active agents (together or alone) to subjects in need are other adjunctive therapies. Any of the active agents provided in this section or elsewhere can exert any one or more of these effects.

[0192] The one or more active agents can be, for example, nucleic acids, proteins, and / or small molecules. Exemplary active agents include, for example, tumor antigens, CD4+ T cell epitopes, cytokines, chemotherapeutic agents, radionuclides, small molecule signal transduction inhibitors, photothermal antennae, immune danger signaling molecules, other immunotherapeutic agents, enzymes, antibiotics, antivirals, antiparasitic (helminth, protozoan) agents, growth factors, growth inhibitors, hormones, hormone antagonists, antibodies and biologically active fragments thereof (including humanized antibodies, single-chain antibodies, and chimeric antibodies), antigens and vaccine formulations (including adjuvants), peptide drugs, anti-inflammatory drugs, immunomodulators (including those that bind to Toll-like receptors (including but not limited to those that bind to IL-1 receptors), and / or IL-6 inhibitors. The present invention also includes ligands that bind to the innate immune system (including but not limited to CpG oligonucleotides), molecules that mobilize and optimize the adaptive immune system, molecules that activate or upregulate the action of cytotoxic T lymphocytes, natural killer cells and helper T cells, and molecules that inactivate or downregulate inhibitory or regulatory T cells), agents that promote the uptake of delivery vectors by cells (including dendritic cells and other antigen-presenting cells), nutritional drugs such as vitamins, and oligonucleotide drugs (including DNA, RNA, antisense, aptamers, small interfering RNA, ribozymes, external guide sequences of ribonuclease P and triplex-forming agents).

[0193] 1. Antigen

[0194] Antigens may be provided as a single antigen or in combination and may be derived from a tumor, an infectious agent or elsewhere. These may be particularly preferred additional agents when targeting antigen presenting cells.

[0195] a.Tumor antigens

[0196] Tumor antigens can be tumor-specific antigens (present only on tumor cells) or tumor-associated antigens (present on some tumor cells and in some normal cells).

[0197] Tumor-associated antigens can include, for example, products encoded by cellular oncogenes or products encoded by abnormally expressed proto-oncogenes (e.g., products encoded by neu, ras, trk, and kit genes), or mutant forms of growth factor receptors or receptor-like cell surface molecules (e.g., surface receptors encoded by the c-erb B gene). Other tumor-associated antigens include molecules that may be directly involved in the transformation event, or molecules that may not be directly involved in the oncogenic transformation event but are expressed by tumor cells (e.g., carcinoembryonic antigen, CA-125, melanoma-associated antigen, etc.) (see, e.g., U.S. Patent No. 6,699,475; Jager et al., Int. J. Cancer, 106:817-20 (2003); Kennedy et al., Int. Rev. Immunol., 22:141-72 (2003); Scanlan et al., Cancer Immun. 4:1 (2004)).

[0198] Genes encoding cell tumor-associated antigens include abnormally expressed cell oncogenes and proto-oncogenes. Generally, the products encoded by cell oncogenes are directly related to the transformation of cells. For example, the tumorigenic neu gene encodes a cell surface molecule involved in oncogenic transformation. Other examples include ras, kit, and trk genes. The products of proto-oncogenes (oncogenes formed after normal gene mutation) may be abnormally expressed (e.g., overexpressed), and this abnormal expression may be relevant to cell transformation. Therefore, the products encoded by proto-oncogenes can be targeted. Some oncogenes encode growth factor receptor molecules or growth factor receptor-like molecules, which are expressed on the surface of tumor cells. The cell surface receptor encoded by the c-erbB gene is an example. Other tumor-associated antigens may be directly involved or may not be involved in malignant transformation. However, some tumor cells express these antigens, and therefore they may be effective targets. Some examples are carcinoembryonic antigen (CEA), CA 125 (associated with ovarian cancer), and melanoma-specific antigens.

[0199] For example, in ovarian and other cancers, tumor-associated antigens can be detected in samples of readily available biological fluids such as serum or mucosal secretions. One such marker is CA125, a cancer-associated antigen that is also shed into the blood and can be detected in serum (e.g., Bast et al., N.Eng. J. Med., 309:883 (1983); Lloyd et al., Int. J. Canc., 71:842 (1997). CA125 levels have been measured in serum and other biological fluids, as well as other markers (e.g., carcinoembryonic antigen (CEA), squamous cell carcinoma antigen (SCC), tissue polypeptide levels of ovarian and other cancers to provide diagnostic and / or prognostic features for ovarian and other cancers (e.g., Sarandakou et al., Acta Oncol., 36:755 (1997); Sarandakou et al., Eur. J. Gynaecol. Oncol., 19:73 (1998); Meier et al., Anticancer Res., 19:74 (1998); Meier et al., Anticancer Res., 19:76 (1998); Meier et al., Anticancer Res., 19:7 ... Elevated serum CA125 may also be associated with neuroblastoma (e.g., Hirokawa et al., Surg. Today, 28:349 (1998), while elevated CEA and SCC may be associated with, among other things, colorectal cancer (Gebauer et al., Anticancer Res., 17(4B):2939 (1997)).

[0200] Mesothelin, a tumor-associated antigen defined by reactivity with the monoclonal antibody K-1, is present on most squamous cell carcinomas (including epithelial ovarian, cervical, and esophageal cancers) and mesotheliomas (Chang et al., Cancer Res, 52:181 (1992); Chang et al., Int. J. Cancer, 50:373 (1992); Chang et al., Int. J. Cancer, 51:548 (1992); Chang et al., Proc. Natl. Acad. Sci. USA, 93:136 (1996); Chowdhury et al., Proc. Natl. Acad. Sci. USA, 95:669 (1998)). Mesothelin can only be detected as a cell-associated tumor marker using MAb K-1 and is not found in soluble form in serum from ovarian cancer patients or in OVCAR-3 cell-conditioned medium (Chang et al., Int. J. Cancer, 50:373 (1992)). However, structurally related human mesothelin polypeptides also include tumor-associated antigen polypeptides, such as the unique mesothelin-related antigen (MRA) polypeptide, which is detected as a naturally occurring soluble antigen in the biological fluids of patients with malignant diseases (see WO 00 / 50900).

[0201] Tumor antigens may include cell surface molecules or cell surface molecules. Tumor antigens with known structures and known or described functions include the following cell surface receptors: HER1 (GenBank Accession No. U48722), HER2 (Yoshino et al., J. Immunol., 152:2393 (1994); Diss et al., Canc. Res., 54:16 (1994); GenBank Accession Nos. X03363 and M17730), HER3 (GenBank Accession Nos. U29339 and M34309), HER4 (Plowman et al., Nature , 366:473 (1993); GenBank accession numbers L07868 and T64105), epidermal growth factor receptor (EGFR) (GenBank accession numbers U48722 and KO3193), vascular endothelial growth factor (GenBank accession number M32977), vascular endothelial growth factor receptor (GenBank accession numbers AF022375, 1680143, U48801, and X62568), insulin-like growth factor-I (GenBank accession numbers X00173, X56774, X56773, X06043, European Patent No. GB 2241703), insulin-like growth factor-II (GenBank accession numbers X03562, X00910, M17863, and M17862), transferrin receptor (Trowbridge and Omary, Proc. Nat. Acad. USA, 78:3039 (1981); GenBank accession numbers X01060 and M11507), estrogen receptor (GenBank accession numbers M38651, X03635, X99101, U47678, and M12674), progesterone receptor (GenBank accession numbers X51730, X6906 8 and M15716), follicle-stimulating hormone receptor (FSH-R) (GenBank Accession Nos. Z34260 and M65085), retinoic acid receptor (GenBank Accession Nos. L12060, M60909, X77664, X57280, X07282, and X06538), MUC-1 (Barnes et al., Proc. Nat. Acad. Sci. USA, 86:7159 (1989); GenBank Accession Nos. M65132 and M64928), NY-ESO-1 (GenBank Accession Nos. AJ003149 and U87459), NA 17-A (PCT Publication No. WO 96 / 40039), Melan-A / MART-1 (Kawakami et al., Proc. Nat. Acad. Sci. USA, 86:7159 (1989); GenBank Accession Nos.Acad. Sci. USA, 91:3515 (1994); GenBank Accession Nos. U06654 and U06452), tyrosinase (Topal ian et al., Proc. Nat. Acad. Sci. USA, 91:9461 (1994); GenBank Accession No. M26729; Weber et al., J. Clin. Invest, 102:1258 (1998)), Gp-100 (Kawakami et al., Proc. Nat. Acad. Sci. USA, 91:3515 (1994); GenBank Accession No. S73003, Adema et al., J. Biol. Chem., 269:20126 (1994)), MAGE (van den Bruggen et al., Science, 254:1643 (1991); GenBank accession numbers U93163, AF064589, U66083, D32077, D32076, D32075, U10694, U10693, U10691, U10690, U10689, U10688, U10687, U10686, U10685, L18877 , U10340, U10339, L18920, U03735, and M77481), BAGE (GenBank Accession No. U19180; U.S. Patent Nos. 5,683,886 and 5,571,711), GAGE ​​(GenBank Accession Nos. AF055475, AF055474, AF055473, U19147, U19146, U19145, U19144, U1914 3 and U19142), any of the CTA receptors, particularly including the HOM-MEL-40 antigen encoded by the SSX2 gene (GenBank accession numbers X86175, U90842, U90841 and X86174), carcinoembryonic antigen (CEA, Gold and Freedman, "Journal of Experimental Medicine (J. Exp. Med)", 121: 439 (1985); GenBank accession numbers M59710, M 59255 and M29540), and PyLT (GenBank accession numbers J02289 and J02038); p97 (melanin transferrin) (Brown et al., J. Immunol., 127:539-46 (1981); Rose et al., Proc. Natl. Acad. Sci. USA, 83:1261-61 (1986)).

[0202] Additional tumor-associated antigens include prostate surface antigen (PSA) (U.S. Pat. Nos. 6,677,157; ​​6,673,545); β-human chorionic gonadotropin β-HCG) (McManus et al., Cancer Res, 36:3476-81 (1976); Yoshimura et al., Cancer, 73:2745-52 (1994); Yamaguchi et al., Br. J. Cancer, 60:382-84 (1989); Alfthan ... Res, 52:4628-33 (1992); glycosyltransferase β-1,4-N-acetylgalactosaminyltransferase (GalNAc) (Hoon et al., Int. J. Cancer, 43:857-62 (1989); Ando et al., Int. J. Cancer, 40:12-17 (1987); Tsuchida et al., J. Nat. J. Cancer, 41:12-17 (1987); atl. Cancer, 78:45-54 (1987); Tsuchida et al., J. Natl. Cancer, 78:55-60 (1987); NUC18 (Lehmann et al., Proc. Natl. Acad. Sci. USA, 86:9891-95 (1989); Lehmann et al., Cancer Research, 86:9891-95 (1989); =Res, 47:841-45 (1987); melanoma antigen gp75 (Vijayasardahi et al., J. Exp. Med, 171:1375-80 (1990); GenBank Accession No. X51455); human cytokeratin 8; high molecular weight melanoma antigen (Natali et al., Cancer, 59:55-63 (1987); keratin 19 (Datta et al., J. Clin. Oncol, 12:475-82 (1994)).

[0203] Tumor antigens of interest include antigens considered in the art to be "cancer / testis" (CT) antigens, which are immunogenic in subjects with malignant disease (Scanlan et al., Cancer Immun., 4:1 (2004)). CT antigens include at least 19 different antigen families, which contain one or more members and the members are capable of inducing an immune response, including but not limited to MAGEA (CT1); BAGE (CT2); MAGEB (CT3); GAGE ​​(CT4); SSX (CT5); NY-ESO-1 (CT6); MAGEC (CT7); SYCP1 (C8); SPANXB1 (CT11.2); NA88 (CT18); CTAGE (CT21); SPA17 (CT22); OY-TES-1 (CT23); CAGE (CT26); HOM-TES-85 (CT28); HCA661 (CT30); NY-SAR-35 (CT38); FATE (CT43) and TPTE (CT44).

[0204] Additional tumor antigens that can be targeted include tumor-associated or tumor-specific antigens, including but not limited to α-actinin-4, Bcr-Abl fusion protein, Casp-8, β-catenin, cdc27, cdk4, cdkn2a, coa-1, dek-can fusion protein, EF2, ETV6-AML1 fusion protein, LDLR-fucosyltransferase AS fusion protein, HLA-A2, HLA-A11, hsp70-2, KIAAO205, Mart2, Mum-1, 2 and 3, neo-PAP, myosin class I, OS-9, pml-RARα fusion protein, PTPRK, K-ras, N-ras, triosephosphate isomerase, Bage-1, Gage 3,4,5,6,7, GnTV, Herv-K-mel, Lage-1, Mage-A1,2,3,4,6,10,12, Mage-C2, NA-88, NY-Eso-1 / Lage-2, SP17, SSX-2 and TRP2-Int2, MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15 (58), CEA, RAGE, NY-ESO (LAGE), SCP-1, Home / Mel-40, PRAME, p53, H-Ras, HER-2 / neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein-Barr virus (Epstein Barrvirus) antigen, EBNA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA19-9, CA72-4, CAM17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p16, TAGE, PSMA, PSCA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, α-fetoprotein, 13HCG, BCA225, BTAA, CA125, CA15-3 (CA 27.29, BCAA), CA195, CA242, CA-50, CAM43, CD68, KP1, CO-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB, 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein, cyclophilin C-related protein), TAAL6, TAG72, TLP, and TPS. Other tumor-associated and tumor-specific antigens are known to those skilled in the art and are suitable for targeting by the disclosed fusion proteins.

[0205] Other examples of cancer-associated antigens include, but are not limited to, mesothelin, EGFRvIII, TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, GD2, GD3, BCMA, Tn Ag, prostate-specific membrane antigen (PSMA), ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, interleukin-11 receptor alpha (IL-11Ra), PSCA, PRSS21, VEGFR2, Lewis Y, CD24, platelet-derived growth factor receptor beta (PDGFRβ), SSEA-4, CD20, folate receptor alpha (FRa), ERBB2 (Her2 / neu), MUC1, epidermal growth factor receptor (EGFR), NCAM, prostate, PAP, ELF2M, ephrin B2 (EphrinB2), I GF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, EphA2, fucose GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor β, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, legumin, HPV E6, E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin and telomerase, PCTA-1 / galectin 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5 and IGLL1.

[0206] In other embodiments, the antigen is an antigen expressed by the neovascular system associated with the tumor. The antigen may be specific to the tumor neovascular system, or the expression level in the tumor neovascular system may be higher than that in the normal vascular system. Compared with the normal vascular system, exemplary antigens overexpressed by the tumor-associated neovascular system include but are not limited to VEGF / KDR, Tie2, vascular cell adhesion molecule (VCAM), endoglin, and α5β3 integrin / vitronectin. Other antigens overexpressed by the tumor-associated neovascular system compared to the normal vascular system are known to those skilled in the art and are suitable for being targeted by the disclosed fusion protein.

[0207] Suitable antigens are known in the art and are available from commercial government and scientific sources. Antigens may be purified or partially purified polypeptides derived from tumors or viral or bacterial sources. Antigens may be recombinant polypeptides produced by expressing DNA encoding the polypeptide antigen in a heterologous expression system.

[0208] b. Viral antigens

[0209] Viral antigens can be isolated from and or derived from viruses, including but not limited to viruses from any of the following virus families: Arenaviridae, Arterivirus, Astroviridae, Baculoviridae, Badnavirus, Barnaviridae, Birnaviridae, Bromoviridae, Bunyaviridae, Caliciviridae, Capillovirus, Caenorhabditis elegans, and Cynoviridae. rlavirus), Caulimovirus, Circoviridae, Closterovirus, Comoviridae, Coronaviridae (e.g., coronaviruses such as severe acute respiratory syndrome (SARS) virus), Corticoviridae, Cystoviridae, Deltavirus, Dianthovirus, Enamovirus, Filoviridae (e.g., Marburg virus, virus and Ebola virus (e.g., Zaire, Reston, Ivory Coast, or Sudan strains)), Flaviviridae (e.g., Hepatitis C virus, Dengue virus 1, Dengue virus 2, Dengue virus 3, and Dengue virus 4), Hepadnaviridae, Herpesviridae (e.g., human herpesviruses 1, 3, 4, 5, and 6, and cytomegalovirus), Hypoviridae, Iridoviridae, Leviviridae, Lipothrixviridae, Microviridae, Orthomyxoviridae (e.g.,The virus families include influenza A and B viruses, and influenza C viruses), Papovaviridae, Paramyxoviridae (e.g., measles, mumps, and human respiratory syncytial virus), Parvoviridae, Picornaviridae (e.g., poliovirus, rhinovirus, hepatovirus, and foot-and-mouth disease virus), Poxviridae (e.g., vaccinia and variola viruses), Reoviridae (e.g., rotavirus), Retroviridae (e.g., lentiviruses, such as human immunodeficiency virus (HIV) 1 and HIV 2), Rhabdoviridae (e.g., rabies virus, measles virus, respiratory syncytial virus, etc.), Togaviridae (e.g., rubella virus, dengue virus, etc.), and Totiviridae. Suitable viral antigens also include all or part of dengue protein M and dengue protein E, dengue D1NS1, dengue D1NS2 and dengue D1NS3.

[0210] Viral antigens can be derived from specific strains, such as papillomaviruses, herpes viruses, e.g., herpes simplex 1 and 2; hepatitis viruses, e.g., hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), delta hepatitis D virus (HDV), hepatitis E virus (HEV), and hepatitis G virus (HGV); tick-borne encephalitis viruses; parainfluenza viruses, varicella-zoster virus, cytomegalovirus, Epstein-Barr virus, rotavirus, rhinovirus, adenovirus, coxsackievirus, equine encephalitis virus, Japanese encephalitis virus, yellow fever virus, Rift Valley fever virus, and lymphocytic choriomeningitis virus.

[0211] c. Bacterial antigens

[0212] Bacterial antigens may be derived from any bacteria, including but not limited to Actinomyces, Anabaena, Bacillus, Bacteroides, Bdellovibrio, Bordetella, Borrelia, Campylobacter, Caulobacter, Chlamydia, Chlorobium, Chromatium, Clostridium, Corynebacterium, acterium), Cytophaga, Deinococcus, Escherichia, Francisella, Halobacterium, Heliobacter, Haemophilus, Haemophilus influenzae type B (HIB), Hyphomicrobium, Legionella, Leptospirosis, Listeria, Meningococcus A, B, and C andC), Methanobacterium, Micrococcus, Mycobacterium, Mycoplasma, Myxococcus, Neisseria, Nitrobacter, Oscillatoria, Prochloron, Proteus, Pseudomonas, Rhodospirillum, Rickettsia ettsia, Salmonella, Shigella, Spirillum, Spirochaeta, Staphylococcus, Streptococcus, Streptomyces, Sulfolobus, Thermoplasma, Thiobacillus, Treponema, Vibrio, and Yersinia.

[0213] d. Parasite antigens

[0214] Parasite antigens can be obtained from parasites, such as, but not limited to, antigens derived from Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia ricketsii, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydial psittaci, Chlamydial trachomatis, Plasmodium falciparum, Trypanosoma brucei, Entamoeba histolytica, Toxoplasma gondii, Trichomonas vaginalis, and Plasmodium falciparum. vaginalis and Schistosoma mansoni. These include sporozoan antigens, Plasmodium antigens, such as all or part of the circumsporozoite protein, sporozoite surface protein, liver stage antigen, apical membrane-associated protein or merozoite surface protein.

[0215] 2. Chemotherapy drugs

[0216] Exemplary active agents include, for example, chemotherapeutic agents, especially antitumor drugs. Most chemotherapeutic drugs can be divided into alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, monoclonal antibodies and other antitumor agents. In certain embodiments, the additional active agent is an alkylating agent (such as temozolomide, cisplatin, carboplatin, oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil, dacarbazine, lomustine, carmustine, procarbazine, chlorambucil and ifosfamide), antimetabolites (such as fluorouracil, gemcitabine, methotrexate, cytosine arabinoside, cytosine ... arabinoside, fludarabine, and floxuridine), antimitotic or vinca alkaloids (such as vincristine, vinblastine, vinorelbine, and vindesine), anthracyclines (including doxorubicin, daunorubicin, valrubicin, idarubicin, and epirubicin), and radiotherapy. actinomycins such as dactinomycin D), cytotoxic antibiotics (including mitomycin, plicamycin, and bleomycin), or topoisomerase inhibitors (including camptothecins such as irinotecan and topotecan, and derivatives of epipodophyllotoxins such as amsacrine, etoposide, etoposide phosphate, and teniposide).

[0217] 3. Immune system modulators

[0218] Immune system modulators are a type of immunotherapy that boosts the body's immune response against cancer. Examples of immune system modulators include cytokines, Bacillus Calmette-Guerin (BCG), and immunomodulatory drugs. Cytokines sometimes used to treat cancer include interferons (IFNs) and interleukins. Researchers have found that a type of interferon called INF-α can boost the immune response against cancer cells by making certain white blood cells, such as natural killer cells and dendritic cells, become active. INF-α can also slow the growth of cancer cells or promote their death. IL-2 increases the number of white blood cells in the body, including killer T cells and natural killer cells. Increasing these cells can cause an immune response against cancer. IL-2 also helps B cells, another type of white blood cell, produce certain substances that can target cancer cells. BCG is used to treat bladder cancer. When inserted directly into the bladder using a catheter, BCG causes an immune response against cancer cells. Immunomodulatory drugs (also called biologic response modifiers) stimulate the immune system. These immunomodulatory drugs include thalidomide. Lenalidomide Pomalidomide and imiquimod

[0219] 4. Immune checkpoint modulators

[0220] The active agent can be an immune checkpoint modulator. Immune checkpoints can be stimulatory or inhibitory, and tumors can use these checkpoints to protect themselves from attack by the immune system. Currently approved checkpoint therapies block inhibitory checkpoint receptors, but research is also underway on therapies that activate stimulatory checkpoints. Therefore, immune checkpoint modulators can be modulators that block inhibitory checkpoints or modulators that activate stimulatory checkpoints. Typically, immune checkpoint modulators induce or otherwise activate or increase an immune response against target cells (such as cancer cells or infected cells).

[0221] In a preferred embodiment, the immune checkpoint regulator blocks inhibitory checkpoints. Thus, blocking negative feedback signaling to immune cells enhances the immune response to tumors. Therefore, in some embodiments, the immune checkpoint regulator is administered to a subject in an effective amount to block inhibitory checkpoints. Exemplary compounds are compounds that block or otherwise inhibit, for example, PD-1, PD-L1, or CTLA4.

[0222] a. PD-1 antagonists

[0223] In some embodiments, the active agent is a PD-1 antagonist. The activation of T cells generally depends on the antigen-specific signal after the T cell receptor (TCR) contacts the antigen peptide presented by the major histocompatibility complex (MHC), and the extent of this reaction is controlled by the positive and negative antigen-independent signals transmitted from a variety of co-stimulatory molecules. The latter is generally a member of the CD28 / B7 family. In contrast, programmed death-1 (PD-1) is a member of the CD28 receptor family that delivers a negative immune response when induced on T cells. The contact between PD-1 and one of its ligands (B7-H1 or B7-DC) induces an inhibitory response that reduces the intensity and / or duration of T cell multiplication and / or T cell response. Suitable PD-1 antagonists are described in U.S. Pat. Nos. 8,114,845, 8,609,089, and 8,709,416, and include compounds or agents that bind to a PD-1 ligand and block the ligand to interfere with or inhibit binding of the ligand to the PD-1 receptor, or that directly bind to the PD-1 receptor and block the receptor without inducing inhibitory signal transduction through the PD-1 receptor.

[0224] In some embodiments, a PD-1 receptor antagonist directly binds to the PD-1 receptor without triggering inhibitory signal transduction and also binds to the ligand of the PD-1 receptor to reduce or inhibit the ligand-triggered signal transduction through the PD-1 receptor. By reducing the number and / or amount of ligands that bind to the PD-1 receptor and trigger the transduction of inhibitory signals, fewer cells are weakened by the negative signals delivered through PD-1 signal transduction and a stronger immune response can be achieved.

[0225] PD-1 signaling is believed to be driven by binding to peptide antigens presented by the major histocompatibility complex (MHC) in close proximity to PD-1 ligands (such as B7-H1 or B7-DC) (see, e.g., Freeman, Proc. Natl. Acad. Sci. USA, 105: 10275-10276 (2008)). Therefore, proteins, antibodies, or small molecules that prevent PD-1 and TCR from co-ligating on the T cell membrane are also useful PD-1 antagonists.

[0226] In a preferred embodiment, the PD-1 receptor antagonist is a small molecule antagonist or antibody that reduces or interferes with PD-1 receptor signaling by binding to PD-1 ligand or PD-1 itself, particularly in cases where co-ligation of PD-1 with the TCR is not followed by such binding, thereby not triggering inhibitory signaling through the PD-1 receptor.

[0227] Other PD-1 antagonists include antibodies that bind to PD-1 or ligands of PD-1, such as PD-L1 (also known as B7-H1) and PD-L2 (also known as B7-DC), and other antibodies.

[0228] Suitable anti-PD-1 antibodies include, but are not limited to, those described in the following patent documents:

[0229] PCT / IL03 / 00425 (Hardy et al., WO / 2003 / 099196)

[0230] PCT / JP2006 / 309606 (Korman et al., WO / 2006 / 121168)

[0231] PCT / US2008 / 008925 (Li et al., WO / 2009 / 014708)

[0232] PCT / JP03 / 08420 (Honjo et al., WO / 2004 / 004771)

[0233] PCT / JP04 / 00549 (Honjo et al., WO / 2004 / 072286)

[0234] PCT / IB2003 / 006304 (Collins et al., WO / 2004 / 056875)

[0235] PCT / US2007 / 088851 (Ahmed et al., WO / 2008 / 083174)

[0236] PCT / US2006 / 026046 (Korman et al., WO / 2007 / 005874)

[0237] PCT / US2008 / 084923 (Terrett et al., WO / 2009 / 073533)

[0238] Berger et al., Clin. Cancer Res., 14:30443051 (2008).

[0239] A specific example of an anti-PD-1 antibody is MDX-1106 (see Kosak, US20070166281 (published July 19, 2007) paragraph 42), a human anti-PD-1 antibody, which is preferably administered at a dose of 3 mg / kg.

[0240] Exemplary anti-B7-H1 antibodies include, but are not limited to, those described in the following patent documents:

[0241] PCT / US06 / 022423 (WO / 2006 / 133396, published on December 14, 2006)

[0242] PCT / US07 / 088851 (WO / 2008 / 083174, published on July 10, 2008)

[0243] US2006 / 0110383 (published on May 25, 2006)

[0244] A specific example of an anti-B7-H1 antibody is MDX-1105 (WO / 2007 / 005874, published on January 11, 2007), a human anti-B7-H1 antibody.

[0245] For anti-B7-DC antibodies, see 7,411,051, 7,052,694, 7,390,888, and U.S. Published Application No. 2006 / 0099203.

[0246] The antibody can be a bispecific antibody comprising an antibody that binds to the PD-1 receptor bridged to an antibody that binds to a PD-1 ligand, such as B7-H1. In some embodiments, the PD-1 binding moiety reduces or inhibits signal transduction through the PD-1 receptor.

[0247] Other exemplary PD-1 receptor antagonists include, but are not limited to, B7-DC polypeptides, including homologs and variants of these polypeptides, as well as any of the aforementioned active fragments, and fusion proteins combining any of these polypeptides. In preferred embodiments, the fusion protein comprises a soluble portion of B7-DC coupled to the Fc portion of an antibody, such as human IgG, and does not bind to all or part of the transmembrane portion of human B7-DC.

[0248] The PD-1 antagonist may also be a fragment of mammalian B7-H1, preferably a fragment from a mouse or primate, preferably a human, wherein the fragment binds to and blocks PD-1 but does not induce inhibitory signal transduction through PD-1. The fragment may also be part of a fusion protein, such as an Ig fusion protein.

[0249] Other available polypeptide PD-1 antagonists include fragments that bind to ligands of PD-1 receptors. These fragments include PD-1 receptor protein or its soluble fragments, which can bind to PD-1 ligands, such as B7-H1 or B7-DC, and prevent binding to endogenous PD-1 receptors, thereby preventing inhibitory signal transduction. B7-H1 is also shown to bind to protein B7.1 (Butte et al., "Immunity", Vol. 27, pp. 111-122, (2007)). Such fragments also include mutations that increase binding to natural ligands, such as the soluble ECD portion of the PD-1 protein of the A99L mutation (Molnar et al., "Proceedings of the National Academy of Sciences (PNAS)", 105: 10483-10488 (2008)). Also suitable are B7-1 or soluble fragments thereof that can bind to the B7-H1 ligand and prevent binding to the endogenous PD-1 receptor, thereby preventing inhibitory signal transduction.

[0250] PD-1 and B7-H1 antisense nucleic acids, both DNA and RNA, and siRNA molecules can also be PD-1 antagonists. Such antisense molecules prevent the expression of PD-1 on T cells and the production of T cell ligands, such as B7-H1, PD-L1 and / or PD-L2. For example, siRNA (e.g., a length of about 21 nucleotides, which is specific to genes encoding PD-1 or encoding PD-1 ligands, and whose oligonucleotides can be easily commercially obtained), is complexed with carriers such as polyethyleneimine (see Cubillos-Ruiz et al., Journal of Clinical Research (J. Clin. Invest) 119 (8): 2231-2244 (2009), which is easily expressed by PD-1 and PD-1 ligands and reduces the expression of these receptors and ligands to achieve cellular uptake of reduced inhibitory signal transduction in T cells, thereby activating T cells.

[0251] Exemplary PD-1 inhibitors include, but are not limited to,

[0252] Pembrolizumab (formerly known as MK-3475 or lambrolizumab, Keytruda) was developed by Merck and first approved by the U.S. Food and Drug Administration in 2014 for the treatment of melanoma.

[0253] Nivolumab (Opdivo) was developed by Bristol-Myers Squibb and first received FDA approval in 2014 for the treatment of melanoma.

[0254] CureTech's pidilizumab

[0255] GlaxoSmithKline and MedImmune's AMP-224

[0256] AMP-514 from GlaxoSmithKline and MedImmune

[0257] Novartis' PDR001

[0258] Regeneron and Sanofi's cemiplimab

[0259] Exemplary PD-L1 inhibitors include, but are not limited to,

[0260] Atezolizumab (Tecentriq) is a fully human IgG1 (immunoglobulin 1) antibody developed by Roche Genentech. In 2016, the FDA approved atezolizumab for urothelial carcinoma and non-small cell lung cancer.

[0261] Avelumab (Bavencio) is a fully human IgG1 antibody co-developed by Merck Serono and Pfizer. Avelumab has been approved by the FDA for the treatment of metastatic Merkel cell carcinoma. However, it failed a Phase III clinical trial for gastric cancer.

[0262] Durvalumab (Imfinzi) is a fully human IgG1 antibody developed by AstraZeneca. It is FDA-approved for the treatment of urothelial carcinoma and unresectable non-small cell lung cancer following chemoradiotherapy.

[0263] Bristol-Myers Squibb's BMS-936559

[0264] Checkpoint Therapeutics' CK-301

[0265] See, e.g., Iwai et al., Journal of Biomedical Science, (2017) 24:26, DOI 10.1186 / s12929-017-0329-9.

[0266] b. CTLA4 antagonists

[0267] Other molecules that can be used to mediate the effects of T cells in an immune response are also contemplated as active agents. For example, in some embodiments, the molecule is an agent that binds to an immune response mediating molecule other than PD-1. In preferred embodiments, the molecule is an antagonist of CTLA4, such as an antagonistic anti-CTLA4 antibody. Examples of anti-CTLA4 antibodies are described in PCT / US2006 / 043690 (Fischkoff et al., WO / 2007 / 056539).

[0268] Dosages for anti-PD-1, anti-B7-H1, and anti-CTLA4 antibodies are known in the art and may range from 0.1 mg / kg to 100 mg / kg, with a narrower range of 1 mg / kg to 50 mg / kg being preferred, and a range of 10 mg / kg to 20 mg / kg being more preferred. Suitable doses for human subjects are between 5 mg / kg and 15 mg / kg, with 10 mg / kg of antibody (e.g., human anti-PD-1 antibody, such as MDX-1106) being most preferred.

[0269] Specific examples of CTLA antagonists include ipilimumab (a human anti-CTLA4 antibody), also known as MDX-010 or MDX-101, which is preferably administered at a dose of about 10 mg / kg; and tremelimumab (a human anti-CTLA4 antibody), which is preferably administered at a dose of about 15 mg / kg. See also Sammartino et al., Clinical Kidney Journal, 3(2):135-137 (2010), published online in December 2009.

[0270] In other embodiments, the antagonist is a small molecule. A series of small organic compounds have been shown to bind to the B7-1 ligand to prevent binding to CTLA4 (see Erbe et al., J. Biol. Chem, 277:7363-7368 (2002). Such small organic compounds can be administered alone or in combination with anti-CTLA4 antibodies to reduce inhibitory signaling of T cells.

[0271] 5. Immune cell regulators

[0272] The active agent can be an immune cell modulator. Immune cell modulators include, but are not limited to, compounds that increase the survival, expansion, activity, and / or persistence of T cells. Such compounds include inhibitors of the PI3K / AT / mTOR pathway, including but not limited to BEZ235, LY294002, GDC-0941, BYL719, GSK2636771, TGX-221, AS25242, CAL-101, IPI-145, MK-2206, GSK690693, GDC-0068, A-674563, CCT128930, AZD8055, INK128, rapamycin, PF-04691502, everolimus, BI-D1870, H89, PF-4708671, FMK, AT7867, NU7441, PI-103, NU7026, PIK-75, ZSTK474, and PP-121. See, for example, WO 2015 / 188119.

[0273] Protein kinase C (PKC) antagonists can further enhance calcium metabolism to improve T cell immunity. Examples include, but are not limited to, phorbol 12-myristate 13-acetate (PMA) (also known as 12-O-tetradecanoylphorbol 13-acetate (TPA), ingenol 3-angelate (I3A), bryostatin, bisindolylmaleimide I (also known as 2-[1-(3-dimethylaminopropyl) indol-3-yl]-3-(indol-3-yl) maleimide or GFX (GF109203X)), calcitonin C, and Go6976 (5,6,7,13-tetrahydro-13-methyl-5-oxo-12H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-12-propionitrile).

[0274] In the following experiments, PMA was incorporated into CCNP-Ab to form PMA@CCNP-Ab. The results showed that PMA@CCNP-Ab significantly increased the CD69+ population in OT-1 CTLs. The frequency of IFN-γ- and TNF-α-positive CTLs, as well as the secretion of these cytokines, increased, further supporting T cell activation.

[0275] III. Pharmaceutical Compositions

[0276] Pharmaceutical compositions comprising the disclosed particles, alone or in combination with additional active agents and / or adjuvants, are provided. Additionally or alternatively, the pharmaceutical compositions can include cells, e.g., immune cells, treated in vitro or ex vivo with the disclosed particles. The pharmaceutical compositions can be administered, e.g., by parenteral (e.g., intramuscular, intraperitoneal, intravenous (IV), intrathecal, or subcutaneous) injection.

[0277] In some embodiments, the composition is administered systemically, for example, by intravenous or intraperitoneal injection, in an amount effective to deliver the composition to the targeted cells.

[0278] In certain embodiments, composition is topically applied, for example, by subcutaneous injection or direct injection into the position to be treated.In certain embodiments, composition is injected or otherwise directly applied to one or more tumors.Generally, injection causes the local concentration of composition to increase, which is greater than the local concentration that can be achieved by systemic administration, and / or the toxicity to other tissues (for example, non-tumor cells) can be reduced.In certain embodiments, composition is topically delivered to suitable cells by using a catheter or syringe.Other modes of such composition topically delivered to cells include using an infusion pump (for example, from Alza Corporation (Alza Corporation, Palo Alto, Calif.) in Palo Alto, California) or composition is incorporated into polymer implants (see, for example, P. Johnson and JG Lloyd-Jones, eds., " Drug Delivery Systems (Drug Delivery Systems)" (Chichester, England: Ellis Horwood Ltd. (Ellis Horwood Ltd.), 1987), which can achieve sustained release of particles to the immediate vicinity of implants.

[0279] Particles, such as nanoparticles, can be provided to cells directly (e.g., by contacting them with the cells) or indirectly (e.g., through the action of any biological process). For example, particles, such as nanoparticles, can be formulated in a physiologically acceptable carrier or vehicle and injected into the tissue or fluid surrounding the cells.

[0280] A. Formulations for Parenteral Administration

[0281] In a preferred embodiment the composition is administered by parenteral injection in the form of an aqueous solution.

[0282] The formulation may be in the form of a suspension or emulsion. Typically, provided pharmaceutical compositions include an effective amount of particles, optionally including a pharmaceutically acceptable diluent, preservative, solubilizer, emulsifier, adjuvant, and / or carrier. Such compositions may include diluents such as sterile water, buffered saline with various buffer contents (e.g., Tris-HCl, acetate, phosphate), pH, and ionic strength; and optionally additives such as detergents and solubilizers (e.g., 20. 80, also known as polysorbate 20 or 80), antioxidants (e.g., ascorbic acid, sodium metabisulfite) and preservatives (e.g., thimerosal, benzyl alcohol) and bulking agents (e.g., lactose, mannitol). Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate. The preparation can be lyophilized and redissolved / resuspended just before use. The preparation can be sterilized, for example, by filtering through a sterilizing filter, by incorporating a sterilizing agent into the composition, by irradiating the composition, or by heating the composition.

[0283] In some embodiments, increasing the temperature of the colloidal solution of the particles is avoided. In some embodiments, the coated nanoparticles can be prepared in a thin film, which can optionally be heat-treated. For example, the phospholipids can be mixed with the nanoparticles in an organic solvent such as chloroform. After the chloroform evaporates, a thin film will remain on the inner surface of the container. The nanoparticles can be transported in this manner. Prior to processing, water / buffer solution is added to the container to redisperse the nanoparticles in the aqueous solution.

[0284] B. Other preparations

[0285] The particles can also be applied topically. Topical application can include application to the lungs, nose, oral cavity (sublingual, buccal), vaginal or rectal mucosa. These modes of administration can be made effective by co-formulating the particles with transdermal or mucosal transport components. In a specific embodiment, the route of administration is nasal administration. A variety of mechanical devices designed for pulmonary delivery of therapeutic products can be used, including but not limited to nebulizers, metered dose inhalers and dry powder inhalers, all of which are familiar to those skilled in the art. Some specific examples of commercially available devices are Nebulizer (Mallinckrodt Inc., St. Louis, Mo.); II nebulizer (Marquest Medical Products, Englewood, Colo.); Metered-dose inhaler (Glaxo Inc., Research Triangle Park, NC) and Nektar, Alkermes, and Mannkind all have approved or are in clinical trials for inhalable insulin powder formulations, and their technologies could be applied to the formulations described herein.

[0286] Formulations for administration to mucosal membranes may be in the form of tablets, gels, capsules, suspensions, or emulsions. Standard pharmaceutical excipients are available from any manufacturer.

[0287] Oral formulations can be in the form of chewing gum, gel strips, tablets, capsules, or lozenges. Oral formulations can include excipients or other modifications to the particles that can provide enteric protection or enhance delivery through the GI tract, including the intestinal epithelium and mucosa (see Samstein et al., Biomaterials, 29(6):703-8 (2008).

[0288] Transdermal formulations can also be prepared. These are typically ointments, lotions, sprays or patches, all of which can be prepared using standard techniques. Transdermal formulations can include penetration enhancers.

[0289] C. Adjuvant

[0290] Adjuvants are known in the art and can be used in the disclosed compositions and methods. The adjuvant can be, but is not limited to, alum (e.g., aluminum hydroxide, aluminum phosphate); saponins purified from the bark of the soap bark tree Q. saponaria, such as QS21 (a glycolipid eluted in peak 21 by HPLC separation; Antigenics, Inc., Worcester, Mass.); poly[di(carboxylic acid phenoxy)phosphazene] (PCPP polymer; Virus Research Institute, USA), Flt3 ligand, Leishmania elongation factor (purified Leishmania protein; Corixa Corporation, Seattle, Wash.), ISCOMS (immunostimulatory complex containing mixed saponins, lipids, and formed into virus-sized particles with pores that can accommodate antigens; CSL, Melbourne, Australia), Pam3Cys, SB-AS4 (SmithKline Beecham) adjuvant system No. 4, which contains alum and MPL; SBB, Belgium), micelle-forming nonionic block copolymers such as CRL 1005 (these contain linear chains of hydrophobic polyoxypropylene pendant with polyoxyethylene chains; Vaxcel, Inc., Norcross, Ga.), and Montanide IMS (e.g., IMS 1312, water-based nanoparticles combined with a soluble immunostimulant; Seppic).

[0291] Adjuvant can be TLR part, as those discussed above.The adjuvant that works by TLR3 includes but is not limited to double-stranded RNA.The adjuvant that works by TLR4 includes but is not limited to the derivative of lipopolysaccharide, such as monophosphoryl lipid A (MPLA; Ribi immunochemistry research company (Ribi ImmunoChem Research, Inc., Hamilton, Mont.) of Montana Hamilton) and muramyl dipeptide (MDP; Ribi company) and threonyl-muramyl dipeptide (t-MDP; Ribi company); OM-174 (the glucosamine disaccharide relevant to lipid A; OM Pharma SA company (OM Pharma SA, Meyrin, Switzerland) of Meilan, Switzerland).The adjuvant that works by TLR5 includes but is not limited to flagellin. Adjuvants that work through TLR7 and / or TLR8 include single-stranded RNA, oligonucleotides (ORN), synthetic low molecular weight compounds, such as imidazoquinoline amines (e.g., imiquimod (imiquimod) (R-837), resiquimod (resiquimod) (R-848)). Adjuvants that work through TLR9 include DNA or synthetic oligodeoxynucleotides (ODN) of viral or bacterial origin, such as CpG ODN. Another class of adjuvants are thiophosphates containing molecules, such as thiophosphate nucleotide analogs and nucleic acids containing thiophosphate backbone bonds.

[0292] Adjuvants can also be oil emulsions (e.g., Freund's adjuvant), saponin preparations; virosomes and virus-like particles; bacterial and microbial derivatives; immunostimulatory oligonucleotides; ADP-ribosylating toxins and detoxified derivatives; alum; BCG; mineral-containing compositions (e.g., mineral salts such as aluminum and calcium salts, hydroxides, phosphates, sulfates, etc.); bioadhesives and / or mucoadhesives; microparticles; liposomes; polyoxyethylene ether and polyoxyethylene ester preparations; polyphosphazenes; muramyl peptides; imidazoquinolone compounds; and surface-active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol).

[0293] Adjuvants can also include immunomodulators, such as cytokines, interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g., interferon-γ), macrophage colony stimulating factor, and tumor necrosis factor.

[0294] IV. How to use

[0295] The disclosed compositions can be used in vitro, in vitro or in vivo, to increase immune response. Calcium signals participate in activating different immune cells, including dendritic cells, T cells, macrophages, natural killer cells and neutrophils. Therefore, the disclosed compositions can be used for targeting these cells, to regulate the immune response produced therefrom, for example, by increasing calcium signal conduction therein. T cells include, for example, effector T cells (for example, cytotoxic T cells, helper T cells, regulatory T cells or a combination thereof), memory T cells, gamma-delta T cells (gamma delta T cells), follicular helper T cells (Tfh), natural killer T cells (NKT cells). In certain embodiments, compositions targeting specific cell types. In certain embodiments, compositions more broadly target immune cells, and therefore target two or more different immune cell types.

[0296] In some embodiments, the composition improves one or more activities of immune cells (e.g., by increasing calcium signaling). These activities include, but are not limited to, T cell activation and / or localization to the tumor site, and / or improving dendritic cell (DC) infiltration and / or DC maturation into the tumor site.

[0297] In some embodiments, the particles are used to activate or prime immune cells in vitro or ex vivo, including but not limited to antigen presenting cells and / or effector immune cells. Such cells can be administered to a subject in need thereof to treat cancer or infection.

[0298] A. In vivo methods

[0299] In some embodiments, the particles are used to activate or prime immune cells in vivo.

[0300] The disclosed compositions can be administered in an effective amount to induce, increase, or enhance an immune response. An "immune response" generally refers to a response that induces, increases, or persists activation or efficiency of innate or adaptive immunity. The compositions can be administered parenterally (by subcutaneous, intradermal, or intramuscular injection) via the lymphatics, or systemically via the circulatory system.

[0301] For example, in some embodiments, the composition is administered to a subject in need thereof to improve infiltration of dendritic cells (DCs) into a tumor site and / or maturation of DCs in the subject.

[0302] Additionally or alternatively, the composition can be administered to a subject in need thereof to increase T cell activation and / or number, particularly cytotoxic T cells, at a tumor in the subject.

[0303] In the following experiments, each mouse was injected with 200 μg / kg, or approximately 4 μg, of AnCHNP. Such a low dose of nanoparticles alone has no tumor-killing effect. Therefore, in some embodiments, the composition is administered in an amount or manner sufficient to induce an immune response without producing a direct anti-tumor effect.

[0304] In certain embodiments, the composition is not systemically delivered. In certain embodiments, the composition is delivered locally, for example, by subcutaneous injection. In certain embodiments, the composition is administered adjacent to or leading to one or more lymph nodes, which are close to the site where an immune response is needed (i.e., near a tumor or site of infection). In certain embodiments, the composition is injected into a muscle. The composition can also be administered directly to the site where an immune response is needed (e.g., a tumor or site of infection).

[0305] The composition can induce, increase or enhance an immune response compared to a control, for example, in the absence of the particles, an immune response in the subject is induced, increased or enhanced. Thus, the compositions and methods can be used to induce or enhance an immune activation immune response.

[0306] The disclosed compositions can enhance the activity of dendritic cells (DCs). In some embodiments, the immune response includes an increase in NF-κB signaling, cytokine activity, and immune response in DCs. In some embodiments, the particles induce DCs to express or secrete chemokines (e.g., CXCL-1, CCL5, CXCL2, and CXCL10) and cytokines (e.g., IL-1β, IL-12, and IL-6), which are known to attract and stimulate immune cells including T cells. Some embodiments include an increase in phospho-NF-κB, indicating activation of the NF-κB pathway, and / or an increase in the expression levels of calcineurin and dephosphorylated NFAT.

[0307] The following experiments show that, in general, sustained calcium release from CHNPs leads to activation of the NF-κB and NFAT pathways, inducing chemokines, cytokines, antigen presentation, and co-stimulatory molecules, thereby enhancing DC-mediated immunity.

[0308] The disclosed compositions can also enhance the activity of T cells. Therefore, in some embodiments, these compositions additionally or alternatively reduce the inactivation of T cells and / or prolong the activation of T cells and / or tumor infiltration and / or the number of T cells (that is, increase the antigen-specific proliferation of T cells, enhance the cytokine production of T cells, stimulate the differentiation effector function of T cells and / or promote T cell survival), or overcome T cell exhaustion and / or allergy and / or improve CTL / Treg ratio. In certain embodiments, compositions increase the expression and / or secretion of CD69, IFN-γ and / or TNF-α of T cells.

[0309] For example, the experiments below also show that T cells efficiently internalize PMA@CCNP-Ab, causing an increase in intracellular calcium levels. Delivery of calcium and PMA to T cells promotes their activation, as evidenced by increased expression or secretion of CD69, IFN-γ, and TNF-α. In vivo testing in C57 / BL6 mice bearing B16-OVA tumors demonstrated that PMA@CCNP-Ab enhanced tumor infiltration by cytotoxic T cells and increased the CTL / Treg ratio. The observed therapeutic benefit correlated with PMA@CCNP-Ab's ability to enhance T cell activation.

[0310] The compositions can be administered as part of a prophylactic vaccine or immunogenic composition, which confers resistance to a subject upon subsequent exposure to a cancer antigen or infectious agent, or as part of a therapeutic vaccine, which can be used to prime or enhance a subject's immune response to a pre-existing antigen, such as an infectious virus or a viral antigen in a subject with cancer.

[0311] The expected outcome of a prophylactic or therapeutic immune response can vary depending on the disease or condition being treated or according to principles known in the art. For example, an immune response against an infectious agent may completely prevent colonization and replication of the infectious agent, thereby effecting "sterile immunity" and the absence of any disease symptoms. However, a vaccine against an infectious agent can be considered effective if it reduces the number, severity, or duration of symptoms, if it reduces the number of individuals in a population with symptoms, or reduces the spread of the infectious agent.

[0312] Similarly, an immune response against cancer or an infectious agent may completely cure the disease, may alleviate symptoms, or may be one aspect of an overall therapeutic intervention against the disease.

[0313] B. In vitro and ex vivo methods

[0314] In some embodiments, the method is one of adaptive cell therapy (ACT). For example, methods of adoptive cell therapy are known in the art and used in clinical practice. Typically, adoptive cell therapy involves separation and ex vivo expansion of tumor-specific cells to achieve a greater number of cells than would be obtained by vaccination alone. The tumor-specific cells are then infused into the cancer patient in an attempt to empower the cancer patient's immune system to clear the remaining tumor by cells that can attack and kill the cancer. Several forms of adoptive T cell therapy can be used for cancer treatment, including but not limited to culturing tumor-infiltrating lymphocytes or TILs; isolating and expanding a specific T cell or clone; using T cells that have been engineered to recognize and attack tumors (i.e., chimeric antigen receptor (CAR) cells. Additionally or alternatively, antigen-presenting cells such as DCs can be used as vaccine vectors or antigen-presenting cells (APCs) to activate naive T cells ex vivo or in vivo. Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells serve as the primary tool effector cells for ACT. See, e.g., Abaksuchina et al., Vaccines (Basel), 2021 Nov 19;9(11):1363. doi:10.3390 / vaccines9111363.

[0315] Thus, in some methods, the disclosed particles are used to prime or activate T cells (e.g., cytotoxic T cells, helper T cells, regulatory T cells, or a combination thereof), memory T cells, gamma-delta T cells (γδ T cells), follicular helper T cells (Tfh), natural killer T cells (NKT cells), dendritic cells, and / or other immune cells in vitro or ex vivo, and then administered to a subject in need thereof, such as a subject with cancer or infection. In some embodiments, cells are collected from the subject, for example, directly from the patient's blood, prior to ex vivo treatment with the disclosed particles. Methods for priming and activating T cells in vitro / ex vivo for adaptive T cell cancer therapy are known in the art. See, for example, Wang et al., Blood, 109(11):4865-4872 (2007) and Hervas-Stubbs et al., J. Immunol, 189(7):3299-310 (2012). The methods can be used in conjunction with the disclosed compositions and methods to increase activation of cells (eg, dendritic cells, T cells, etc.) for adoptive therapy.

[0316] Antigen-specific T cell lines can be generated by in vitro stimulation with antigen, followed by non-specific expansion on CD3 / CD28 beads. The ability to expand antigen-specific T cells can be assessed using IFN-γ and granzyme B enzyme-linked immunosorbent assays. The phenotype of the resulting T cell lines can be assessed by flow cytometry. Expansion of antigen-specific T cell populations from peripheral blood mononuclear cells (PBMCs) is typically performed by repeated in vitro stimulation with an antigenic peptide of optimal length in the presence of IL-2. Traditionally, low doses of IL-2 (10 U / ml to 50 U / ml) are used to avoid activation / expansion of lymphokine-activated killer cells, as revealed by chromium release assays, which are commonly used to monitor specific T cell expansion. The concentration of the antigenic peptide can be 0.1-10 μM.

[0317] Historically, adoptive T cell therapy strategies have focused primarily on infusing tumor antigen-specific cytotoxic T cells (CTLs) that can directly kill tumor cells. However, CD4+ T helper (Th) cells can also be used. Th can activate antigen-specific effector cells and recruit cells of the innate immune system such as macrophages and dendritic cells to assist in antigen presentation (APC), and antigen-sensitized Th cells can directly activate tumor antigen-specific CTLs. Due to the activation of APCs, antigen-specific Th1 has been shown to be an initiator of epitope or determinant expansion, which expands the immune response to other antigens in the tumor. The ability to trigger epitope expansion broadens the immune response to many potential antigens in the tumor and may lead to more effective tumor cell killing due to the ability to generate heterogeneous responses. In this way, adoptive cell therapy can be used to stimulate endogenous immunity.

[0318] Thus, in some embodiments, the composition administered to a subject in need thereof (e.g., a subject suffering from cancer or infection) is a population of cells treated in vitro or ex vivo with the disclosed particles.

[0319] In some embodiments, ex vivo activated dendritic cells are administered as part of a dendritic cell vaccine. Dendritic cell vaccines are a combination of vaccines and cell therapies. DCs have become important regulators of inducing anti-tumor immunity due to their skilled antigen presentation ability. Dendritic cells can be used as vaccines by preparing dendritic cells with polypeptides or a small portion of tumor antigens and then injecting them into the body. When DC vaccines are injected into tumors, DC activation may be particularly strong, and data show that the combination of DC-based vaccination and other cancer therapies can further increase the potential of DC-based cancer vaccines and improve patient survival rates. See also, for example, Calmeiro et al., Pharmaceutics. February 2020; 12(2): 158.

[0320] C. Combination therapy

[0321] For example, the composition comprising particles and / or cells can be administered before, during, or after cancer therapy. The subject can have a benign or malignant tumor. In some embodiments, the subject has cancer and is receiving cancer treatment, e.g., vaccination, radiation therapy, chemotherapy, or immunotherapy.

[0322] In certain embodiments, the composition enhances the treatment of cancer compared to administering vaccination, radiotherapy, chemotherapy or immunotherapy alone. Compared to administering radiotherapy and chemotherapy without administering the composition, the composition is administered in combination with radiotherapy and / or chemotherapy to enhance the treatment of cancer. Compared to administering radiotherapy and immunotherapy without administering the composition, the composition is administered in combination with radiotherapy and immunotherapy to enhance the treatment of cancer. Compared to administering immunotherapy and chemotherapy without administering the composition, the composition is administered in combination with immunotherapy and chemotherapy to enhance the treatment of cancer. Compared to administering radiotherapy, chemotherapy and immunotherapy without administering the composition, the composition is administered in combination with radiotherapy, chemotherapy and immunotherapy to enhance the treatment of cancer.

[0323] Thus, in some embodiments, the subject is a subject receiving radiation-based therapy, including but not limited to ionizing radiation therapy, phototherapy, or proton therapy.

[0324] Thus, the method includes administering one or more doses of ionizing radiation therapy, phototherapy, or proton therapy to the subject. Typically, a dose of ionizing, phototherapy, or proton therapy radiation is administered (e.g., minutes, hours, or days) after administering a pharmaceutical composition including the disclosed compositions. For example, in exemplary embodiments, a dose of radiation is administered 1 hour to 48 hours, or 1 hour to 24 hours, or 1 hour to 12 hours, or 1 hour to 6 hours, or 2 hours to 6 hours, or 1 hour, 2 hours, 3 hours, 4 hours, or 5 hours prior to administering the disclosed pharmaceutical composition.

[0325] In some embodiments, the subject is a subject receiving chemotherapy.

[0326] In some embodiments, the subject is a subject who is receiving immunotherapy.The subject can be a subject who is receiving only one therapy or a combination of these therapies.

[0327] In some embodiments, the subject is one who is receiving a vaccination, eg, an antigen alone or in combination with an adjuvant.

[0328] D. Subjects to be treated

[0329] The disclosed compositions comprising, for example, particles and / or cells can be administered to a subject in need thereof. In some embodiments, the methods are used to treat cancer or infection. Thus, in some embodiments, the subject suffers from cancer or infection.

[0330] 1. Cancer

[0331] The compositions can be used to treat cancer. In mature animals, a balance is typically maintained between cell regeneration and cell death in most organs and tissues. Each type of mature cell in the body has a given lifespan; as these cells die, new cells are generated by the proliferation and differentiation of various stem cell types. Under normal circumstances, the production of new cells is regulated so that the number of cells of any particular type remains constant. However, cells occasionally emerge that no longer respond to normal growth control mechanisms. Cell clones generated by these cells can expand to considerable size, giving rise to tumors or neoplasms. Tumors that do not grow indefinitely and do not extensively invade surrounding healthy tissue are benign. Tumors that continue to grow and become increasingly invasive are malignant. The term cancer specifically refers to malignant tumors. In addition to uncontrolled growth, malignant tumors also exhibit metastasis. During this process, small clusters of cancer cells break off from the tumor, invade blood or lymphatic vessels, and are carried to other tissues, where they continue to proliferate. In this way, a primary tumor in one location may give rise to secondary tumors in another location.

[0332] The compositions and methods described herein can be used to treat a subject with a benign or malignant tumor by delaying or inhibiting the growth of the subject's tumor, reducing the growth or size of the tumor, inhibiting or reducing the metastasis of the tumor, and / or inhibiting or reducing symptoms associated with the development or growth of the tumor. The following examples demonstrate that these viruses and methods can be used to treat cancer in vivo, particularly brain tumors.

[0333] Malignant tumors that can be treated are classified herein according to the embryonic origin of the tissue from which the tumor originates. Carcinomas are tumors that arise from endoderm or ectoderm tissues, such as the skin or the epithelial linings of internal organs and glands. The compositions are particularly effective in treating carcinomas. Less frequently occurring sarcomas originate from mesodermal connective tissues, such as bone, fat, and cartilage. Leukemias and lymphomas are malignant tumors of the hematopoietic cells of the bone marrow. Leukemias proliferate as single cells, while lymphomas tend to grow as tumor masses. Malignant tumors may arise in many organs or tissues of the body, thereby forming cancers.

[0334] Cancer types that can be treated with the provided compositions and methods include, but are not limited to, cancers such as vascular cancers, multiple myeloma, adenocarcinomas, and sarcomas, as well as bone cancer, bladder cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, uterine cancer, and germ cell cancer. In some embodiments, the compositions are used to treat multiple cancer types in parallel. These compositions can also be used to treat metastatic lesions or tumors in multiple locations.

[0335] A representative but non-limiting list of cancers that can be treated using the disclosed compositions includes cancers of the blood and lymphatic system (including leukemias, Hodgkin's lymphomas, non-Hodgkin's lymphomas, These include cancers of the genitourinary system (including prostate, bladder, kidney, urethra, penis, and testicle), nervous system (including meningioma, glioma, glioblastoma, and ependymoma), head and neck (including squamous cell carcinoma of the oral cavity, nasal cavity, nasopharynx, oropharynx, pharynx, and paranasal sinuses), lung (including small cell lung cancer and non-small cell lung cancer), gynecological (including cervical, endometrial, vaginal, vulvar, ovarian, and fallopian tube), gastrointestinal (including gastric, small intestine, colorectal, liver, hepatobiliary, and pancreatic) cancers, skin (including melanoma, squamous cell carcinoma, and basal cell carcinoma), breast (including ductal and lobular carcinomas, as well as triple-negative breast cancer), and pediatric (including neuroblastoma, Ewing's sarcoma, Wilms' tumor, and medulloblastoma).

[0336] 2. Infection

[0337] Composition is also used to treat acute or chronic infectious diseases. Because viral infection is mainly removed by T cells, therefore, in the case where animal or human experimenter benefits from faster or more thoroughly removing viral infection original, improving T cell activity has therapeutic effect. Therefore, composition can be applied to treat local or systemic viral infection, including but not limited to immunodeficiency (for example, HIV), papilloma (for example, HPV), herpes (for example, HSV), encephalitis, influenza (for example, human influenza A virus) and common cold (for example, human rhinovirus) virus infection. For example, the pharmaceutical preparation comprising composition can be topically applied to treat viral skin diseases, such as herpes lesions or herpes zoster or genital warts. Composition can also be applied to treat systemic viral diseases, including but not limited to AIDS, influenza, common cold or encephalitis.

[0338] Representative infections that can be treated include, but are not limited to, infections caused by microorganisms including, but not limited to, Actinomyces, Anabaena, Bacillus, Bacteroides, Bdellovibrio, Bordetella, Borrelia, Campylobacter, Caulobacter, Chlamydia, Chlorobacter, Chromobacterium, Clostridium, Corynebacterium, Cytophaga, Deinococcus, Escherichia, Francisella, Halobacter, Helicobacter, Haemophilus, Haemophilus influenzae type B (HIB), Histoplasma, Mycobacterium, Legionella, Leishmania, Leptospira, Listeria, Neisseria meningitidis A, B, and C, Methanobacterium, Micrococcus, Mycobacterium, Mycoplasma, Myxococcus, Neisseria, Nitrobacter, Oscillatoria, Prochlorococcus, Proteus, Pseudomonas, Rhodospirillum, Rickettsia, Salmonella, Shigella, Spirillum, Spirochete, Staphylococcus, Streptococcus, Streptomyces, Sulfolobus, Thermoplasma, Thiobacillus and Treponema, Vibrio, Yersinia, Cryptococcus neoformans, Histoplasma capsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsia rickettsii, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydia psittaci, Chlamydia trachomatis, Plasmodium falciparum, Plasmodium vivax, Trypanosoma brucei, Entamoeba histolytica, Toxoplasma gondii, Trichomonas vaginalis, and Schistosoma mansoni.

[0339] In some embodiments, the type of disease to be treated or prevented is a chronic infectious disease caused by bacterial, viral, protozoan, helminthic or other microbial pathogens that gain intracellular entry and are attacked by, for example, cytotoxic T lymphocytes.

[0340] In preferred embodiments, the infection to be treated is a chronic infection caused by a hepatitis virus, human immunodeficiency virus (HIV), human T-lymphoproliferative virus (HTLV), herpes virus, Epstein-Barr virus, or human papillomavirus.

[0341] The present invention can be further understood through the following numbered paragraphs:

[0342] 1. A nanoparticle comprising a calcium core and a shell and / or coating.

[0343] 2. The nanoparticle according to paragraph 1, wherein the core further comprises a hydroxide, and optionally calcium hydroxide (Ca(OH)2).

[0344] 3. The nanoparticle according to paragraph 1, wherein the core further comprises a carbonate, and optionally calcium carbonate (CaCO3).

[0345] 4. Nanoparticles according to paragraph 1, wherein the core is selected from calcium citrate (CaCit), calcium phosphate (Ca3(PO4)2), CaCL2, calcium sulfate (CaSO4), CaC2O4, Ca(NO3)2, calcium silicate (Ca2SiO4), calcium fluoride (CaF2), CaBr2 and CaI2.

[0346] 5. The nanoparticle of any one of paragraphs 1 to 4, comprising the shell.

[0347] 6. The nanoparticle of paragraph 5, wherein the shell reduces, prevents, or otherwise delays degradation of the nanoparticle.

[0348] 7. The nanoparticles according to paragraphs 5 or 6, wherein the shell comprises one or more of the following: silica, mesoporous silica, carbon, a sulfide, wherein the sulfide is optionally ZnS, CoS, CuS, Cu2S, FeS, MoS, Al2S3, Y2S3 or MnS; an oxide, wherein the oxide is optionally Fe3O4, Fe2O3, Gd2O3, TiO2, Al2O3 or MnO2; a fluoride, wherein the fluoride is optionally NaYF4, YF3, La F3, CeF3, PrF3 or GdFe3; a fatty acid, optionally oleic acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, arachidic acid, eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA); an alkylamine, optionally octylamine, nonylamine, decylamine, undecylamine, laurylamine, tridecylamine, tetradecylamine, pentadecylamine, hexadecylamine, heptadecylamine, octadecylamine, oleylamine; MgO, CuO or ZnO.

[0349] 8. The nanoparticle of any one of paragraphs 1 to 7, wherein the nanoparticle comprises the coating.

[0350] 9. Nanoparticles according to paragraph 8, wherein the coating improves dispersion in aqueous solution and / or delays core release and / or extends half-life.

[0351] 10. The nanoparticle of paragraphs 8 and 9, wherein the coating comprises one or more polymers, peptides, proteins, lipids, or a combination thereof.

[0352] 11. The nanoparticle of any of paragraphs 8 to 10, wherein the coating comprises PEG.

[0353] 12. A nanoparticle according to any one of paragraphs 1 to 11, comprising a targeting agent, optionally wherein the targeting agent targets one or more immune cells, optionally wherein the one or more immune cells are selected from dendritic cells, T cells, macrophages, natural killer cells, neutrophils and combinations thereof, optionally wherein the T cells are selected from cytotoxic T cells, helper T cells, regulatory T cells, memory T cells, γ-δ T cells (γδ T cells), follicular helper T cells (Tfh), natural killer T cells (NKT cells) and combinations thereof.

[0354] 13. The nanoparticle of paragraph 12, wherein the targeting agent targets dendritic cells.

[0355] 14. The nanoparticle of paragraphs 12 and 13, wherein the targeting agent targets CD205 and is optionally an anti-CD205 antibody.

[0356] 15. The nanoparticle of paragraph 12, wherein the targeting agent targets T cells.

[0357] 16. The nanoparticle of paragraph 15, wherein the T cells comprise cytotoxic T cells or are cytotoxic T cells.

[0358] 17. The nanoparticle of paragraph 15 or 16, wherein the targeting agent targets CD3 or PD-1, and is optionally an anti-CD3 or anti-PD-1 antibody.

[0359] 18. The nanoparticle of any one of paragraphs 1 to 17, further comprising an active agent, optionally selected from an antigen, a chemotherapeutic drug, an immune system modulator, an immune checkpoint modulator, or an immune cell modulator.

[0360] 19. The nanoparticle of paragraph 18, comprising an immune cell modulator, optionally wherein the immune cell modulator is a protein kinase C (PKC) antagonist, optionally wherein the PKC antagonist is phorbol 12-myristate 13-acetate (PMA).

[0361] 20. A pharmaceutical composition comprising the nanoparticles according to any one of paragraphs 1 to 19.

[0362] 21. The pharmaceutical composition according to paragraph 20, further comprising an adjuvant.

[0363] 22. The pharmaceutical composition of paragraph 20 or 21, further comprising an antigen, a chemotherapeutic drug, an immune system modulator, an immune checkpoint modulator, or an immune cell modulator.

[0364] 23. A pharmaceutical composition comprising immune cells treated in vitro or ex vivo with the nanoparticles according to any one of paragraphs 1 to 19, optionally wherein the immune cells are selected from dendritic cells, T cells, macrophages, natural killer cells, neutrophils and combinations thereof, optionally wherein the T cells are selected from cytotoxic T cells, helper T cells, regulatory T cells, memory T cells, gamma-delta T cells (gamma delta T cells), follicular helper T cells (Tfh), natural killer T cells (NKT cells) and combinations thereof.

[0365] 24. A method of increasing calcium signaling in an immune cell, the method comprising contacting the immune cell with an effective amount of a pharmaceutical composition according to any one of paragraphs 20 to 22 to increase calcium signaling therein, optionally wherein the immune cell is selected from dendritic cells, T cells, macrophages, natural killer cells, neutrophils, and combinations thereof, optionally wherein the T cells are selected from cytotoxic T cells, helper T cells, regulatory T cells, memory T cells, gamma-delta T cells (gamma delta T cells), follicular helper T cells (Tfh), natural killer T cells (NKT cells), and combinations thereof.

[0366] 25. A method of enhancing an immune response in a subject in need thereof, the method comprising administering to the subject an effective amount of the pharmaceutical composition of any one of paragraphs 20 to 23.

[0367] 26. The method of paragraph 25, wherein the immune response comprises one or more of: increasing NF-κB signaling and / or cytokine activity in dendritic cells, enhancing dendritic cell infiltration into tumor sites, and / or promoting dendritic cell maturation.

[0368] 27. The method of paragraph 25 or 26, wherein the immune response comprises one or more of: inducing dendritic cells to express or secrete chemokines (e.g., CXCL-1, CCL5, CXCL2, and / or CXCL10), cytokines (e.g., IL-1β, IL-12, and / or IL-6), or a combination thereof.

[0369] 28. The method of paragraph 26 or 27, wherein the immune response comprises one or more of: increased T cell activation, increased T cell localization to tumor sites, increased T cell expression and / or secretion of CD69, IFN-γ, and / or TNF-α.

[0370] 29. The method of any one of paragraphs 24 to 28, wherein the subject has cancer or an infection.

[0371] 30. A method of treating or preventing cancer, comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition according to any one of paragraphs 20 to 23.

[0372] 31. The method of paragraph 30, wherein the amount or mode of administration is effective to induce an immune response against the cancer but does not have a direct anti-cancer effect.

[0373] 32. A method of treating or preventing an infection, comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition of any one of paragraphs 20 to 23.

[0374] 33. The method of any one of paragraphs 25 to 32, further comprising administering to the subject one or more of the following treatments: surgery, radiotherapy, chemotherapy, or immunotherapy, optionally an immune checkpoint regulator, an immune system regulator, or an immune cell regulator.

[0375] The present invention can be further understood by reference to the following non-limiting examples.

[0376] Example

[0377] Example 1: Calcium nanoparticles stimulate dendritic cells and enhance anti-tumor immunity

[0378] Materials and methods

[0379] Synthesis of calcium hydroxide or Ca(OH)2 nanoparticles (CHNPs)

[0380] In a typical synthesis, 443.92 mg of calcium chloride (CaCl2, anhydrous, 97%, Sigma-Aldrich, lot SLBQ3073V) was first dissolved in 18.571 mL of Milli Q H2O. 1.429 mL of 6 M sodium hydroxide (NaOH, Fisher, lot 166374) was added dropwise to the solution. The resulting solution was magnetically stirred at 90°C for 5 minutes. The crude product was collected by centrifugation and then redispersed in ethanol (200 proof, Koptec, lot 274014) and briefly sonicated. The washing step was repeated 3 times to remove unreacted precursors.

[0381] Synthesis of silica-coated calcium hydroxide nanoparticles (SCHNPs)

[0382] 50 mg of CHNPs were dispersed in a mixed solvent containing 40 mL of ethanol and 0.4 mL of ammonia (28.0%-30.0%, JTBaker, batch number 0000010971). The solution was stirred vigorously for 30 minutes. After ultrasonic treatment for 30 seconds, 300 μL of TEOS (tetraethyl orthosilicate, 98%, Sigma-Aldrich, batch number STBJ8253) was added dropwise to the solution, followed by 180 μL of APTES ((3-aminopropyl) triethoxysilane, 98%, Sigma-Aldrich, batch number MKCM7627). The resulting solution was stirred at room temperature for 20 hours. The CHNPs were collected by centrifugation and washed three times with ethanol.

[0383] Synthesis of PEG-diacid-coated calcium hydroxide nanoparticles (PCHNPs)

[0384] 20mg of SCHNPs were dispersed in 10mL DMSO (dimethyl sulfoxide, 99.9%, Sigma-Aldrich, batch number MKBF8194V) and transferred to a 20mL glass bottle. Under magnetic stirring, 200mg PEG-diacid (MW 2,000, JenKem tech, batch number ZZ192P158), 20mg EDC (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide, 97%, Sigma-Aldrich, batch number 507429) and 15mg NHS (N-hydroxysuccinimide, 98%, Sigma-Aldrich, batch number 130672) dissolved in 10mL DMSO were added to the nanoparticle suspension. The resulting solution was magnetically stirred at 60°C for 20 hours. PCHNPs were collected by centrifugation and washed twice with Milli Q HO.

[0385] Synthesis of anti-CD205 conjugated calcium hydroxide nanoparticles (AnCHNPs)

[0386] PCHNPs (0.5 mg) were dispersed in 1 mL of cold sterile PBS and maintained under magnetic stirring at 4°C. 10 μL of anti-CD205 antibody (mouse monoclonal HD30, Sigma-Aldrich, lot number 531834) was added to the PCHNP solution. After 25 minutes, 2 μL of ethanolamine (99%, Sigma-Aldrich, lot number 398136) was added to the solution. After an additional 5 minutes of reaction, AnCHNPs were collected by centrifugation and washed once with PBS. Unless otherwise stated, freshly prepared AnCHNPs were used in subsequent in vitro and in vivo studies. Unless otherwise stated, all nanoparticle doses are expressed as Ca concentrations.

[0387] Physical and chemical characterization of nanoparticles

[0388] Scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS) elemental mapping images were obtained on a FEI Teneo field emission SEM equipped with an Oxford EDS system. Transmission electron microscopy (TEM) analyses were performed on a FEI Tecnai20 transmission electron microscope operated at an accelerating voltage of 200 kV. High-resolution TEM analyses were performed on a Hitachi transmission electron microscope H9500 operated at an accelerating voltage of 300 kV. The images were taken with Cu Kα1 radiation. X-ray diffraction (XRD) analysis was performed on a Bruker D8-Advance system using dried samples placed on cut glass slides. Dynamic light scattering (DLS) and zeta potential measurements were performed on a Malvern Zetasizer Nano ZS system. Fourier transform infrared (FT-IR) spectra were recorded on a Nicolet iS10 FT-IR spectrometer.

[0389] Nanoparticle stability and calcium release

[0390] CHNP and PCHNP are dispersed in 100 μ L ammonium acetate buffer solution (pH=5.5 or 7.4), and are loaded into Slide-A-Lyzer™ MINI dialysis device (MWCO=2K, product number 69550, Thermo Fisher Scientific Inc., USA).The dialysis unit is placed in the 5mL Eppendorf pipe containing the identical ammonium acetate buffer of 4.5mL.Pipe is at room temperature placed on oscillator (20rpm).At different time points (0 hour, 0.25 hour, 0.5 hour, 1 hour, 2 hours, 4 hours, 8 hours, 10 hours and 24 hours), from Eppendorf pipe, get 500 μ L solution, and measure its Ca by calcium ion selective electrode (HORIBALAQUAtwin Ca-11). 2+ 500 μL of fresh buffer was added back to the Eppendorf tube to maintain a total volume of 4.5 mL. All samples were analyzed in triplicate. In addition, TEM images of PCHNPs were obtained at 0, 2, 4, 8, 12, and 24 hours.

[0391] Cell culture

[0392] B16F10-OVA cells (mouse melanoma) were cultured in high-glucose DMEM supplemented with G418. B16F10 cells (mouse melanoma) were grown in high glucose DMEM ( 30-2002TM). Bone marrow-derived dendritic cells (BMDCs) were established from progenitor cells extracted from the bone marrow of C57BL / 6 mice and cultured in RPMI-1640 (Corning, 10-040-CV) containing GM-SCF according to a published protocol (Jiang et al., Advanced Materials 2019, 31(46), 1904058). MB49 cells (mouse bladder cancer) were grown in RPMI-1640 (Corning, 10-040-CV). All cell culture media were supplemented with 10% fetal bovine serum (FBS), 100 units / mL of penicillin and 100 units / mL of streptomycin (MediaTech, USA). All cells were maintained in a humidified environment of 5% carbon dioxide at 37°C.

[0393] Cytotoxicity

[0394] ATPlite-1-step luminescent detection kit (PerkinElmer, lot number 107-21051) was used to determine the cellular ATP content according to the manufacturer's protocol. BMDCs were plated at 1×10 4 Cells were seeded into 96-well plates at a density of 10 cells / mL and incubated overnight. The cells were then treated with CaCl2 solution, AnCHNPs, and SiO2-PEG shells at doses ranging from 0.05 to 100 μg / mL for 24 hours. The luminescence intensity of each well was measured on a microplate reader (Synergy Mx, BioTeK) and normalized to that of control cells.

[0395] Cellular uptake

[0396] BMDCs were plated at 1 × 10 per well. 6 Cells were seeded into 6-well plates at a density of 100 cells / mL and incubated overnight. Then, the cells were treated with Cy-5 labeled PCHNPs and AnCHNPs (5 μg / mL) for 2 hours. In addition, different endocytosis inhibitors, sodium azide (NaN3, 99.5%, Sigma-Aldrich, batch number S2002), Dynasore (C 18 H 14 N2O4, 98%, Sigma-Aldrich, lot number 324410), nystatin (Sigma-Aldrich, lot number N4014), chlorpromazine (C 17 H 19 The fluorescence of Cy-5 uptake by DCs was measured by flow cytometry.

[0397] Lysosomal pH

[0398] LysoSensor TM Yellow / blue DND-160 (PDMPO) kit (Invitrogen, lot number 2174576) was used to study the changes in lysosomal pH after BMDC uptake of AnCHNPs. Briefly, BMDCs were plated at 1×10 4 The cells were seeded into 96-well plates at a density of 100 cells / mL and incubated overnight. At different time points (0 h, 1 h, 2 h, 4 h, 8 h and 24 h), the incubation medium was removed and supplemented with preheated (37 ° C) medium containing the probe (1 μM). The cells were incubated for 5 minutes under the same growth conditions. Then, the loading solution was replaced with fresh culture medium, and fluorescence was measured on a microplate reader (SynergyMx, Boten) (329 nm and 384 nm dual excitation and 440 nm and 540 nm dual emission). In acidic organelles, LysoSensorTM Yellow / Blue DND-160 (PDMPO) mainly shows yellow fluorescence, and in less acidic organelles it emits blue fluorescence. Lysosomal pH can be estimated based on the blue / yellow fluorescence ratio.

[0399] [Ca2+]int measurement

[0400] Fluo-3 AM kit (Cayman, 14960) was used to measure [Ca2+]int in BMDCs treated with AnCHNPs. Briefly, BMDCs were plated at 1×10 4 The cells were seeded into 96-well plates at a density of 100 cells / well and incubated overnight. At different time points (0 h, 1 h, 2 h, 4 h, 8 h and 24 h), the culture medium was removed from the wells and supplemented with preheated (37 ° C) probe-containing culture medium (to a final concentration of 5 μM). The cells were incubated for 30 minutes under the same growth conditions. Then, the loading solution was replaced with fresh culture medium to remove the dye molecules non-specifically attached to the cell surface. The cells were incubated for another 30 minutes to completely deesterify the acetoxymethyl ester. Fluorescence (excitation / emission: 485 / 520 nm) was recorded on a microplate reader (Synergy Mx, Berton).

[0401] [Na+]int and [K+]int measurements

[0402] SBFI-AM (sodium-binding benzofuran isophthalate acetoxymethyl ester, Setareh Biotech, lot number: 50609) and PBFI-AM (potassium-binding benzofuran isophthalate acetoxymethyl ester, Setareh Biotech, lot number: 5027) were used to measure [Na+]int and [K+]int in BMDCs after treatment with AnCHNPs according to the manufacturer's protocol. Briefly, BMDCs were plated at 1×10 cells / well. 4 The cells were seeded into 96-well plates at a density of 100 cells / well and incubated overnight. At different time points (0 hour, 1 hour, 2 hours, 4 hours, 8 hours and 24 hours), the culture medium was removed from the wells and a loading solution containing the probe (final concentration of 10 μM) was added to the wells. The cells were incubated for 30 minutes under the same growth conditions. The loading solution was then replaced with fresh culture medium to remove the dye molecules nonspecifically attached to the cell surface. Fluorescence (excitation: 340 / 380 nm, emission: 505 nm) was recorded on a microplate reader (Synergy Mx, Berton), and the ratio was used to determine the concentrations of Na+ and K+, respectively.

[0403] Studying BMDC maturation, migration, and antigen presentation in vitro

[0404] One day before the experiment, mature BMDCs were plated at 1 × 10 per well. 6 Cells were seeded onto 6-well plates at a density of 100 cells / well. BMDCs were treated with PBS, CaCl2 solution (5 μg / mL or 10 μg / mL), and AnCHNPs (5 μg / mL or 10 μg / mL). After 24 hours of incubation, the supernatant was removed and BMDCs were harvested using a cell scraper. BMDCs were subsequently stained with MHCII-FITC (No. 107616) and CD205-APC (No. 138206) and analyzed by flow cytometry. Similarly, BMDCs were treated with SiO2-PEG shell (10 μg / mL), collected after 24 hours of incubation, stained with MHCII-FITC (No. 107616), CD80-PerCP-Cy5.5 (No. 560526), ​​CD86-BV605 (No. 563055), CD40-PE (No. 12-0401-83) and OVA-APC (No. 17-5743-82), and analyzed by flow cytometry.

[0405] Migrated B16F10-OVA cells after 100 Gy irradiation (320 kV) were transferred to 6-well plates. In the lower chamber of the permeability support system, the density was 1×10 5 For the control, non-irradiated B16F10-OVA cells were used. 1×10 cells were added to each well.6 CFSE-labeled BMDCs were seeded into the upper chamber of the wells at a density of 10 cells. BMDCs were treated with PBS, CaCl2 solution (5 μg / mL or 10 μg / mL), and AnCHNPs (5 μg / mL or 10 μg / mL). LPS (1 μg / mL) was tested as a positive control (Supporting Information). After 24 hours of incubation, cells in the lower chamber were harvested using a cell scraper and prepared for flow cytometry. The percentage of CFSE-positive cells was quantified.

[0406] Activation and antigen presentation

[0407] Irradiated B16F10-OVA cells (100 Gy, 320 kV) were placed at 1×10 5 For comparison, non-irradiated B16F10-OVA cancer cells were also tested. 6 BMDCs were seeded into each well at a density of 100 cells. Co-culture was performed using PBS, CaCl solution (5 μg / mL or 10 μg / mL), AnCHNP (5 μg / mL or 10 μg / mL). After 24 hours of incubation, cells were collected by a cell scraper and stained with MHCII-FITC (numbering 107616), CD80-PerCP-Cy5.5 (numbering 560526), ​​CD86-BV605 (numbering 563055), CD40-PE (numbering 12-0401-83) and OVA-APC (numbering 17-5743-82). Flow cytometry was used to analyze the supernatant. In addition, supernatant was collected and IL-6, IL-10, IL-12, TNF-α DuoSet test kit (Minneapolis, Minnesota) from R&D Systems was used to measure IL-6, IL-10, IL-12, and TNF-α contents by ELISA. The results were analyzed using the four-parameter logistic curve method of Myassay.com.

[0408] RNA sequencing (RNA-seq) and data analysis

[0409] BMDCs were plated at 1 × 10 per well. 6 Cells were seeded into 100 mm Petri dishes at a density of 100 cells and incubated overnight. Cells were treated with OVA (10 μg / mL) or OVA (10 μg / mL) + AnCHNP (5 μg / mL). After incubation for 12 hours, cells were harvested using a cell scraper. RNA was extracted from three independent BMDC samples treated with different methods using a miRNA kit (Takara, lot 2010 / 002). RNA quality was analyzed using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Purified RNA samples were sent to Novogene Corporation (Sacramento, CA) for library construction and sequenced using an Illumina HiSeq TM 2000 platform to carry out sequencing, to obtain the expression library of 50-nt read length.Analyze RNAseq data as previously described.In brief, differentially expressed genes (DEGs) were identified using DESeq R package function estimateSizeFactors and nbinomTest.P value<0.05, and change fold>1.5 or change fold<0.5 were set as the threshold value of significant differential expression.DEGs were subjected to hierarchical cluster analysis, to explore transcript expression pattern, and all DEGs were subjected to gene ontology (Gene Ontology, GO) analysis, to identify the potential function of all DEGs.GSEA was carried out using the annotation gene set of GSEA desktop application software and molecular signature database v6.2.The detailed RNA-seq information of this assay can be consulted and deposited in GSE208276 in NIH gene expression comprehensive database (Gene Expression Omnibus, GEO).

[0410] RT-qPCR

[0411] RT-qPCR was performed on a QuantStudio 3 system using SYBR Green as an indicator. A PCR reaction mixture comprising 10 ng of cDNA, 500 nM of each primer (synthesized by Sigma, St. Louis, MO), 5 μL of 2x SYBR Green PCR Master Mix (Quantabio, product number 101414-284) and RNase-free water was added to increase the final volume to 10 μL. The qRT-PCR reaction was performed at 95°C for 15 seconds and at 60°C for 1 minute for 40 cycles. GAPDH and histones were used as internal standards for normalization, and the data were quantified based on the ΔΔCt method. Melting curve analysis was performed on all qRT-PCR products, and the results showed a single DNA double-strand peak. The primer sequences are:

[0412] NOS2: forward 5'-AGAGCCACAGTCCTCTTTGC-3' (SEQ ID NO: 1); reverse 5'-GCTCCTCTTCCAAGGTGCTT-3' (SEQ ID NO: 2).

[0413] CCL5: forward 5'-CTGCTGCTTTGCCTACCTCT-3' (SEQ ID NO: 3); reverse 5'-CGAGTGACAAACACGACTGC-3' (SEQ ID NO: 4).

[0414] CXCL1: forward 5'-CTGGGATTCACCTCAAGAACATC-3' (SEQ ID NO: 5); reverse 5'-CAGGGTCAGGCAAGCCTC-3' (SEQ ID NO: 6).

[0415] IL-12b: forward 5'-ATGAGAACTACAGCACCAGCTTC-3' (SEQ ID NO: 7); reverse 5-ACTTGAGGGAGAAGTAGGAATGG-3' (SEQ ID NO: 8).

[0416] IL-1b: forward 5'-TCGTGCTGTCGGACCCATAT-3' (SEQ ID NO: 9); reverse 5'-GTCGTTGCTTGGTTCTCCTTGT-3' (SEQ ID NO: 10).

[0417] Western blotting

[0418] BMDCs were cultured at a rate of 1 × 10 per cell. 6The density of cells was seeded into 100mm petri dishes and incubated overnight. Then, cells were treated with OVA (10 μg / mL) or OVA (10 μg / mL) plus AnCHNP (5 μg / mL). After incubation for 24 hours, cells were collected and lysed with RIPA buffer supplemented with 1x protease inhibitor cocktail (Amresco (cocktail)). Protein concentration was determined using bicinchoninic acid (BCA) protein assay (Thermo Fisher Scientific). Protein lysate was loaded onto 10% SDS-PAGE and transferred to a PVDF membrane. Nonspecific binding to the membrane was blocked by incubation at room temperature for 1 hour with 5% skim milk. The membrane was incubated overnight at 4°C with a primary antibody of a dilution specified by the manufacturer. Subsequently, the membrane was incubated at room temperature for 1 hour with a secondary antibody and then treated with ECL reagent (Thermo Fisher Scientific). Then, the membrane was exposed to X-ray film (Santa Cruz (Santa Cruz)). All imaging results were analyzed by ImageJ. Antibodies used were: NFAT1 (Cell Signaling, product number 4389S); pan-calcineurin A (Cell Signaling, product number 2614S); IκBα, phospho-IκBα, NF-κB p65, phospho-NF-κB p65 (Cell Signaling, product number 9936T); GAPDH (Cell Signaling, product number 5174S).

[0419] Animal models

[0420] All experimental procedures were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Georgia. C57BL / 6 mice (female, 4 weeks old) were purchased from Envigo Laboratories and housed under pathogen-free conditions. After 2 weeks of adaptation (6 weeks old), the mice were placed in a 2×10 5 Animal models were established by subcutaneously injecting 50 μL of PBS with 10 B16F10-OVA, B16F10, or MB49 cells into the right hind limb of each mouse.

[0421] Flow cytometry analysis of immune cell phenotypes

[0422] C57BL / 6 mice bearing B16F10-OVA tumors were randomly divided into three groups (n = 10 per group) and treated with the following: (1) 10 Gy X-ray irradiation (320 kV) + PBS (50 μL), (2) 10 Gy X-ray irradiation + CaCl2 solution (200 μg / kg, intratumoral injection), (3) 10 Gy X-ray irradiation + AnCHNP (200 μg / kg, intratumoral injection). When the tumor size reached approximately 100 mm, the mice were randomly divided into three groups (n = 10 per group) and treated with the following: (1) 10 Gy X-ray irradiation (320 kV) + PBS (50 μL), (2) 10 Gy X-ray irradiation + CaCl2 solution (200 μg / kg, intratumoral injection), (3) 10 Gy X-ray irradiation + AnCHNP (200 μg / kg, intratumoral injection). 3At day 0 (day 0), treatment was started. All injections were performed at five locations of the tumor to ensure good coverage. One hour after radiation, 50 μL PBS containing CaCl2 and AnCHNP was injected. On the 3rd day, 5 mice in each group were euthanized. On the 7th day, the remaining animals were euthanized. Tumors, spleens, and tumor-draining lymph nodes were collected for immune response analysis. The tumor was cut into small pieces with scissors and digested by incubation at 37°C for 45 minutes with DMEM containing 1 mg / mL type V collagenase (Worthington Biochemical Corporation). The digested tissue was gently filtered through a 250 μm cell sieve (Thermo Fisher Scientific, lot number UB2685874A). According to the manufacturer's instructions, red blood cells were lysed with Ack lysis buffer (Gibco). The single cell suspension was washed with cold sterile PBS and resuspended in staining buffer. After counting and aliquoting, cells were stained with fluorophore-conjugated antibodies for 30 minutes at 4°C. Spleen and lymph nodes were processed according to similar procedures, except that a 70 μm cell sieve (Corning Falcon, reference number 352235) was used and type V collagenase was not used. The following anti-mouse antibodies from BD Biosciences were used: CD45-APC-Cy7 (number 557659), CD4-BV605 (number 563151), FoxP3-PE (number 563101), CD11c-PE-Cy7 (number 558079), CD86-BV605 (number 563055), CD80-PerCP-Cy5.5 (number 560526). CD40-PE (number 12-0401-83) was purchased from Invitrogen. OVA-APC (No. 17-5743-82) was purchased from eBioscience. MHCII-FITC (No. 107616), CD205-APC (No. 138206), IFN-γ-APC (No. 505810), CD3-FITC (No. 100206), and CD8-BV510 (No. 100752) were purchased from BioLegend. Live / Dead DAPI was purchased from Thermo Fisher Scientific.Multiparameter staining was used to identify the following target cell populations: (a) CD8+ T cells (CD45+CD3+CD8+), (b) CD8+IFNγ+ T cells (CD45+CD3+CD8+IFNγ+), (c) CD4+ T cells (CD45+CD3+CD4+), (d) Treg cells (CD45+CD3+CD4+FoxP3+), (e) MHC-II+ DCs (CD11c+MHC-II+), (f) CD80+ DCs (CD11c+MHC-II+CD80+), (g) CD86+ DCs (CD11c+MHC-II+CD86+), (h) CD40+ DCs (CD11c+MHC-II+CD40+), (i) OVA+ DCs (CD11c+MHC-II+SIINFEKL-H-2Kb+). In order to carry out intracellular FoxP3 and IFN-γ staining, cells are fixed and permeabilized using a permeabilization solution kit (BD company (BD), 554714) and washed before performing flow cytometry (Quanteon, Agilent (Agilent)). In order to assess tumor-specific T cell responses, splenocytes were co-cultured with B16F10-OVA cells for 6 hours before staining and flow cytometry. Data were processed by FlowJo 10.0. Cell doublets were excluded based on forward and side scatter. Dead cells were excluded based on DAPI staining positivity. In addition, blood samples were collected on the 3rd and 7th days for cytokine analysis. Specifically, according to the manufacturer's protocol, IL-1β, IL-6, IL-10, IL-12, TNF-α and IFN-γ in serum were measured using R&D Systems company mouse DuoSet ELISA kit (Minneapolis, Minnesota). The results were analyzed using the four-parameter logistic curve method from Myassay.com.

[0423] Therapy research

[0424] Combined with radiation therapy (RT): Studies were conducted in C57BL / 6 mice bearing B16F10-OVA or MB49 tumors. For the B16F10-OVA tumor model, when tumors reached approximately 50 mm in size, 3At the same time, animals were randomly assigned to receive the following treatments (n = 5 per treatment group): (1) PBS (intratumoral injection, 50 μL*2, day 0 and day 2), no irradiation; (2) AnCHNP (intratumoral injection, 200 μg / kg*2, day 0 and day 2); (3) RT (10 Gy*2, day 0 and day 2) + PBS (intratumoral injection, 50 μL*2, day 0 and day 2); (4) RT (10 Gy*2, day 0 and day 2) + AnCHNP (intratumoral injection, 200 μg / kg *2, day 0 and day 2); (5) RT (10 Gy*2, day 0 and day 2) + AnCHNP (intraditumoral injection, 200 μg / kg*2, day 0 and day 2) + anti-CD8 antibody (intraperitoneal injection, 10 mg / kg*2, day 0 and day 4); (6) RT (10 Gy*2, day 0 and day 2) + AnCHNP (intraditumoral injection, 200 μg / kg*2, day 0 and day 2) + anti-CD4 antibody (intraperitoneal injection, 10 mg / kg*2, day 0 and day 4). All intratumoral injections were performed at five sites of the tumor to ensure good coverage. Antibodies and AnCHNPs were injected in 100 μL and 50 μL PBS, respectively. If RT was applicable, AnCHNPs were injected 1 hour after radiation. Tumor size and body weight were checked every day. The two-dimensional dimensions of the tumor were measured with a caliper, and the tumor volume was calculated as (length) × (width)2 / 2. After treatment, tumors and major organs were collected and cut into 4 μm thick sections for H&E and Ki-67 staining. For the MB49 tumor model, animals received the following treatments (n = 5 per group): (1) PBS (intramedullary injection, 50 μL*2, day 0 and day 2), no irradiation; (2) RT (10 Gy*2, day 0 and day 2) + PBS (intramedullary injection, 50 μL*2, day 0 and day 2); (3) RT (10 Gy*2, day 0 and day 2) + AnCHNP (intramedullary injection, 200 μg / kg*2, day 0 and day 2). The treatment regimen was similar to that described in the B16F10-OVA study.

[0425] Combination with chemotherapy: Studies were conducted in C57BL / 6 mice bearing B16F10-OVA tumors. 3Animals were randomly assigned to receive the following treatments (n = 5 per group): (1) PBS (intraperitoneal injection, 50 μL*2, day 0 and day 2); (2) carboplatin (intraperitoneal injection, 40 mg / kg, 100 μL, day 0); (3) carboplatin (intraperitoneal injection, 40 mg / kg, 100 μL, day 0) + AnCHNP (intraperitoneal injection, 200 μg / kg*2, 50 μL, day 0 and day 2). Tumor size and body weight were examined daily. Tumors were measured two-dimensionally with a caliper, and tumor volume was estimated as (length) × (width)2 / 2.

[0426] Combination with immunotherapy: The study was conducted in C57BL / 6 mice bearing B16F10 tumors. 3 Animals were randomly assigned to receive the following treatments (n=5 per group): (1) PBS (intraperitoneal injection, 50 μL, day 0 and day 2); (2) anti-PD-L1 antibody (intraperitoneal injection, 10 mg / kg, day -2, day 0, day 2, and day 4); (3) anti-PD-L1 antibody (intraperitoneal injection, 10 mg / kg, day -2, day 0, day 2, and day 4) + AnCHNP (intraperitoneal injection, 200 μg / kg, day 0 and day 2). Antibodies were injected intraperitoneally on day 0 and day 2, and AnCHNPs were injected intratumorally. All injections were performed at five sites in the tumor to ensure good coverage. Antibodies and AnCHNPs were injected in 100 μL and 50 μL PBS, respectively. Tumor size and body weight were examined every other day. Tumors were measured two-dimensionally with calipers, and tumor volume was estimated as (length) × (width)2 / 2.

[0427] Statistical analysis

[0428] All in vitro studies were performed in triplicate unless otherwise stated. The half-maximal inhibitory concentration (IC50) was determined by Doseresp using Origin 9. 50 ). For in vivo studies, all measurements were performed in triplicate unless otherwise noted. All data are expressed as mean ± SD. Multiple determinations were compared using a one-way ANOVA test, and two groups were compared using a paired t-test, with a P value of 0.05 or less indicating statistical significance.

[0429] result

[0430] Nanoparticle synthesis, surface modification and physicochemical characterization

[0431] Calcium hydroxide nanoparticles (CHNPs) were synthesized by a coprecipitation method using CaCl2 and NaOH as precursors ( Figure 1AScanning electron microscopy (SEM) (Figure 1B) and transmission electron microscopy (TEM) (Figure 1C) revealed that the CHNPs were hexagonal in shape with an average diameter (the long diagonal of the hexagon) of 219.9 ± 17.8 nm. X-ray powder diffraction (XRD) confirmed that the nanocrystals were hexagonal platelets of Ca(OH)2 (PDF No. 01-073-5492, Figure 1E ).

[0432] Then, the CHNPs were coated with silica ( Figure 1A ). A mixture of tetraethyl orthosilicate (TEOS) and (3-aminopropyl)triethoxysilane (APTES) was used as a silane precursor to present amine groups on the surface of the resulting nanoparticles. Subsequently, polyethylene glycol (PEG) dibasic acid (mw=2000) was conjugated to the surface of silica via EDC / NHS coupling. SEM and energy dispersive spectroscopy (EDS) confirmed the success of the coating (Figures 1d and f). TEM revealed that the coating thickness was approximately 20 nm (Figure 1C). XRD showed that the coating did not negatively affect the crystallinity of the Ca(OH)2 core ( Figure 1E ).

[0433] PEGylated Ca(OH)2 / SiO2 core / shell nanoparticles (PCHNPs) can be well dispersed in water. Their hydrodynamic size is 245.2±30.26nm, compared to 227.3±27.02nm for bare Ca(OH)2 / SiO2 nanoparticles ( Figure 1G The surface of PCHNP is almost neutral (-4.91mV, Figure 1H In contrast, bare Ca(OH)2 / SiO2 nanoparticles were slightly positively charged (+16.4 mV) due to the surface amine groups. Fourier transform infrared (FT-IR) also confirmed the success of PEGylation, and found that PCHNPs had characteristic CH stretching vibration peaks (2882 cm-1) and bending vibration peaks (1467 cm-1). -1 and 1341cm -1 ), and COC stretching vibration peak (1033cm -1 )( Figure 9A ).

[0434] Finally, anti-CD205 antibody was coupled to PSCHNP using EDC / NHS chemistry. The resulting conjugate (i.e., AnCHNP) was stable in aqueous solution ( Figure 1I By quantifying protein and calcium, it was estimated that each nanoparticle carried an average of 27 antibody molecules. Coupling with antibodies increased the hydrodynamic size of the nanoparticles to 295.3 ± 46.7 nm ( Figure 1G). Meanwhile, the surface charge slightly increased to -2.83 mV during conjugation ( Figure 1H ).

[0435] In summary, Ca(OH)2 nanoparticles were synthesized, coated with silica and PEGylated on the surface. Anti-CD205 antibody was successfully conjugated to the nanoparticles.

[0436] AnCHNP uptake by DCs and its effect on [Ca 2+ ] int Impact

[0437] The silica coating slowed down but did not prevent the degradation of the Ca(OH)2 core. Sustained release of calcium ( Figure 2A The cumulative release reaches about 80% in 24 hours, and the half-life is about 7 hours ( Figure 2A ). When the pH of the solution dropped to 5.5, the degradation rate remained almost unchanged. Samples extracted from the PCHNP solution at different times were also examined under TEM. Consistent with the release results, the Ca(OH)2 core gradually dissolved ( Figure 2B Meanwhile, the silica shell remained largely intact, effectively functioning as a calcium capsule.

[0438] Subsequent experiments investigated the cellular uptake of AnCHNPs by BMDCs. To this end, AnCHNPs were labeled with Cy5 and incubated with BMDCs (bone marrow-derived dendritic cells) at 5 μg / mL or 10 μg / mL (based on Ca, the same below). For comparison, Cy5-labeled PCHNPs were also tested. Flow cytometry revealed that the nanoparticle uptake of AnCHNPs was significantly increased compared to PCHNPs ( Figure 2C When AnCHNPs were co-incubated with azide, a common endocytosis inhibitor, uptake was reduced. Chlorpromazine and dynasore also inhibited uptake ( Figure 2D), which blocked clathrin-dependent and dynamin-dependent endocytosis, respectively. Meanwhile, nystatin, which inhibits the caveolae endocytosis pathway, had no effect on particle uptake. These results suggest that AnCHNPs enter DCs via receptor-mediated endocytosis, as has been observed by others using anti-CD205 antibodies (Tel et al., European Journal of Immunology 2011, 41(4), 1014-1023, Schreibelt et al., Blood, The Journal of the American Society of Hematology 2012, 119(10), 2284-2292).

[0439] Incubation with AnCHNPs caused an increase in lysosomal pH ( Figure 2E ), which may be due to the neutralization of protons by Ca(OH)2. At the same time, Fluo-3AM assay found [Ca2 + ] int The time-dependent increase in Figure 2F This is due to the degradation of Ca(OH)2 particles and, at the same time, the release of calcium into the cytosol. [Ca 2+ The increase in [Ca]int persisted for more than 24 hours, which is consistent with what was observed in solution. In contrast, the increase in [Ca]int induced by CaCl2 salt at the same calcium dose was not significant. 2+ ]int increases very little ( Figure 2F ). At the same time, according to SBFI-AM and PBFI-AM determination, [Na + ] int and [K + ] int Levels remain largely unchanged ( Figure 2G and 2H ).

[0440] In conclusion, the results demonstrated that AnCHNPs were taken up by DCs via clathrin-dependent and dynein-dependent endocytosis and were gradually degraded intracellularly, resulting in [Ca 2+ ] int Effects of AnCHNP on DC maturation and migration

[0441] AnCHNPs were first incubated with 5 μg / mL or 10 μg / mL BMDCs in the absence of cancer cells, and surface MHC-II ( Figure 3A Compared with untreated DCs, when BMDCs were treated with AnCHNPs, MHC-II+ Both the population and expression level (MFI) of DCs increased significantly ( Figure 3B ), indicating that DC maturation was enhanced. AnCHNP also induced CD205 expression in DC ( Figure 3C ), which may create a positive feedback loop, leading to more AnCHNP uptake, thereby promoting cell maturation. It should be noted that others have also reported upregulation of CD205 in activated DCs (Butler et al., Immunology 2007, 120(3), 362-371). In contrast, CaCl2 had no effect on the expression of MHC-II or CD205 ( Figures 3A-3C Silica nanoparticles of similar size to AnCHNPs also showed no positive effect on MHC-II expression ( Figure 3D ).

[0442] Subsequent studies examined the effects of AnCHNPs on DC migration in a transwell assay, in which B16F10-OVA cells that had or had not been irradiated (100 Gy) were seeded onto the lower chamber, and CFSE-labeled BMDCs were loaded onto the insert. Irradiated B16F10-OVA cells resulted in enhanced transwell migration of DCs compared to non-irradiated B16F10-OVA cells ( Figure 3E ), which is due to the radiation-induced release of DAMPs and chemokines that promote chemotactic migration (Randolph et al., Annual review of immunology 2008, 26(1), 293-316). Incubation with AnCHNPs significantly increased the number of DCs migrating to the bottom chamber, suggesting that nanoparticles can enhance the ability of DCs to perceive chemotactic signals and migrate toward the source. In contrast, CaCl2 had the least effect on DC migration.

[0443] Next, we examined the maturation and activation of DCs when they were co-cultured with pre-irradiated (100 Gy) B16F10-OVA cells. In this case, treatment with AnCHNPs significantly increased CD80 + CD86 + The frequency of DC ( Figure 4A Other maturation markers, including CD40 and MHC-II, were also elevated ( Figure 4B In addition, surface SIINFEKL-H-2Kb increased significantly, indicating enhanced antigen presentation by DCs when treated with AnCHNPs. Notably, AnCHNPs were more effective at 5 μg / mL than at 10 μg / mL, likely due to the negative impact of the nanoparticles on cell viability at higher concentrations ( Figure 10CIn contrast, calcium salts and silica nanoparticles had no positive effect on DC activation ( Figure 3D The cytokines in the supernatants of the co-cultures were also measured. DCs treated with AnCHNPs showed increased secretion of pro-inflammatory cytokines, including IL-6, IL-12, and TNF-α, relative to DCs treated with vehicle or CaCl2 alone ( Figure 4C ), but the secretion of the anti-inflammatory cytokine IL-10 was reduced (although not significant, p = 0.3307).

[0444] Taken together, these in vitro results support the conclusion that AnCHNPs effectively promote DC maturation, migration, and antigen presentation.

[0445] Mechanism of AnCHNP activation of DCs

[0446] To explore the gene expression changes that occurred in DC cells with or without AnCHNPs, a whole transcriptome sequencing study was performed. DEG analysis showed that in mouse BMDCs treated with AnCHNPs, 1325 genes (fold change > 1.5 and P < 0.05) were upregulated, and 3049 genes (fold change < 0.5 and P < 0.05) were downregulated. Interestingly, the reactive free radical nitric oxide synthase 2 (Nos2), which acts as a biological mediator of antitumor activity, was the most upregulated gene in BMDCs after AnCHNP treatment ( Figure 5B GO enrichment analysis revealed that gene signatures of NF-κB signaling, cytokine activity, and immune response were among the top 10 most upregulated GO terms in AnCHNP-treated BMDCs compared with controls ( Figure 5C Consistent with this, GSEA analysis also showed that in the presence of AnCHNPs, I_κB_kinase_NF_κB_signaling, response to cytokines, regulation of immune system processes, and regulation of immune responses in BMDCs were most enriched ( Figure 5D ). qPCR validated these observations and found that treatment with AnCHNPs induced chemokines (e.g., CXCL-1, CCL5, CXCL2, and CXCL10) and cytokines (e.g., IL-1β, IL-12, and IL-6), which are known to attract and stimulate immune cells, including T cells ( Figure 5F Western blotting was also performed to investigate the activation pathways of BMDCs. BMDCs treated with AnCHNPs showed increased expression of phospho-NF-κB relative to controls, indicating activation of the NF-κB pathway. Simultaneously, AnCHNP treatment also resulted in increased expression levels of calcineurin and dephosphorylated NFAT, indicating activation of the NFAT axis ( Figure 5E ).

[0447] Overall, sustained calcium release from AnCHNPs leads to activation of the NF-κB and NFAT pathways, inducing chemokines, cytokines, antigen presentation, and co-stimulatory molecules, thereby enhancing DC-mediated immunity.

[0448] Effects of AnCHNP on immune response in vivo

[0449] Subsequent studies set out to investigate the effects of AnCHNPs in vivo. This was tested in C57BL / 6 mice bearing B16F10-OVA tumors. Radiation (10 Gy) was applied to the tumors, potentially triggering antigen / DAMP release. Subsequently, an intratumoral (it) injection of AnCHNPs (200 μg / kg) was administered at 1 hour. For comparison, either CaCl2 alone or vehicle (PBS) was injected. Animals were euthanized on day 3 or day 7, and tumors, spleens, and tumor-draining lymph nodes (TDLNs) were harvested for flow cytometric analysis (Figure 6a).

[0450] Mice treated with AnCHNPs showed a significant increase in CD11c+ cells in tumors compared to PBS or CaCl2 controls on both days 3 and 7, indicating increased tumor infiltration of DCs ( Figure 6B Specifically, the populations of MHC-II+, CD80+CD86+, and CD40+ DCs increased significantly ( Figure 6C ), indicating enhanced DC maturation. In addition, AnCHNPs caused an increase in SIINFEKL-H-2Kb+ DCs in tumors on day 3, indicating improved antigen presentation ( Figure 6C Similarly, on day 3, an increase in the population of MHC-II+, CD80+CD86+, CD40+, and SIINFEKL-H-2Kb+ DCs was observed in TDLN ( FIG6 c ), which was attributed to enhanced DC migration after AnCHNP treatment.

[0451] T lymphocytes in the tumor were also examined. On day 7, AnCHNPs significantly promoted tumor infiltration of cytotoxic T cells (CTL, CD45+CD3+CD8+). Specifically, the population of effector T cells (IFN-γ+CTL) increased on both day 3 and day 7 ( Figure 6D ). At the same time, the frequency of Treg (CD45+CD3+CD4+Foxp3+) was significantly reduced. In the AnCHNP group, the tumor CTL / Treg ratio increased by about 2 times, indicating that intratumoral injection immunity was strongly enhanced. A similar trend was also observed in T lymphocytes in the spleen ( Figure 6D In contrast, CaCl2 had minimal effect on CTLs or Tregs in tumors.

[0452] Antigen-specific cellular immunity was also examined by co-incubating splenocytes and B16F10-OVA cells in vitro. In splenocytes obtained from the AnCHNP-treated group, the frequency of IFN-γ+ CTLs was significantly increased ( Figure 12 ), indicating that the nanoparticles induced a systemic anti-tumor immune response. In contrast, splenocytes obtained from the CaCl2 group showed weak T cell activation during co-incubation.

[0453] Cytokine levels in serum from the different treatment groups were examined. Animals treated with AnCHNPs, but not CaCl2, showed elevated levels of IL-1β, IL-6, TNF-α, IFN-γ, and IL-12, but decreased IL-10 levels, at both day 3 and day 7 relative to PBS controls. Figure 6E ), these results are consistent with the analysis of leukocytes.

[0454] In conclusion, the results indicate that AnCHNPs can promote DC maturation and migration, thereby enhancing innate and cellular immunity against cancer.

[0455] To evaluate the efficacy of AnCNHP when combined with RT

[0456] Next, the therapeutic benefit of AnCNHP when used in combination with other treatments, starting with RT, was evaluated. This was also tested in the B16F10-OVA tumor model. Specifically, one hour after radiation (10 Gy) was applied to the tumor, AnCNHP was injected intratumorally (50 μL, 200 μg / kg in PBS, and the rest of the body was lead shielded. A total of two treatments were performed, each with an interval of two days (RT+AnCHNP). For comparison, animals were treated with only vehicle, only RT, or only AnCHNP ( Figure 7A ).

[0457] Tumors in the PBS group grew rapidly, and all animals were either moribund or reached the humane endpoint within 2 weeks ( Figure 7B RT modestly inhibited tumor growth, but all animals in this group died within 3 weeks. In contrast, AnCHNP plus RT significantly improved tumor suppression. Eighty percent of the animals in the combined group experienced tumor regression within the first three weeks. All animals in this group were still alive after 5 weeks, with 20% of the animals being tumor-free. Notably, AnCHNP alone had no effect on tumor growth ( Figure 7B and 7C), suggesting that the therapeutic benefit is due to the immunomodulatory effects of the nanoparticles. This idea is supported by results from T cell depletion studies in which animals received anti-CD4 or anti-CD8 antibodies in addition to the AnCHNP-RT combination. Depletion of either CD4 or CD8 T cells worsened the therapeutic effect. Of the two antibodies, the anti-CD8 antibody more significantly attenuated the therapeutic benefit. These results support the conclusion that enhancing cellular immunity is the primary factor behind the radiosensitizing effect of AnCHNPs ( Figure 7B and 7C ).

[0458] Postmortem histopathological examination of tumor and major organ samples was performed. Hematoxylin / eosin (H&E) staining revealed extensive areas of nuclear shrinkage and fragmentation in tumors treated with AnCHNP plus radiation. Concomitantly, Ki-67 staining levels decreased in the combination group, indicating reduced cell proliferation. Furthermore, no signs of toxicity were observed in any major organ tissues.

[0459] For validation, AnCHNP plus RT was also tested in C57BL / 6 mice bearing MB49 tumors ( Figure 7D In this model, RT alone was more effective, extending the average survival from 17 days to 40 days. Adding AnCHNP to the treatment regimen significantly improved efficacy. The combination group showed a 65.9% improvement in tumor growth inhibition at day 40 compared to RT alone. After 7 weeks, 60% of the animals were still alive, while all animals in the PBS and RT groups had died at this time ( Figure 7E and 7F ).

[0460] In conclusion, our in vivo studies demonstrated that low-dose AnCHNPs could effectively enhance RT-induced immunity, thereby improving tumor control and animal survival.

[0461] To evaluate the efficacy of AnCNHP when used in combination with chemotherapy or immunotherapy

[0462] The next study evaluated whether AnCNHP could enhance the efficacy of chemotherapy such as carboplatin. The combination of carboplatin (40 mg / kg, intraperitoneal injection) and AnCNHP (200 μg / kg, intratumoral injection) was first tested in the B16F10 tumor model ( Figure 8A Carboplatin is a known ICD agent, but as a monotherapy, it is not effective in stimulating a strong immune response (Ho et al., Critical reviews in oncology / hematology 2016, 102, 37-46). In fact, carboplatin alone can only slightly slow tumor growth ( Figure 8B), all animals died within 3 weeks. Adding AnCHNP significantly improved treatment efficacy, extending median survival from 15 days in the carboplatin group to 23 days in the combination group. No additional toxicity was observed.

[0463] The study also investigated whether AnCHNP could enhance the efficacy of immune checkpoint blockade, also in mice bearing B16F10 tumors ( Figure 8D B16F10 is a poorly immunogenic tumor model (Yang et al., Journal of nanobiotechnology 2021, 19(1), 1-11), and anti-PD-L1 antibody alone (10 mg / kg, 4 times) showed only moderate therapeutic benefit. Adding AnCNHP (200 μg / kg, intratumoral injection) to the treatment regimen improved the efficacy ( Figure 8E and 8F), and this combination was well tolerated.

[0464] Taken together, the results suggest that AnCHNPs can also enhance the efficacy of chemotherapy and immunotherapy without causing additional toxicity.

[0465] Summarize

[0466] In this study, AnCHNPs were investigated as an immunomodulator. AnCHNPs enter cells via endocytosis and are degraded within lysosomes, releasing calcium into the cytosol. Typically, DCs are activated by sensing external stimuli such as pathogens or damaged tissues through pattern recognition receptors (e.g., Toll-like receptors). This triggers a cascade of events that leads to calcium store depletion, Ca 2+ Release of activated Ca 2+ Activation of NFAT and NF-κB pathways, leading to increased calcium influx (Shumilina et al., American Journal of Physiology-Cell Physiology 2011, 300(6), C1205-C1214). In contrast, AnCHNPs directly activate NFAT and NF-κB pathways, leading to DC maturation, even in the absence of external stimulation ( Figure 3A ).

[0467] Although calcium phosphate nanoparticles have long been used for gene delivery, the effects of calcium nanoparticles on the function of immune cells, especially DCs, have been rarely studied. Several recent studies have shown that calcium carbonate nanoparticles can enhance immune responses, but the activation mechanism remains largely elusive. In addition, few attempts have been made to selectively deliver calcium nanoparticles to DCs. This is important because calcium released into the extracellular environment has a significant effect on [Ca 2+ ]int The increase in Figure 2F In other words, delivering calcium nanoparticles to the TME rather than directly into DCs did not activate immune cells. Here, the calcium nanoparticles were coupled to anti-CD205 antibodies, promoting receptor-mediated endocytosis. The nanoparticles were also coated with silica, which rapidly dissolves while providing sustained calcium release. Both designs contribute to highly effective immune modulation.

[0468] One advantage of calcium-based immunomodulators is their high biocompatibility. As shown in this study, after 24 hours, the calcium core of AnCHNPs is largely degraded. The resulting calcium ions, which are unable to freely cross the plasma membrane, are safely excreted. The risk of local and systemic toxicity is low or even non-existent. For the therapy study, each mouse was injected with 200 μg / kg, or approximately 4 μg, of AnCHNPs. Such a low dose of nanoparticles itself has no tumoricidal effect ( Figure 7B and 7C ). On the other hand, the data showed that at the tested doses, AnCHNPs alone can overcome immunosuppressive factors in the TME, triggering both innate and adaptive immunity. However, higher doses of AnCHNPs can stimulate robust immunity without causing calcium overload. Tumor antigens and / or conventional immunomodulators can be loaded onto the disclosed calcium nanoparticles, effectively creating vaccines that promote DC-mediated anti-tumor immunity. In summary, the current study introduces a nanoplatform that presents opportunities for safe and efficient immunomodulation and cancer management.

[0469] Example 2: Calcium Nanoparticles for Enhancing T Cell Immunity and Promoting Cancer Therapy

[0470] Materials and methods

[0471] Nanoparticle synthesis and characterization

[0472] Synthesis of CaCO3 nanoparticles

[0473] To synthesize calcium carbonate (CaCO ) nanoparticles, a calcium chloride and ammonium bicarbonate coprecipitation method was used. Specifically, in a 1000mL glass beaker, 1359mg of CaCl was dissolved in 900mL of ethanol. To promote dissolution, a water bath sonication process can be used. The beaker was carefully covered with plastic wrap and the plastic wrap was evenly pierced with a 29G needle to allow CO to pass through. The beaker was then placed in a 3L plastic beaker containing 36g of NH HCO . The entire reaction system was sealed with plastic wrap. After approximately 60 hours, particles began to form. Dynamic light scattering (DLS) was used to measure particle size until the desired diameter of 150nm to 160nm was reached. CaCO nanoparticles were collected by centrifugation at 12,096g for 10 minutes. After centrifugation, the nanoparticle precipitate was washed three times with 20mL of ethanol. The CaCO nanoparticles were dispersed in 10mL of ethanol and stored at room temperature for future use.

[0474] Synthesis of CaCO3@OA nanoparticles

[0475] 10 mg of CaCO nanoparticles were dispersed in 20 mL of ethanol, and 20 mg of oleic acid was added. The mixture was allowed to react overnight at room temperature with constant stirring. CaCO@OA nanoparticles were obtained by centrifugation at 12,096 g for 10 minutes. To remove unreacted oleic acid, the particles were washed three times with a mixture of 5 mL of ethanol and 10 mL of hexane.

[0476] Synthesis and characterization of CaCO3@OA@lipid (CCNP)

[0477] The previously described CaCO3@OA nanoparticles are hydrophobic and dispersible in hexane. They are coated with a PEGylated phospholipid, such as 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (DSPE-PEG-COOH). The coating is introduced through hydrophobic-hydrophobic interactions. After application, the nanoparticles (CCNPs) become hydrophilic and readily disperse in aqueous solutions.

[0478] In a typical reaction, 20 mg of DSPE-PEG-COOH (dissolved in chloroform) was mixed with 10 mg of CaCO3@OA in a 50 mL round-bottom flask and the mixture was sonicated until the particles were completely dispersed. Chloroform was then removed at room temperature using a rotary evaporator to form a thin film at the bottom of the flask. 20 mL of HEPES buffer (0.01 M) was added to the flask and immediately sonicated in a water bath for 5 minutes. CCNPs were concentrated by centrifugation at 9,400 g for 10 minutes. A second round of centrifugation was performed to collect additional CCNPs.

[0479] Synthesis and characterization of CCNP-Ab

[0480] Anti-PD1 antibodies were conjugated to CCNPs using EDC / NHS chemistry. Briefly, 10 mg of CCNPs were dispersed in 2.4 mL of HEPES buffer, and 2 mg of EDC (5 mg / mL, HEPES) and 4 mg of NHS sulfo groups (5 mg / mL, HEPES) were added to the mixture. The solution was vortexed at 220 rpm for 20 minutes and subsequently centrifuged at 9,400 g for 10 minutes. The particle pellet was then redispersed in 0.75 mL of HEPES. 200 μg of anti-PD-1 antibodies were added to the solution and stirred for 30 minutes. To quench the reaction, 28 μL of ethanolamine solution (200 mg / mL) was added, and the mixture was vortexed for another 10 minutes. CCNP-Ab nanoparticles were collected by centrifugation at 9,400 g for 10 minutes, redispersed in 350 μL of HEPES, and stored at 4 ° C for future use.

[0481] Synthesis of PMA@CCNP-Ab

[0482] To incorporate PMA into CCNP-Ab, CCNP-Ab was prepared in aqueous solution at the desired calcium concentration. PMA (5 mg / mL, acetonitrile) was then added to achieve a final concentration of 50 ng / mL. The solution was sonicated for 1 minute to complete the loading process. The loading rate was calculated by HPLC.

[0483] result

[0484] The role of the immune system, particularly cytotoxic T cells, in combating cancer is now well established. Cancer cells possess tumor-associated antigens (TAAs) that, like viruses and bacteria, can be recognized by the immune system and killed by cytotoxic T cells (CTLs) in an antigen-specific manner. However, solid tumors are often characterized by an immunosuppressive environment that inhibits or inactivates T cell activation and proliferation. Various strategies, including immune checkpoint inhibitors (ICIs), are being developed to directly or indirectly enhance the function of endogenous T cells. However, a significant proportion of patients do not respond to ICIs. Alternatively, antigen-specific T cells can be expanded or engineered outside the patient's body and reintroduced into the host. These include adoptive T cell transfer and CAR-T therapy, which have made significant progress and are entering the clinic. However, the efficacy of these therapies may still be limited by issues such as toxicity and the harsh tumor microenvironment. New immunotherapy options are needed that can be used as monotherapy or in combination to enhance existing immunotherapies.

[0485] To address these problems, innovative immunomodulators based on calcium nanoparticles have been developed that specifically target T cells and enhance T cell function. Calcium plays a central role in T cell activation as a second messenger. Calcium signaling begins by stimulating the TCR pathway and ultimately causes activation of the transcription factor NFAT through activation of the calcium-sensitive phosphatase calcineurin. This technology can deliver calcium directly to the cytosol of T cells in the form of calcium nanoparticles to regulate T cell function. In order to achieve controlled calcium release, which is very important for T cell activation, calcium nanoparticles are coated with a lipid layer. This coating also allows loading of additional immunomodulators, such as PKC antagonists (such as phorbol 12-myristate 13-acetate (PMA)), which together with calcium enhance T cell immunity. In addition, targeting ligands such as anti-PD1 antibodies can also be conjugated to nanoparticles to direct nanoparticles to T cells.

[0486] Nanoparticle synthesis and characterization

[0487] As a representative example, calcium carbonate (CaCO3) nanoparticles loaded with PMA and surface-conjugated with anti-PD1 antibodies were prepared, referred to as PMA@CCNP-Ab. First, calcium carbonate (CaCO3) nanoparticles were prepared by a coprecipitation method using calcium chloride and ammonium bicarbonate. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) revealed that the obtained nanoparticles exhibited good uniformity ( Figures 16A-16D ). The elemental composition of CaCO3 nanoparticles was studied by energy dispersive X-ray spectroscopy (EDX), such as Figure 16I As shown. The peaks of calcium (Ca), carbon (C) and oxygen (O) were identified. X-ray diffraction (XRD) analysis ( Figure 16J ) further confirmed that the nanoparticles were made of CaCO3.

[0488] Next, CaCO3 particles were coated with oleic acid to form CaCO3@OA nanoparticles. TEM and SEM images ( Figure 16E 、 16F and 16G) reveal the morphology of CaCO3@OA. The surface of the nanoparticles is obviously smoother. Figure 16H The size distribution of CaCO3@OA is shown. Infrared (IR) spectroscopy measurements provided additional evidence for the conjugation of oleic acid on CaCO3 nanoparticles.

[0489] The previously described CaCO3@OA nanoparticles are hydrophobic and dispersible in hexane. The CaCO3@OA nanoparticles are coated with PEGylated phospholipids, such as 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (DSPE-PEG-COOH), so they can be more easily dispersed in aqueous solutions.

[0490] Anti-PD1 antibodies were conjugated to CCNPs using EDC / NHS chemistry. Zeta potential ( Figure 16L ) decreased after conjugation, indicating successful antibody conjugation. After lipid coating and antibody conjugation, the nanoparticle size increased ( Figure 16M ). When tested in solution, it was found that at pH 5.0, Ca 2+ Slow release over 96 hours ( Figure 16N ). In contrast, at neutral pH, the release leveled off after 24 hours.

[0491] PMA was incorporated into CCNP-Ab to form PMA@CCNP-Ab.

[0492] Cytotoxicity of CCNPs

[0493] The cytotoxicity of PMA-loaded nanoparticles (PMA@CCNP-Ab) was evaluated using the EL4 cell line. PMA@CCNP-Ab was well tolerated by the cells, but when Ca 2+ When the concentration exceeds 12.5 μg / mL, obvious toxicity will occur ( Figure 17A This may be due to T cells being overloaded with calcium at higher concentrations. In contrast, CaCl2 salts cannot penetrate the cell membrane and are 2 + There was no significant effect on cell viability until the concentration reached 100 μg / mL ( Figure 17B ). The calcium dose used in subsequent in vitro studies was 10 μg / mL.

[0494] Cellular uptake and effects on cytosolic calcium levels

[0495] PMA@CCNP and PMA@CCNP-Ab were labeled with Cy5, and the intracellular uptake of the nanoparticles was evaluated using PD-1-positive EL4 cells. For comparison, the nanoparticles were also incubated with endocytosis inhibitors such as dynasore or nystatin. Compared with PMA@CCNP, PMA@CCNP-Ab showed a significant increase in nanoparticle uptake ( Figure 17C ), which was attributed to PD-1-mediated endocytosis of PMA@CCNP-Ab. Co-incubation with dynasore reduced PMA@CCNP-Ab uptake, indicating that PMA@CCNP-Ab internalization involves a dynamin-dependent pathway. In contrast, nystatin had no inhibitory effect on cellular uptake, indicating that internalization is independent of lipid-mediated uptake processes. Incubation at 4°C effectively reduced nanoparticle uptake, indicating that nanoparticle uptake is mediated by endocytosis rather than diffusion.

[0496] Fluo-3AM was used as an indicator to measure changes in intracellular calcium levels. PMA@CCNP-Ab significantly increased the intracellular calcium level ( Figure 17D ), which was attributed to the degradation of the nanoparticles within the cells and the release of calcium. In contrast, CaCl2 salt at the same calcium concentration did not increase cellular calcium levels.

[0497] The ability of PMA@CCNP-Ab to activate T cells was also evaluated by Western blotting. PMA@CCNP-Ab nanoparticles effectively activated the NF-κB pathway, as evidenced by increased expression of phosphorylated p65 and IκBα ( Figure 17E In addition, PMA@CCNP-Ab also activated the NFAT pathway, as evidenced by increased dephosphorylation of NFAT ( Figure 17F Both pathways are known to be involved in calcium signaling in T cell activation.

[0498] Effects on T cell activation

[0499] The effects of PMA@CCNP-Ab on T cells from the spleen of OT-1 mice were evaluated ( Figure 17G 、 17H ). Cytotoxic T cells (CTLs) from OT-1 mice recognize OVA and are widely used as a tool to study antigen-specific immunity. OT-1 T cells were primed with anti-CD3 and anti-CD28 antibodies before incubation with PMA@CCNP-Ab. PBS, ION / PMA, CaCl2, and CCNP-Ab were tested for comparison. After 48 or 72 hours, the cells were harvested, stained for CD8, CD69, IFN-γ, and TNF-α, and analyzed by flow cytometry. The results showed that PMA@CCNP-Ab could significantly increase the CD69+ population in OT-1 CTLs, and the efficacy was comparable to that of ION / PMA ( Figure 17G 、 17H The frequency of IFN-γ-positive CTLs and TNF-α-positive CTLs increased, further supporting T cell activation. The stimulatory effect was more pronounced at 72 hours.

[0500] T cell activation was assessed by analyzing cytokine release from OT-1 cells after incubation with PMA@CCNP-Ab. This was assessed by ELISA using co-cultures of OT-1 splenocytes and irradiated (100 Gy) B16-OVA cancer cells ( Figure 17I 、 17J ). The results showed that the secretion of IFN-γ and IL-2 increased when cells were incubated with PMA@CCNP-Ab. Together, these results indicate that PMA@CCNP-Ab nanoparticles are able to enhance T cell activation.

[0501] Effects on cellular immunity in vivo

[0502] B16-OVA cells were inoculated into C57BL / 6 mice. When the tumor size reached 100 mm 3 At day 1 (day 1), X-rays (15 Gy) were applied to trigger an intratumoral immune response. On days 2, 5, and 8, PMA@CCNP-Ab was injected intratumorally (it) at a dose of 5 μg calcium and 10 ng PMA per mouse. PBS, ION / PMA, CaCl2, and CCNP-Ab were injected intratumorally for comparison. Mice that did not receive irradiation were also examined. On day 13, all mice were sacrificed. Tumors, spleens, and lymph nodes were harvested, processed into single cells, and stained for CD45, CD3, CD8, CD4, IFN-γ, and FoxP3.

[0503] Flow cytometry analysis revealed that CD8 + Increased infiltration of T cells (CTL) into tumors ( Figure 18A The tumor CTL / Treg ratio in the PMA@CCNP-Ab group was also significantly higher than that in the other treatment groups. Similar patterns were observed in the spleen and lymph nodes ( Figure 18B 、 18C ). These results indicate that PMA@CCNP-Ab enhances T cell activation and proliferation, thereby enhancing cellular immunity.

[0504] Splenocytes from different groups were co-cultured with B16-OVA in vitro to evaluate the effects of treatment on cellular immunity ( Figure 18D The number of activated CTLs (CD8+IFN-γ+) in splenocytes from the PMA@CCNP-Ab group was significantly increased, confirming that the nanoparticles enhanced the antigen-specific immune response against tumors.

[0505] Therapy efficacy

[0506] Next, the efficacy of PMA@CCNP-Ab was evaluated in C57BL / 6 mice bearing B16 tumors. PMA@CCNP-Ab nanoparticles were injected intratumorally at a dose of 5 μg calcium and 10 ng PMA per mouse. A total of three doses were administered, with two days between each dose. For comparison, CaCl2 salt was injected intratumorally at the same calcium dose. PMA@CCNP-Ab effectively inhibited tumor growth and significantly improved animal survival ( Figures 19A-19C ). Also, the therapeutic benefit was lost when anti-CD8 antibodies were injected to deplete CTLs in the animals ( Figures 19A-19C), indicating that activation of cellular immunity is the main reason for the tumor inhibition of PMA@CCNP-Ab nanoparticles. No acute or chronic toxicity was observed in animals treated with PMA@CCNP-Ab.

[0507] Together, these results demonstrate that T cells efficiently internalized the PMA@CCNP-Ab, leading to elevated intracellular calcium levels. Delivery of calcium and PMA to T cells promoted their activation, as evidenced by increased expression or secretion of CD69, IFN-γ, and TNF-α. This was observed in both the EL4 cell line and primary T cells from OT1 mice. In vivo testing in C57 / BL6 mice bearing B16-OVA tumors demonstrated that the PMA@CCNP-Ab enhanced tumor infiltration by cytotoxic T cells and increased the CTL / Treg ratio. The observed therapeutic benefit correlated with the PMA@CCNP-Ab's ability to enhance T cell activation. Furthermore, the PMA@CCNP-Ab could also be used to enhance cell-based therapies, including adoptive T cell transfer and CAR-T therapy.

[0508] The disclosed nanotechnology offers several features that make it unique:

[0509] Controlled calcium release: T cell activation requires intracellular calcium concentration ([Ca 2+ ] int ) continued to increase. [Ca] was not possible using calcium salts (due to the ion-impermeable plasma membrane) or bare calcium nanoparticles (due to the rapid dissolution of the particles in the TME). 2+ ] int To address this issue, a lipid coating was used that prevented the nanoparticles from rapid degradation, allowing them to enter cells via endocytosis and gradually release calcium ions within the cells.

[0510] Low toxicity: Unlike cytokine- or interferon-based immunomodulators, calcium nanoparticles have low toxicity and can be administered repeatedly without causing systemic toxicity. 2+ and CO3 2- , these components can be safely excreted, metabolized or absorbed by the host.

[0511] Targeted delivery: Nanoparticles can be conjugated with T cell targeting ligands (such as anti-PD1 or anti-CD3 antibodies) to achieve targeted delivery of calcium and PKC antagonists to T cells. In contrast, conventional stimulation tools (such as ionomycin-PMA combination) are effective in vitro but are not effective in vivo due to rapid clearance and lack of specificity.

[0512] Unique mechanism of action: Normally, engagement of the T cell receptor (TCR) with antigens presented by MHC-I molecules activates phospholipase Cγ1 (PLCγ1) and produces inositol 1,4,5-triphosphate (IP3). IP3 binds to its receptor on the endoplasmic reticulum (ER) membrane of T cells, causing calcium to flow from the ER into the cytosol. Luminal calcium depletion is sensed by STIM1 / 2 and triggers their translocation to the plasma membrane, where they activate Orai 1 / 2 to form Ca 2+ selective pores (i.e., CRAC channels) and induces Ca 2+ Influx (i.e., store-operated calcium entry, SOCE). Activation can be inhibited or blocked at multiple stages, thereby impairing cellular immunity. In the disclosed method, calcium delivery bypasses upstream signaling, which is believed to enable T cell activation even in an immunosuppressive environment.

Claims

1. A nanoparticle comprising a calcium core and a shell and / or coating.

2. The nanoparticle of claim 1, wherein the core further comprises a hydroxide, and optionally calcium hydroxide (Ca(OH)2).

3. The nanoparticle of claim 1, wherein the core further comprises a carbonate, and optionally calcium carbonate (CaCO3).

4. The nanoparticle of claim 1 , wherein the core is selected from the group consisting of calcium citrate (CaCit), calcium phosphate (Ca3(PO4)2), CaCL2, calcium sulfate (CaSO4), CaC2O4, Ca(NO3)2, calcium silicate (Ca2SiO4), calcium fluoride (CaF2), CaBr2, and CaI2.

5. The nanoparticle according to any one of claims 1 to 4, comprising the shell.

6. The nanoparticle of claim 5, wherein the shell reduces, prevents, or otherwise delays degradation of the nanoparticle.

7. The nanoparticles according to claim 5 or 6, wherein the shell comprises one or more of the following: silica, mesoporous silica, carbon, a sulfide, optionally ZnS, CoS, CuS, Cu2S, FeS, MoS, Al2S3, Y2S3 or MnS; an oxide, optionally Fe3O4, Fe2O3, Gd2O3, TiO2, Al2O3 or MnO2; a fluoride, optionally NaYF4, YF3, LaF3, CeF3, PrF3 or GdFe3; a fatty acid, optionally oleic acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, arachidic acid, eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA); an alkylamine, optionally octylamine, nonylamine, decylamine, undecylamine, laurylamine, tridecylamine, tetradecylamine, pentadecylamine, hexadecylamine, heptadecylamine, octadecylamine, oleylamine; MgO, CuO or ZnO.

8. The nanoparticle according to any one of claims 1 to 7, wherein the nanoparticle comprises the coating.

9. Nanoparticles according to claim 8, wherein the coating improves dispersion in aqueous solution and / or delays core release and / or increases half-life.

10. The nanoparticle according to claims 8 and 9, wherein the coating comprises one or more polymers, peptides, proteins, lipids or a combination thereof.

11. The nanoparticle of any one of claims 8 to 10, wherein the coating comprises PEG.

12. The nanoparticle of claim 1 , comprising a targeting agent, optionally wherein the targeting agent targets one or more immune cells, optionally wherein the one or more immune cells are selected from dendritic cells, T cells, macrophages, natural killer cells, neutrophils, and combinations thereof, optionally wherein the T cells are selected from cytotoxic T cells, helper T cells, regulatory T cells, memory T cells, gamma-delta T cells (gamma delta T cells), follicular helper T cells (Tfh), natural killer T cells (NKT cells), and combinations thereof.

13. The nanoparticle of claim 12, wherein the targeting agent targets dendritic cells.

14. The nanoparticle according to claims 12 and 13, wherein the targeting agent targets CD205, and is optionally an anti-CD205 antibody.

15. The nanoparticle of claim 12, wherein the targeting agent targets T cells.

16. The nanoparticle of claim 15, wherein the T cell comprises or is a cytotoxic T cell.

17. The nanoparticle of claim 15 or 16, wherein the targeting agent targets CD3 or PD-1, and is optionally an anti-CD3 or anti-PD-1 antibody.

18. The nanoparticle of any one of claims 1 to 17, further comprising an activating agent optionally selected from an antigen, a chemotherapeutic drug, an immune system modulator, an immune checkpoint regulator, or an immune cell regulator.

19. The nanoparticle of claim 18, comprising an immune cell modulator, optionally wherein the immune cell modulator is a protein kinase C (PKC) antagonist, optionally wherein the PKC antagonist is phorbol 12-myristate 13-acetate (PMA).

20. A pharmaceutical composition comprising the nanoparticles according to any one of claims 1 to 19.

21. The pharmaceutical composition according to claim 20, further comprising an adjuvant.

22. The pharmaceutical composition according to claim 20 or 21, further comprising an antigen, a chemotherapeutic drug, an immune system modulator, an immune checkpoint modulator or an immune cell modulator.

23. A pharmaceutical composition comprising immune cells treated in vitro or ex vivo with the nanoparticles according to any one of claims 1 to 19, optionally wherein the immune cells are selected from dendritic cells, T cells, macrophages, natural killer cells, neutrophils and combinations thereof, optionally wherein the T cells are selected from cytotoxic T cells, helper T cells, regulatory T cells, memory T cells, gamma-delta T cells (gamma delta T cells), follicular helper T cells (Tfh), natural killer T cells (NKT cells) and combinations thereof.

24. A method of increasing calcium signaling in an immune cell, the method comprising contacting the immune cell with an effective amount of a pharmaceutical composition according to any one of claims 20 to 22 to increase calcium signaling therein, optionally wherein the immune cell is selected from dendritic cells, T cells, macrophages, natural killer cells, neutrophils and combinations thereof, optionally wherein the T cell is selected from cytotoxic T cells, helper T cells, regulatory T cells, memory T cells, gamma-delta T cells (gamma delta T cells), follicular helper T cells (Tfh), natural killer T cells (NKT cells) and combinations thereof.

25. A method of enhancing an immune response in a subject in need thereof, the method comprising administering to the subject an effective amount of the pharmaceutical composition of any one of claims 20 to 23.

26. The method of claim 25, wherein the immune response comprises one or more of: increased NF-κB signaling and / or cytokine activity in dendritic cells, increased infiltration of dendritic cells into tumor sites, and / or enhanced maturation of dendritic cells.

27. The method of claim 25 or 26, wherein the immune response comprises one or more of the following: inducing dendritic cells to express or secrete chemokines (e.g., CXCL-1, CCL5, CXCL2 and / or CXCL10), cytokines (e.g., IL-1β, IL-12 and / or IL-6), or a combination thereof.

28. The method of claim 26 or 27, wherein the immune response comprises one or more of the following: increased T cell activation, increased T cell localization to tumor sites, increased expression and / or secretion of CD69, IFN-γ and / or TNF-α by T cells.

29. The method of any one of claims 24 to 28, wherein the subject has cancer or an infection.

30. A method for treating or preventing cancer, comprising administering an effective amount of the pharmaceutical composition according to any one of claims 20 to 23 to a subject in need thereof.

31. The method of claim 30, wherein the amount or mode of administration is effective to induce an immune response against the cancer but does not have a direct anti-cancer effect.

32. A method of treating or preventing infection, comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition according to any one of claims 20 to 23.

33. The method of any one of claims 25 to 32, further comprising treating the subject with one or more of: surgery, radiotherapy, chemotherapy, or immunotherapy, optionally with an immune checkpoint regulator, an immune system regulator, or an immune cell regulator.

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