Nanoconstructs and nanoparticle-mediated delivery of immunogenic cell death inducers to enhance cancer immunotherapy

Abstract

Nanoconstructs and compositions comprising nanoparticles coated with an immune adjuvant (e.g., ATP) and having one or more therapeutic agents (e.g., ICD-inducing agents) encapsulated therein; and methods for treating cancer in a subject using such nanoconstructs and compositions, and combination immunotherapy.

Description

[Technical field]

[0001] Priority This application is related to and claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 224,009, filed July 21, 2021, the contents of which are hereby expressly incorporated by reference in their entirety into this disclosure.

[0002] Government Rights This invention was made with Government support under CA232419 and CA258737 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] The present disclosure relates generally to nanoparticles, specifically poly(lactic-co-glycolic acid) nanoparticles that are surface-modified with adenosine triphosphate and loaded with anti-cancer drugs (e.g., immunogenic cell death inducers), and methods for the treatment of cancer using such nanoparticles. [Background technology]

[0004] Cancer is the second leading cause of death in the United States, claiming the lives of more than 600,000 people each year. In 2022, it is expected that more than 1.9 million patients will be diagnosed with cancer in the United States. Although the lethality of cancer tumors has been on a downward trend over the past few years, this is likely due to the development of early detection techniques and lifestyle improvements rather than increased therapeutic efficacy. Although various types of antitumor therapeutic treatments have been developed to treat cancer, limitations still exist, due at least in part to patient variability and tumor heterogeneity.

[0005] Cancer immunotherapy has been explored in hopes of overcoming existing limitations by harnessing the patient's own immune system, leading to the development of various therapeutic agents. In general, immunotherapy works by selectively exploiting the host's immune defenses against targets such as cancerous tumors. Immune checkpoint blockade immunotherapy is one such type of immunotherapy that uses antibodies and other compounds to block T cell negative regulatory molecules (e.g., cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and anti-programmed cell death protein 1 (PD-1)), and has produced positive results in some patients. However, due to the complex network of immunosuppressive pathways present in advanced tumors, only a small number of patients respond to this therapy.

[0006] Other cancer immunotherapy involves utilizing immunogenic cell death (ICD) inducers. ICD is a form of cancer cell death that can activate dendritic cells, leading to tumor-specific T cell immunity. It was long thought that cell death could simply be divided into only two different types of cell death, apoptosis and necrosis, but recent findings suggest that there is much more complexity. A third type of cell death, immunogenic apoptotic cell death, differs from the well-established concept of apoptosis (removal of cellular silence) because it alarms the host's immune system and triggers a cascade of immunological responses. Thus, ICD inducers induce tumor cell death to expose tumor antigens, and are accompanied by endoplasmic reticulum stress and reactive oxygen species production in cancer cells, which in turn induce the shedding of signaling molecules known as damage-associated molecular patterns (DAMPs).

[0007] DAMPs act as immune adjuvants that activate antigen-presenting cells (APCs), immune cells specialized in presenting antigens to T cells. Release of DAMPs can trigger the host immune system, which can confer robust adjuvanticity to dying cancer cells. ICD-associated DAMPs can include surface-exposed calreticulin (CRT), as well as secreted adenosine triphosphate (ATP), annexin A1 (ANXA1), type I interferons, and chromatin-binding high mobility group B1 (HMGB1). Additional hallmarks of ICD include phosphorylation of eukaryotic translation initiation factor 2 subunit-α (EIF2S1 or eIF2α), activation of autophagy, and global cessation of transcription and translation.

[0008] Some chemotherapeutic agents (e.g., oxaliplatin, mitoxantrone, doxorubicin, bortezomib, and cyclophosphamide) can be ICD inducers. These substances have the characteristics of ICD, so when used to treat tumors, they provide additional therapeutic effects due to persistent immune cell activation. However, so far, chemotherapeutic agents have limited therapeutic efficacy due to poor retention in tumors. In addition, ICD inducers exhibit high immunotoxicity, which remains a significant challenge when used alone or in combination with immune checkpoint inhibitors.

[0009] In addition, the tumor microenvironment (TME) can be regulated by multiple immunosuppressive cells, including tumor-associated macrophages (TAMs), myeloid-derived suppressor cells, cancer-associated fibroblasts, tumor-associated neutrophils, and regulatory T cells. For example, TAMs can comprise up to 50% of solid tumor mass and can promote tumor growth by interacting with cancer cells and other immune cells to promote angiogenesis, immunosuppression, and inflammation. The immunosuppressive TME can further complicate the efficacy of ICD-induced chemotherapy.

[0010] In view of the above, the present disclosure seeks to provide nanoparticles or compounds that can encapsulate ICD-inducing agents (i.e., as cargo), increase the antigenicity of tumor cells, recruit immune cells, and / or be used in conjunction with other immunotherapies such as, for example, immune checkpoint blockade therapy. [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] Yu et al., Pharmacokinetics, biodistribution and in vivo efficacy of cisplatin loaded poly(L-glutamic acid)-g-methoxy poly(ethylene glycol) complex nanoparticles for tumor therapy, J Control Release 205:89~97 (2015) [Non-Patent Document 2] Li et al., Efficient delivery of docetaxel for the treatment of brain tumors by cyclic RGD-tagged polymeric micelles, Molecular Med Reports 11:3078~3086 (2015) [Non-Patent Document 3] Park et al., Polydopamine-based simple and versatile surface modification of polymeric nano drug carriers, ACS Nano 8:3347~3356 (2014) [Non-Patent Document 4] Raymond et al., Oxaliplatin: mechanism of action and antineoplastic activity, Semin Oncol 25:4~12 (1998) [Non-Patent Document 5] Rafiei and Haddadi, Docetaxel-loaded PLGA and PLGA-PEG nanoparticles for intravenous application:pharmacokinetics and biodistribution profile, Int J Nanomedicine 12:935~947 (2017) [Non-Patent Document 6] Liu et al., Mixed Liposome Approach for Ratiometric and Sequential Delivery of Paclitaxel and Gemcitabine, AAPS PharmSciTech, 19:693~699 (2018) [Non-Patent Document 7] Park and Yeo, Albumin-coated nanocrystals for carrier-free delivery of paclitaxel, J. Controlled Release 263:90~101 (2017) [Non-Patent Document 8] Park et al., A Comparative In Vivo Study of Albumin-Coated Paclitaxel Nanocrystals and Abraxane, Small 14:e1703670 (2018) [Non-Patent Document 9] Jorgovanovic et al., Roles of IFN-gamma in tumor progression and regression: a review, Biomark Res 8:49 (2020) [Non-Patent Document 10] Han et al., Turning the Tide Against Regulatory T Cells, Front Oncol 9:279 (2019) [Non-Patent Document 11] Yoshida et al., Anti-PD-1 antibody decreases tumor-infiltrating regulatory T cells, BMC Cancer 20:25 (2020) [Non-Patent Document 12] Ravelli et al., Immune-related strategies driving immunotherapy in breast cancer treatment: a real clinical opportunity, Expert Rev Anticancer Ther 15:689~702 (2015) [Non-Patent Document 13] Messenheimer et al., Timing of PD-1 Blockade Is Critical to Effective Combination Immunotherapy with Anti-OX40, Clin Cancer Res 23:6165–6177 (2017) [Non-Patent Document 14] Kim et al., Sequential and Timely Combination of a Cancer Nanovaccine with Immune Checkpoint Blockade Effectively Inhibits Tumor Growth and Relapse, Angew Chem Int Ed Engl 59:14628~14638 (2020) [Non-Patent Document 15] Rajabnia and Meshkini, Fabrication of adenosine 5'-triphosphate-capped silver nanoparticles: Enhanced cytotoxicity efficacy and targeting effect against tumor cells, Process Biochemistry 65:186~196 (2018) [Non-Patent Document 16] Hyun et al., Surface modification of polymer nanoparticles with native albumin for enhancing drug delivery to solid tumors, Biomaterials 180:206~224 (2018) [Non-Patent Document 17] Xu et al., Quinic Acid-Conjugated Nanoparticles Enhance Drug Delivery to Solid Tumors via Interactions with Endothelial Selectins, Small 14:e1803601 (2018) Summary of the Invention

[0012] Nanoconstructs are provided. In certain embodiments, the nanoconstructs include a nanoparticle having an exterior surface, one or more therapeutic agents encapsulated within the nanoparticle, and an immunoadjuvant-modified polyphenol compound bound to the exterior surface of the nanoparticle.

[0013] The polyphenolic compound may be selected from the group consisting of polymerized dopamine (pD), tannic acid, tannic acid-iron complex, gallic acid, ellagic acid, hydroxyhydroquinone, epigallocatechin, epicatechin gallate, epigallocatechin gallate, and pyrogallol. In certain embodiments, the polyphenolic compound is pD.

[0014] The immune adjuvant may be selected from the group consisting of adenosine triphosphate (ATP), calreticulin, high mobility group box 1, deoxyribonucleic acid, annexin A1, type I interferon, heat shock protein 70, and heat shock protein 90. In certain embodiments, the immune adjuvant is ATP. In certain embodiments, the immune adjuvant is calreticulin. In certain embodiments, the immune adjuvant is high mobility group box 1. The immune adjuvant may be, for example, about 10.5 wt% to about 12.5 wt% of the nanoconstruct, or any other wt% of the construct as deemed desirable.

[0015] The nanoparticles may be selected from the group consisting of poly(lactic-co-glycolic acid) (PLGA), polycaprolactone, D-α-tocopherol polyethylene glycol 1000 succinic acid-PLGA conjugate, polylactic acid, PLGA-methoxy-polyethylene glycol, ethylene vinyl acetate, mesoporous silica, liposomes, nanocrystals, and polyphenol aggregates. In certain embodiments, the nanoparticles are PLGA nanoparticles. In certain embodiments, the nanoparticles are PLGA-methoxy-polyethylene glycol nanoparticles. In certain embodiments, the nanoparticles are liposomes. In certain embodiments, the nanoparticles are nanocrystals.

[0016] The one or more therapeutic agents may be one or more chemotherapeutic agents. In certain embodiments, at least one of the one or more therapeutic agents is an immunogenic cell death (ICD) inducer. In certain embodiments, the one or more therapeutic agents may be oxaliplatin, carfilzomib, paclitaxel, mitoxantrone, bleomycin, doxorubicin, epirubicin, idarubicin, cyclophosphamide, or cardiac glycoside (or any combination of the foregoing). In certain embodiments, the one or more therapeutic agents are carfilzomib. In certain embodiments, the one or more therapeutic agents are paclitaxel.

[0017] Compositions comprising the nanoconstructs are also provided. In certain embodiments, such compositions comprise the nanoconstructs and a pharma- ceutically acceptable carrier.

[0018] Further provided is a method for treating cancer in a subject. In certain embodiments, the method for treating cancer in a subject comprises administering to the subject a therapeutically effective amount of a nanoconstruct described herein or a composition described herein. The administering step can be performed, for example, subcutaneously, intravenously, intramuscularly, intraperitoneally, intratumorally, or locally.

[0019] The method may further include administering an immunotherapy to the subject (e.g., a combination therapy). In certain embodiments, the immunotherapy is an immune checkpoint inhibitor therapy. The therapeutically effective amount of the nanoconstruct may be administered to the subject prior to administration of the immunotherapy to the subject. Alternatively, the therapeutically effective amount of the nanoconstruct may be administered to the subject after administration of the immunotherapy to the subject.

[0020] Also provided is a method for treating cancer in a subject, the method comprising administering to the subject a priming dose comprising a therapeutically effective amount of a nanoconstruct comprising nanoparticles having an outer surface, one or more therapeutic agents encapsulated within the nanoparticles, and an immune adjuvant-modified polyphenol compound bound to the outer surface of the nanoparticle; and an immune checkpoint inhibitor, a tumor-targeting antibody, or a cancer vaccine, wherein the priming dose induces or enhances an anti-tumor immune response in the subject at a target site. The target site can be a solid tumor. The target site is a cancerous tissue or cell.

[0021] The immune adjuvant may be selected from the group consisting of adenosine triphosphate, calreticulin, high mobility group box 1, deoxyribonucleic acid, annexin A1, type I interferon, heat shock protein 70, and heat shock protein 90. In certain embodiments, the one or more therapeutic agents are selected from the group consisting of oxaliplatin, carfilzomib, paclitaxel, mitoxantrone, bleomycin, doxorubicin, epirubicin, idarubicin, cyclophosphamide, and cardiac glycosides. In certain embodiments, the nanoparticles are selected from the group consisting of PLGA, polycaprolactone, D-α-tocopherol polyethylene glycol 1000 succinic acid-PLGA conjugate, polylactic acid, PLGA-methoxy-polyethylene glycol, ethylene vinyl acetate, mesoporous silica, liposomes, nanocrystals, and polyphenol aggregates.

[0022] The nanoparticles can be PLGA. The one or more therapeutic agents can be chemotherapeutic agents. At least one of the therapeutic agents can be an ICD inducer. The polyphenolic compound of the method can be pD, tannic acid, tannic acid-iron complex, gallic acid, ellagic acid, hydroxyhydroquinone, epigallocatechin, epicatechin gallate, epigallocatechin gallate, or pyrogallol. In certain embodiments, the polyphenolic compound of the method can be selected from the group consisting of pD, tannic acid, tannic acid-iron complex, gallic acid, ellagic acid, hydroxyhydroquinone, epigallocatechin, epicatechin gallate, epigallocatechin gallate, and pyrogallol. The immune checkpoint inhibitor can be an antibody or antibody fragment targeting PD-1 (e.g., nivolumab or pembrolizumab) or PD-L1 (e.g., atezolizumab, avelumab, or durvalumab), CTLA-4 (e.g., tremelimumab or ipilimumab), an anti-CD25 antibody (e.g., basiliximab), or decitabine (e.g., a demethylating agent that controls T cell exhaustion).

[0023] In certain embodiments, the priming dose of the method is administered at least 4 days prior to the immune checkpoint inhibitor, thereby treating cancer in the subject.

[0024] Also provided are methods for enhancing an anti-cancer immune response in a subject, in certain embodiments, such methods comprise administering to a subject a therapeutically effective amount of a nanoconstruct herein or a composition herein.

[0025] Still further, methods of enhancing infiltration of immune cells to a target site are provided, such methods comprising delivering one or more therapeutic agents to a target site in a subject, the delivery comprising administering a therapeutically effective amount of a nanoconstruct or composition under conditions to deliver the nanoconstruct to the target site. In certain embodiments, the target site is a solid tumor, and the nanoconstruct enhances an immune response (e.g., an anti-tumor specific immune response) at the target site.

[0026] In certain embodiments, administering a therapeutically effective amount of the nanoconstruct or composition induces ICD at the target site. In certain cases, the target site is a solid tumor and administering a therapeutically effective amount of the nanoconstruct or composition results in a reduction in the volume of the solid tumor.

[0027] Administration of a therapeutically effective amount of the nanoconstruct or composition can result in tumor regression. In certain embodiments, the administering step is performed subcutaneously, intravenously, intramuscularly, intraperitoneally, intratumorally, or topically.

[0028] The disclosed embodiments, as well as other features, advantages, and aspects contained herein and the implementations thereof, will become apparent in light of the following detailed description of various exemplary embodiments of the present disclosure, which will be better understood when taken in conjunction with the accompanying drawings. [Brief description of the drawings]

[0029] [Figure 1A]Figure 1 shows data on damage-associated molecular patterns (DAMPs) release from CT26 cells treated with cytotoxic agents at IC50. Figure 1A shows data on calreticulin (CRT) exposure on the CT26 cell surface. *: p<0.05, ***: p<0.001, ns: not significant by one-way ANOVA combined with Dunnett's multiple comparison test. [Figure 1B] Figure 1B shows data on the release of damage-associated molecular patterns (DAMPs) from CT26 cells treated with cytotoxic agents at IC50. Figure 1B shows data on HMGB1 measured in the medium. *: p<0.05, ***: p<0.001, ns: not significant by one-way ANOVA combined with Dunnett's multiple comparison test. [Figure 1C] Figure 1C shows data on the release of damage-associated molecular patterns (DAMPs) from CT26 cells treated with cytotoxic agents at IC50. Figure 1C shows data on adenosine triphosphate (ATP) measured in the medium. *: p<0.05, ***: p<0.001, ns: not significant, by one-way ANOVA combined with Dunnett's multiple comparison test. [Figure 2A] Figure 2A shows data on DAMP release from 4T1 cells treated with cytotoxic drugs at IC50, while Figure 2B shows data on CRT exposure at the 4T1 cell surface. *: p<0.05, ***: p<0.001, ns: not significant by one-way ANOVA combined with Dunnett's multiple comparison test. [Figure 2B] FIG. 2B shows data on DAMP release from 4T1 cells treated with cytotoxic agents at IC50, while FIG. 2B shows data on HMGB1 measured in the medium. *: p<0.05, ***: p<0.001, ns: not significant by one-way ANOVA combined with Dunnett's multiple comparison test. [Figure 2C]FIG. 2C shows data on the release of DAMPs from 4T1 cells treated with cytotoxic drugs at IC50, and FIG. 2C shows data on ATP measured in the medium. *: p<0.05, ***: p<0.001, ns: not significant by one-way ANOVA combined with Dunnett's multiple comparison test. [Figure 3A] Figure 3 shows data on DAMP release from B16F10 cells treated with cytotoxic drugs at IC50. Figure 3A shows data on CRT exposure on the B16F10 cell surface. *: p<0.05, ***: p<0.001, ns: not significant by one-way ANOVA combined with Dunnett's multiple comparison test. [Figure 3B] FIG. 3B shows data on the release of DAMPs from B16F10 cells treated with cytotoxic agents at IC50, while FIG. 3B shows data on HMGB1 measured in the medium. *: p<0.05, ***: p<0.001, ns: not significant by one-way ANOVA combined with Dunnett's multiple comparison test. [Figure 3C] FIG. 3C shows data on the release of DAMPs from B16F10 cells treated with cytotoxic agents at IC50, and FIG. 3C shows data on ATP measured in the medium. *: p<0.05, ***: p<0.001, ns: not significant, by one-way ANOVA combined with Dunnett's multiple comparison test. [Figure 4] Figure 1 shows the percentage of CT26 cells phagocytosed by JAWSII DCs after treatment with CFZ or PTX for 6 or 24 hours (by one-way ANOVA with Dunnett's multiple comparison test; *: p<0.05, ***: p<0.001, ****: p<0.0001, ns: not significant). [Diagram 5] FIG. 1 shows data from an in vivo vaccination study, where phosphate buffered saline (PBS) is the control, gemcitabine (GEM) is the negative control, oxaliplatin (OXA) is the positive control, and paclitaxel (PTX) and carfilzomib (CFZ) are drug candidates. [Figure 6]FIG. 1 shows data on interferon gamma (IFN-γ) secretion from splenocytes following stimulation with AH1 peptide, a CT26 immunodominant MHC class I-restricted antigen. [Figure 7A] 1 is a bar graph showing the size and zeta potential of NP-pD-ATP measured in 10 mM phosphate buffer, pH 7.4. [Figure 7B] 1 is a plot of the change in ATP conjugation based on ATP to nanoparticle (NP) ratio (w / w). [Figure 7C] FIG. 13 is a transmission electron micrograph (TEM) of NP-pD-ATP (visualized by negative staining with 1% uranyl acetate; scale bar: 100 nm). [Figure 8A] Schematic of the Transwell study set-up with NP-pD-ATP or ATP. [Figure 8B] FIG. 8B is a graph of the data collected from the study in FIG. 8A, showing the percentage of THP-1 cells that migrated across the Transwell (***: p<0.001, ns: not significant, by one-way ANOVA with Dunnett's multiple comparison test). [Figure 8C] FIG. 8C is a graph of the data collected from the study in FIG. 8A, showing the percentage of JAWSII cells that migrated across the Transwell (***: p<0.001, ns: not significant, by one-way ANOVA with Dunnett's multiple comparison test). [Figure 8D] FIG. 1 shows the percentage of JAWSII cells that migrated across the Transwell after various incubation times. [Figure 8E] Figure 1 shows the stability of PLGA-pD-ATP as estimated by the percentage of JAWSII cells that migrated across the Transwell in response to each treatment. Supernatant & Pellet: Nanoparticles were pre-incubated in 10% FBS containing medium for 24 hours and separated into supernatant and pellet (gray bars). The lack of JAWSII cell migration in response to the supernatant indicates that ATP remained bound to the nanoparticles and was not released into the supernatant. [Figure 9A] Schematic of the Transwell probe set-up with NP-pD-ATP or ATP, with or without apyrase. [Figure 9B] FIG. 9B is a graph of the data collected from the study shown in FIG. 9A. FIG. 9B shows the percentage of JAWSII cells that migrated across the Transwell after treatment with apyrase (***: p<0.001, ns: not significant, by one-way ANOVA with Dunnett's multiple comparison test). [Figure 9C] 1 is a bar graph of ATP levels measured using an ATP bioluminescence assay after co-incubation with apyrase. [Figure 10] Schematic diagram of ATP conjugation to PLGA nanoparticles. PLGA-NPs were coated with a polydopamine (pD) layer at pH 8.5 to produce PLGA-pD. The amine groups of ATP were further conjugated to the hydroxyl groups of pD. [Figure 11A] 1 is a line graph showing the rate of PTX release from nanoparticles performed in PBS containing 0.2% Tween 80 with constant stirring at 37° C. [Figure 11B] FIG. 1 is a line graph showing the rate of CFZ release from nanoparticles performed in PBS containing 0.2% Tween 80 with constant stirring at 37° C. [Figure 12A] 1 is a line graph of the release rate of CFZ from nanoparticles of the present disclosure made from PLGA with the indicated lactic acid to glycolic acid (LA:GA) ratios and molecular weights (kDa). The study was carried out in PBS containing 0.2% Tween 80 with constant stirring at 37° C. [Figure 12B] FIG. 1 shows the release rate of CFZ from CFZ-loaded PLGA / PLGA-mPEG-NPs with various PLGA:PLGA-mPEG ratios (e.g., PLGA:PLGA-mPEG-100:0, 90:10, 50:50, and 0:100). [Figure 12C]FIG. 1 shows the rate of CFZ release from liposomes loaded with CFZ and produced at various pressures (with pressure settings at 5000 psi and 20000 psi) in a high pressure homogenizer as described herein. [Figure 12D] FIG. 13 shows the liposomally encapsulated CFZ release rate mass balance at various times during the study. [Figure 13A] Figure 13A shows data plots from a cytotoxicity study assessing PTX (both free and in various encapsulated forms) in two different cell types (CT26 in Figure 13A). PTX is the free drug, PTX@NP is PTX encapsulated in uncoated nanoparticles, PTX@NP-pD is PTX encapsulated in pD-coated nanoparticles, and PTX@NP-pD-ATP is PTX encapsulated in pD-coated nanoparticles decorated with ATP. [Figure 13B] Figure 13B shows data plots from a cytotoxicity study assessing PTX (both free and in various encapsulated forms) in two different cell types (JAWSII dendritic cells (DCs) in Figure 13B). PTX is the free drug, PTX@NP is PTX encapsulated in uncoated nanoparticles, PTX@NP-pD is PTX encapsulated in pD-coated nanoparticles, and PTX@NP-pD-ATP is PTX encapsulated in pD-coated nanoparticles decorated with ATP. [Figure 14A] Figure 14A shows data plots from a cytotoxicity study assessing CFZ (both free and in various encapsulated forms) in two different cell types (CT26 in Figure 14A). CFZ is the free drug, CFZ@NP is CFZ encapsulated in uncoated nanoparticles, CFZ@NP-pD is CFZ encapsulated in pD-coated nanoparticles, and CFZ@NP-pD-ATP is CFZ encapsulated in pD-coated nanoparticles decorated with ATP. [Figure 14B]Figure 14B shows data plots from a cytotoxicity study assessing CFZ (both free and in various encapsulated forms) in two different cell types (JAWSII DC in Figure 14B). CFZ is the free drug, CFZ@NP is CFZ encapsulated in uncoated nanoparticles, CFZ@NP-pD is CFZ encapsulated in pD-coated nanoparticles, and CFZ@NP-pD-ATP is CFZ encapsulated in pD-coated nanoparticles decorated with ATP. [Figure 15A] FIG. 13 is a graph of the change in CT26 tumor size following intratumoral injection of each treatment with measurements taken at various time points over an 80 hour period post-injection (equivalent to 100 μg PTX; n=3 per group). [Figure 15B] Bar graphs representing the percentage and cell ratio of CD11c+CD86+ DC in tumors after day 3 of each treatment, *: p<0.05, **: p<0.01 by one-way ANOVA coupled with Tukey's multiple comparison test. TME: tumor microenvironment. [Figure 16] Data plots showing the change in Cy7 radiance from tumors over time following a single intratumoral injection with each treatment (n=5 per group). [Figure 17] Images taken with an AMI whole body imager (Spectral Instruments, Inc., Tuscon, Arizona) at various time points after a single intratumoral injection of Cy7 dye showing the change in tumor radiance over time. [Figure 18] 13A-13D are images taken with an AMI whole body imager at various time points after a single intratumoral injection of NP-pD-Cy7 showing the change in tumor radiance over time. [Figure 19A] Schematic representation of the procedure for how CT26 tumors were inoculated and the study treatments were administered. [Figure 19B] 1 is a graph showing the change in CT26 tumor size after intravenous injection in each group (n=8 per group). [Figure 19C] 1 is a graph of mouse survival over time after treatment. [Figure 20A] Schematic representation of the procedure for how B16F10 tumors were inoculated and the study treatments were administered. [Figure 20B] This is a graph showing the change in tumor size after intravenous injection in each group (n=8 per group). [Figure 20C] 1 is a graph of mouse survival over time after treatment. [Figure 21A] Schematic representation of the procedure for how CT26 tumors were inoculated and the study treatments were administered (including additional control groups of Abxtal, and Abxtal + free ATP). [Figure 21B] FIG. 1 is a graph of the change in CT26 tumor size following intravenous injection of each treatment group (n=5 per group; CR=complete response). [Figure 21C] 1 is a graph of percent survival of mice following treatment (or lack thereof). [Figure 22] Schematic diagram of how CT26 tumors were inoculated and the investigational treatments were administered (immune cells were analyzed 7 days after intratumoral (IT) injection of each treatment). [Figure 23] Representative data plots for percentage and cell ratio of immune cells in tumors at 7 days after treatment (described in connection with FIG. 22), n=5; Mix: PTX@NP-pD+ATP; ATP-NP: PTX@NP-pD-ATP; *: p<0.05, **: p<0.01, ***: p<0.001, and ****: p<0.0001 by one-way ANOVA coupled with Tukey's multiple comparison test; and ns=not significant. [Figure 24]Representative data plots for percentage of immune cells and cell ratios in tumor-draining lymph nodes (TDLN) at 7 days after treatment (described in conjunction with FIG. 22), n=5, Mix=PTX@NP-pD+ATP; ATP-NP: PTX@NP-pD-ATP, *p<0.05, **p<0.01; **p<0.01, ***p<0.001, ****p<0.00001, ns=not significant by one-way ANOVA coupled with Tukey's multiple comparison test. [Diagram 25] Representative data plots for percentage of immune cells and cell ratios in spleen at 7 days after treatment (described in connection with FIG. 22), n=5; Mix: PTX@NP-pD+ATP; ATP-NP: PTX@NP-pD-ATP; *, p<0.05; **, p<0.01; **, p<0.01; ***, p<0.001; ****, p<0.00001 by one-way ANOVA coupled with Tukey's multiple comparison test, ns=not significant. [Figure 26A] Representative data plots for the percentage and ratio of immune cells in the tumors and their correlation with tumor size at day 25 after IT injection of each treatment. [Figure 26B] Representative data plots for the percentage and cell ratios of immune cells in the spleen at day 25 after IT injection of each treatment, and their correlation with tumor size. [Figure 26C] Representative data plots for the percentage and cell ratios of immune cells in the spleen at day 25 after IT injection of each treatment, and their correlation with tumor size. [Figure 27] Schematic diagram of how CT26 tumors were inoculated and the study treatments were administered (anti-programmed cell death protein 1 (PD-1) antibody was given 7 days after the first treatment). IV: intravenous injection; IP: intraperitoneal injection. [Figure 28A]FIG. 27 is a graph of the change in CT26 tumor size after intravenous injection of PTX@NP-pD-ATP with (groups 2 and 4) or without (groups 1 and 3) intraperitoneal injection of anti-PD-1 antibody according to the protocol (n=8 per group). [Figure 28B] 28 shows individual growth curves for tumors following the treatment protocol of FIG. 27. [Figure 29A] Graph of change in CT26 tumor size following intraperitoneal injection of anti-PD-1 antibody (n=8 per group) (treatment was initiated when tumors were approximately 50-100 mm3 in volume). [Figure 29B] Individual growth curves for tumors following treatment with anti-PD-1 antibodies, where such treatment begins when the tumors are small sized tumors (volume approximately 50-100 mm3). [Diagram 30] Graph of percent tumor-free mice after rechallenge with CT26 tumor cells. Mice tested in this study included age-matched tumor-naive mice (tumor naive) and mice treated with PTX@NP-pD-ATP and anti-PD-1 antibodies (NP+Abs) in the combination therapy study of FIG. 27-FIG. 29B that achieved complete remission. [Figure 31A] FIG. 13 is a graph of the change in CT26 tumor size after intravenous injection of PBS, PTX@NP-pD+ATP, or PTX@NP-pD-ATP in nude mice (n=8 per group). [Figure 31B] Graph of specific growth rate of tumors treated with PBS (control group), mixed treatment (MIX: PTX@NP-pD+ATP), and ATP-modified nanoparticle treatment (NP-ATP: PTX@NP-pD-ATP) (ΔlogV / Δt). [Figure 31C] Individual growth curves for tumors in the different treatment groups after treatment (**** p<0.0001, ns=not significant by one-way ANOVA coupled with Tukey's multiple comparison test). [Figure 32A]Confocal microscopy images locating rhodamine B-labeled nanoparticles coated with polyethyleneimine (PEI) or ATP in CT26 cells; green label: wheat germ agglutinin (cell membrane), red label: rhodamine B-labeled nanoparticles, and blue label: DAPI (nuclei); scale bar: 50 μm. [Figure 32B] Confocal microscopy images locating rhodamine B-labeled nanoparticles coated with polyethyleneimine (PEI) or ATP in B16F10 cells; green label: wheat germ agglutinin (cell membrane), red label: rhodamine B-labeled nanoparticles, and blue label: DAPI (nuclei); scale bar: 50 μm. [Diagram 33] FIG. 1 is a graph of a quantitative measurement showing the amount of Rhodamine B-labeled particles coated with PEI or ATP taken up by CT26 and B16F10 cells (measured by flow cytometry). [Diagram 34] PTX content in CT26 tumors after a single intravenous injection of PTX@NP-pD+ATP or PTX@NP-pD-ATP (PTX equivalent 20 mg / kg) for 24 h (n=5; ns: not significant by Student's t test). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] While the present disclosure is susceptible to various modifications and alternative forms, exemplary embodiments thereof have been shown by way of example in the drawings and are herein described in detail.

[0031] For the purpose of promoting understanding of the principles of the present disclosure, reference will now be made to the embodiments illustrated in the drawings and specific language will be used to describe them. Nevertheless, it will be understood that no limitation of scope is intended by the description of these embodiments. On the contrary, the present disclosure is intended to cover alternatives, modifications, and equivalents as may be included within the spirit and scope of the present application as defined by the appended claims. As previously noted, although the present technology may be illustrated and described in one or more preferred embodiments, the nanoparticles, compositions, and methods herein may include many different configurations, forms, materials, and accessories.

[0032] All patents, patent applications, articles, textbooks, and other publications mentioned in this specification are indicative of the level of those skilled in the art to which this disclosure pertains. All such publications are incorporated by reference herein to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0033] Nanoconstruct delivery platforms and compositions comprising such nanoconstructs are provided. In certain embodiments, the nanoconstructs are carriers of one or more therapeutic compounds (e.g., drugs or chemotherapeutic agents) and can enhance the retention and stability of such therapeutic compounds by surface modification with immune adjuvants, target solid tumors for cancer treatment, and / or enhance the infiltration of immune cells into tumors (e.g., thus eliciting increased anti-tumor specific immune responses). Thus, the nanoconstructs can be used to sensitize tumors to anti-cancer therapies.

[0034] In certain embodiments, the nanoconstruct comprises a nanoparticle having an outer surface, one or more therapeutic agents (e.g., cargo) encapsulated within the nanoparticle, and an immunoadjuvant-modified polyphenol compound bound to the outer surface of the nanoparticle.

[0035] In certain embodiments, the nanoconstruct comprises poly(lactic-co-glycolic acid) (PLGA) nanoparticles that are surface-modified with an immunoadjuvant and encapsulate (e.g., carry) one or more chemotherapeutic agents. An exemplary embodiment of such an immunoadjuvant is adenosine triphosphate (ATP).

[0036] In use, the immune adjuvant (e.g., ATP) decorating the nanoparticles can recruit immune cells, such as antigen-presenting cells (APCs), natural killer (NK) cells, and T cells, to the target site. Thus, administration of the nanoconstruct can result in enhanced infiltration of immune cells to the target site (e.g., solid tumors), thereby activating (or enhancing) a specific immune response in the subject due to immune cell activation.

[0037] When used to treat cancer in a subject, such nanoconstructs can additionally deliver chemotherapeutic agents to the tumor site. The encapsulated therapeutic agent can be an immunogenic cell death (ICD) inducer. Thus, the ICD inducer can not only deliver anti-cancer drug therapy, but it can also increase the antigenicity of dying tumor cells at the target site, thus sensitizing them to other therapies (e.g., other immunotherapies). In certain embodiments, the nanoconstructs are administered in combination with immune checkpoint inhibitor therapy to increase anti-tumor activity.

[0038] Nanostructures Nanoconstructs include nanoparticles (NPs) coated with one or more substances, such as polymers, targeting molecules, labels, and / or small molecules. The coated surface of the nanoparticles may be further decorated with one or more substances, such as targeting molecules, labels, immune adjuvants, and the like (e.g., ATP). One or more therapeutic agents may be encapsulated within the nanoparticles.

[0039] In use, the nanoparticles may increase retention of the cargo (e.g., drug / therapeutic agent) at the target site (e.g., tumor), may exhibit enhanced pharmacokinetics, and may be surface modified with a high surface-to-mass ratio to enhance delivery of the cargo. See, e.g., Yu et al., Pharmacokinetics, biodistribution and in vivo efficacy of cisplatin loaded poly(L-glutamic acid)-g-methoxy poly(ethylene glycol) complex nanoparticles for tumor therapy, J Control Release 205:89-97 (2015); Li et al., Efficient delivery of docetaxel for the treatment of brain tumors by cyclic RGD-tagged polymeric micelles, Molecular Med Reports 11:3078-3086 (2015); Park et al., Polydopamine-based simple and versatile surface modification of polymeric nano drug carriers, ACS Nano 8:3347-3356 (2014); Raymond et al., Oxaliplatin: mechanism of action and antineoplastic activity, Semin Oncol 25:4-12 (1998).

[0040] The nanoparticles may include polymers such as biodegradable polymers. In certain embodiments, the nanoparticles include one or more of poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), PLGA-methoxy-polyethylene glycol (mPEG), D-α-tocopherol polyethylene glycol 1000 succinate (TPGS)-PLGA conjugate, polylactic acid (PLA), mesoporous silica, and ethylene vinyl acetate. In certain embodiments, the nanoparticles are liposomes and / or include polyphenol aggregates. In certain embodiments, the nanoparticles are drug crystals or nanocrystals made up mostly of drug molecules.

[0041] The nanoparticles may be polymeric selected to enhance delivery of hydrophobic molecules by surface functionalization of the vehicle (i.e., nanoparticle). In certain embodiments, the nanoparticles are PLGA nanoparticles.

[0042] Additionally, the nanoparticles may contain more than one type of polymer, for example, a combination of PLGA and PLGA-mPEG in a w / w ratio of PLGA / PLGA-mPEG of about 90:10, about 85:15, about 50:50, about 35:65, about 65:35, about 15:85, or about 10:90. The ratio of the polymer components may be adjusted to optimize the release rate of the resulting nanoparticles, as is known in the relevant art.

[0043] Nanoparticles can be porous or non-porous. In certain embodiments, nanoparticles are porous. In certain embodiments, nanoparticles are substantially non-porous, but become porous over time (e.g., in use, when in the circulation in vivo). The size of the nanoparticle core can be about 5 nm to about 200 nm, about 5 nm to about 20 nm, about 30 nm to about 100 nm, about 30 nm to about 80 nm, about 30 nm to about 60 nm, about 40 nm to about 80 nm, about 70 nm to about 90 nm, or about 5 nm, about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, or about 100 nm. In certain embodiments, the nanoparticle core size is suitable for systemic injection into a subject. Generally, the nanoparticle core is spherical, although other shapes such as rods and disks can be used.

[0044] Additional moieties can be attached to the nanoparticles by a variety of mechanisms, either covalently or non-covalently. In certain embodiments, the surface of the nanoparticle (or the coated surface) is covalently modified by chemical reaction (e.g., Michael addition or Schiff base reaction). Alternatively, the surface of the nanoparticle can be modified to include reactive moieties for conjugation to ligands or other moieties, as desired.

[0045] In certain embodiments, nanoparticles, such as PLGA nanoparticles, are coated with one or more polymers or other compounds, which can be useful for functionalization purposes.

[0046] The polymer may be attached to the surface of the nanoparticles using any suitable means. In some embodiments, the polymer is attached to the nanoparticles via physical or chemical interactions. The polymer may be a positively charged polymer, such as, but not limited to, polythylenimine (PEI). In some embodiments, the polymer is a polyphenolic compound. In some embodiments, the polymer is polymerized dopamine (pD) or other polyphenols, such as tannic acid, tannic acid-iron complex, gallic acid, ellagic acid, hydroxyhydroquinone, epigallocatechin, epicatechin gallate, epigallocatechin gallate, and pyrogallol. In certain embodiments, the polymer is a cationic polymer (e.g., polyethyleneimine).

[0047] In some embodiments, the nanoparticles comprise a pD coating. In some embodiments, the nanoparticles comprise a pD coating as a primer coating onto which subsequent coatings are applied.

[0048] In certain embodiments, the nanoconstruct is a PLGA nanoparticle having a pD coating.

[0049] The polymer may be linear or branched. The polymer may be present at about 1 wt.% to about 50 wt.%, such as about 1 wt.% to 50 wt.%, or 1 wt.% to about 50 wt.% polymer per nanoconstruct, e.g., 5-40%, 10-30%, 20-30%, 5-15%, 5-20%, 5-25%, 5-30%, 10-20%, 10-25%, or 25-40%, e.g., about 5, about 10, about 15, about 20, about 25, about 30, or about 35%.

[0050] The nanoconstructs may be surface modified (or functionalized) with targeting moieties, imaging agents, immunological adjuvants, and / or other moieties to impart specific characteristics to the nanoconstructs. pD-coated nanoparticles may, in particular, provide nucleophilic functional groups to the ligands via Michael addition and / or Schiff base reactions.

[0051] Examples of imaging agents include, but are not limited to, fluorescent dyes such as FITC, Cy dyes, and amine-reactive Alexa Fluor dyes. Other imaging agents that may be useful for decorating the surface of nanoparticles will be apparent to those of skill in the art.

[0052] The nanoconstruct may be configured for targeted delivery and / or controlled release of a therapeutic agent encapsulated therein. The nanoconstruct may include a targeting moiety (e.g., a cell-interactive ligand that facilitates cell-nanoparticle interaction) for targeted delivery of the nanoconstruct to a specific cell or tissue, for example. In certain exemplary embodiments, the nanoconstruct is configured for delivery to a specific site, such as a solid tumor, for therapy or treatment. The site may be in vivo, for example, in the case of a solid tumor present in a subject. A targeting agent may be used to target the site and, optionally, aid or induce internalization into the cell.

[0053] The nanoparticles may be surface-functionalized with immune adjuvants. In certain embodiments, the nanoparticles are modified with one or more cytokines useful for recruiting dendritic or other activated immune cells, including, but not limited to, ATP, [N-[N-(4-methoxy-2,3,6-trimethylphenylsulfonyl)-L-aspartyl]-D-[4-amidino-phenylalanyl]-piperidine (CCR), CXC motif ligands (CXCL), and [(1R,4S,6S)-4-(4-amino-2-oxopyrimidin-1(2H)-yl)-6-hydroxycyclohex-2-en-1-yl]methyl dihydrogen phosphate (XCR). In certain embodiments, the immune adjuvant may be ATP, calreticulin (CRT), high mobility group box 1 (HMGB1), deoxyribonucleic acid (DNA), annexin A1 (ANXA1), type I interferon, heat shock protein 70 (HSP70), and heat shock protein 90 (HSP90).

[0054] In certain embodiments in which the immune adjuvant comprises ATP, the ATP can be oriented in a manner that preserves the triphosphate group (eg, for chemotactic activity) (see, eg, Examples 4-6).

[0055] In certain embodiments, the nanoparticles have the following structure:

[0056] [ka]

[0057] The surface is functionalized with CCR having the formula:

[0058] In certain embodiments, the nanoparticles have the following structure:

[0059] [ka]

[0060] The surface is functionalized with XCR having the formula:

[0061] In certain embodiments, the nanoparticles have the following structure:

[0062] [ka]

[0063] The surface is functionalized with ATP having the formula:

[0064] ATP is a chemoattractant for immune cells (e.g., dendritic cells and macrophages) and promotes, among other functions, phagocytic clearance of dying cells. ATP contains amine groups that can be conjugated to the surface of pD-coated nanoparticles.

[0065] ATP-decorated nanoparticles provide stronger chemoattractant activity than free ATP (see Example 16) and maintain the chemotactic ability of ATP to recruit dendritic cells and macrophages to target sites (e.g., tumors) (see Example 5), which leads to stronger activation of adaptive immune cells. Furthermore, conjugation of ATP significantly improves the stability of ATP against apyrase (see Example 6), which prevents the degradation of ATP into the immunosuppressive products ADP, AMP, and adenosine. Thus, conjugation of ATP to nanoparticles not only maintains the activity of ATP, but also improves its stability in vivo.

[0066] ATP can be present at about 1% to about 15% by weight (about 1% to 15%, 1% to about 15%, about 9% to about 11.5%, about 9% to 11.5%, or 9% to about 11.5%, etc.). For example, ATP per PLGA can be used in a weight ratio ranging from about 10:1 to about 100:1 (about 10:1 to 100:1, or 10:1 to about 100:1, or about 9:1 to about 99:1, or about 15:1 to about 95:1, or about 15:1 to about 105:1, etc.) during the conjugation process to achieve complete conjugation.

[0067] In certain embodiments, the amount of conjugated ATP on the nanoparticles is about 10.5 wt% to about 11.2 wt%, such as 10.5% to 11.2% of the nanoconstruct. In certain embodiments, the amount of conjugated ATP on the nanoparticles is about 10.5 wt% to about 12.5 wt%, such as 10.5% to 12.5% ​​of the nanoconstruct. In certain embodiments, the PLGA per ATP may be used at a mass ratio of about 50:1, such as 50:1, during the conjugation process.

[0068] The nanoparticles may carry one or more therapeutic agents. Such therapeutic agents may include, but are not limited to, chemotherapeutic agents, such as ICD inducers (or compounds having characteristics of ICD inducers), stabilizers, targeting agents, small molecules or proteins, labels, and / or oligonucleotides. Combinations of various additional agents are also contemplated. The nanoparticles may also include more than one type of polymer, stabilizer, targeting agent, small molecule, protein, label, oligonucleotide, mitoxantrone, bleomycin, doxorubicin, epirubicin, idarubicin, cyclophosphamide, and cardiac glycosides.

[0069] The nanoparticles may be loaded with one or more therapeutic agents, including ICD inducers, non-limiting examples of which are carfilzomib, paclitaxel, oxaliplatin, mitoxantrone, bleomycin, doxorubicin, epirubicin, idarubicin, cyclophosphamide, and cardiac glycosides.

[0070] ICD inducers can be identified by detecting the release of immunostimulatory damage-associated molecular patterns (DAMPs), such as (i) surface exposure of CRT, (ii) passively released chromatin-bound HMGB1, and (iii) extracellularly secreted ATP. These molecules serve as "danger signals" to the immune system, directing APCs to recognize and process antigens to elicit adaptive immunity. CRT is a protein chaperone present in the endoplasmic reticulum. Upon damage, CRT translocates to the membrane and acts as an "eat me" signal to dendritic cells. HMGB1 is a non-histone chromatin-binding protein that mediates DNA repair, recombination, and transcription. Extracellular HMGB1 binds to various types of pattern recognition receptors to promote cancer antigen processing by dendritic cells and presentation to T cells. ATP is the major energy source in cells, actively secreted during apoptosis. It acts as a "find me" signal to APCs to promote phagocytic clearance of dying cells.

[0071] The mechanism of action does not determine the ICD potential of a compound. For example, oxaliplatin appears to be an ICD inducer, whereas cisplatin is not. Both are platinum compounds with the same mechanism of action (alkylating DNA to form intrastrand and interstrand crosslinks). Cisplatin differs from oxaliplatin by having an additional 1,2-diaminocyclohexane carrier ligand. A potential feature of ICD inducers is the ability to induce endoplasmic reticulum stress, which may be a primary indicator of ICD inducers. Furthermore, ICD inducers may be hydrophobic drugs that may benefit from encapsulation in nanoparticle platforms in certain cases.

[0072] Carfilzomib (CFZ) and paclitaxel (PTX) are two such ICD inducers (see at least Examples 1-3). PTX is a microtubule inhibitor with low aqueous solubility (8.5-17 μg / mL) and has the following structure:

[0073] [ka]

[0074] has.

[0075] PTX causes cell cycle arrest at the G2 / M phase by interfering with spindle formation, which induces cell death that may involve extensive endoplasmic reticulum stress.

[0076] CFZ is a second-generation epoxyketone proteasome inhibitor with low aqueous solubility (0.7-3.6 μg / mL) and has the following structure:

[0077] [ka]

[0078] When administered, CFZ can establish an irreversible and covalent interaction with the 20S proteasome of cancer cells, which can lead to the production of misfolded proteins and cause endoplasmic reticulum stress.

[0079] ICD-inducing chemotherapy has recently attracted interest as a way to enhance cancer immunotherapy. ICD-inducing agents increase the antigenicity of dying tumor cells, sensitizing them to, for example, immune checkpoint blockade therapy. However, the immunotoxicity of ICD-inducing agents (which may impair the host's ability to mount antitumor immunity) and their poor adjuvanticity are still significant challenges in combination therapy of ICD-inducing agents with immune checkpoint inhibitors so far. Nanoconstructs may alleviate these issues by encapsulating ICD-inducing agents within nanoparticles.

[0080] Nanoarchitectural synthesis Nanoconstructs can be prepared by conventional methods of organic synthesis practiced by those of skill in the art. The general reaction sequences outlined in the examples below are general methods useful for preparing nanoconstructs and are not intended to be limiting in scope or utility.

[0081] The components can be attached to the nanoparticles or other components by any means, including covalent and non-covalent bonds. A variety of conjugation chemistries are known in the art and are described herein. In some embodiments, one or more of the components are attached to the surface of the nanoparticle (e.g., pD). In further embodiments, one or more of the components are attached to each other. For example, in some embodiments, the targeting moiety can be covalently attached to a stabilizer, which is covalently attached (e.g., via an amine) to a polymer, which in turn is electrostatically attached to the exterior of the nanoparticle, and the therapeutic agent is attached to the interior surface of the pore.

[0082] Although nanoparticles such as PLGA can be commercially obtained or created by any method, in some embodiments, the nanoparticles are formed by dissolving the polymer in an organic solvent such as dichloromethane (DCM). The polymer solution is emulsified in an aqueous solution containing an emulsifier such as polyvinyl alcohol, which helps to reduce the size of the PLGA / DCM droplets. The particle size can be further reduced by the use of a probe sonicator or high-pressure homogenizer. In certain embodiments, the emulsion containing the PLGA / DCM nanodroplets is subjected to agitation evaporation (e.g., rotary evaporation) to remove the DCM, resulting in solidified PLGA nanoparticles. The PLGA nanoparticles can be isolated by centrifugation and washed with DI water.

[0083] In some embodiments, the nanoparticles are coated with at least a polymer. The nanoparticles can be incubated with dopamine, for example under oxidizing conditions, to produce pD-coated nanoparticles (nanoparticles-pD).

[0084] In some embodiments, functional groups and / or other moieties, such as, without limitation, ATP, can be added to the nanoparticles during synthesis, as desired, by further incubating the coated nanoparticles with one or more reagents having the moiety of interest (e.g., ATP).

[0085] Therapeutic agents may also be encapsulated within the nanoparticles during or after synthesis. In certain embodiments, one or more therapeutic agents may be encapsulated within the coated and decorated nanoparticles by the single emulsion method with a target loading efficiency of 5 wt%, 10 wt%, 15 wt%, 20 wt%, 25 wt%, 30 wt%, 35 wt%, 40 wt%, 45 wt%, or 50 wt%, or any other wt% achievable and / or desired using the nanoconstructs herein.

[0086] Compositions, routes of administration, and dosages The nanoconstructs can be formulated for therapeutic or research use. Typically, such formulations for therapy include the nanoconstructs suspended in a pharma- ceutically acceptable carrier.

[0087] The nanoconstructs may be administered in unit dosage form and / or in compositions containing one or more pharma- ceutically acceptable carriers, adjuvants, diluents, excipients, and / or vehicles, and combinations thereof. As used herein, the term "administering" and its elements generally refer to any and all means of introducing a compound into a host subject, including, but not limited to, intravenous, intratumoral, intramuscular, subcutaneous, transdermal, and similar routes of administration.

[0088] As used herein, the term "composition" generally refers to any product that contains more than one component, including a nanoconstruct.

[0089] The nanoconstructs may be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient, in a variety of forms adapted to the chosen route of administration. For example, the pharmaceutical compositions may be formulated for and administered via oral or parenteral, intravenous, intraarterial, intraperitoneal, intrathecal, epidural, intraventricular, intraurethral, ​​intrasternal, intracranial, intratumoral, intramuscular, topical, inhalation, and / or subcutaneous routes. In at least one embodiment, the nanoconstructs, such as a portion of the composition, may be administered into the bloodstream, intramuscularly, into a solid tumor, or directly into an internal organ.

[0090] For example, in at least one embodiment, the nanoconstructs may be administered systemically in combination with a pharma- ceutically acceptable vehicle. The percentages of the components of the compositions and preparations may vary and may be from about 1 to about 99% by mass of active ingredient, as well as binders, excipients, disintegrants, lubricants, and / or sweeteners (as known in the art). The amount of active compound (e.g., therapeutic agent) in such therapeutically useful compositions is such that an effective dosage level can be obtained.

[0091] Preparation of parenteral nanoconstructs / compositions under sterile conditions, for example by lyophilization, can be easily accomplished using standard pharmaceutical techniques well known to those skilled in the art. In at least one embodiment, the solubility of the compounds used in the preparation of parenteral compositions can be increased by the use of appropriate formulation techniques, such as the incorporation of solubility enhancers.

[0092] As previously noted, the nanoconstructs / compositions may also be administered by infusion or injection (e.g., using needle (including fine needle) and / or needleless injectors). Solutions of the active compositions may be aqueous, optionally mixed with non-toxic surfactants, and / or may contain carriers or excipients such as salts, carbohydrates, and buffers (preferably at a pH of 3-9), although for some applications, they may be more appropriately formulated as sterile non-aqueous solutions or in dry form to be used in conjunction with an appropriate vehicle such as sterile pyrogen-free water or phosphate buffered saline (PBS). For example, dispersions may be prepared in glycerol, liquid PEG, triacetin, and mixtures thereof, as well as in oils. Under ordinary conditions of storage and use, these preparations may further contain a preservative to prevent the growth of microorganisms.

[0093] Pharmaceutical dosage forms suitable for injection or infusion may include sterile aqueous solutions or dispersions or sterile powders containing the active ingredient, optionally encapsulated in liposomes, nanocrystals, or polymeric nanoparticles, suitable for extemporaneous preparation of sterile injectable or infusible solutions or dispersions. In all cases, the final dosage form should be sterile, fluid, and stable under the conditions of manufacture and storage. The liquid carrier or vehicle may be, for example, a solvent or liquid dispersion medium, including, but not limited to, water, electrolytes, sugars, ethanol, polyols (e.g., glycerol, propylene glycol, liquid PEG, etc.), vegetable oils, non-toxic glyceryl esters, and / or suitable mixtures thereof. In at least one embodiment, the proper fluidity may be maintained by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions, or by the use of surfactants.

[0094] Sterile injectable solutions can be prepared by incorporating the nanoconstructs and / or compositions in the required amount of an appropriate solvent with one or more of the other ingredients specified above, as required, followed by filtered sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred method of preparation is vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient plus any additional desired ingredients present in a previously sterile-filtered solution.

[0095] For topical administration, it may be desirable to administer the nanoconstructs directly to the tumor site as a composition or formulation in combination with an acceptable carrier, which may be solid or liquid. For example, in certain embodiments, solid carriers may include finely divided solids, such as talc, clay, microcrystalline cellulose, silica, alumina, and the like. Similarly, useful liquid carriers may include water, alcohol, or glycol, or water-alcohol / glycol hybrids, in which the compounds may be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Additionally or alternatively, adjuvants such as antimicrobial agents may be added to optimize the properties for a given use. The resulting liquid composition may be applied from an absorbent pad, may be used to impregnate bandages and / or other dressings, may be sprayed onto the target area using a pump-type or aerosol sprayer, or may simply be applied directly to the desired area of ​​the subject.

[0096] Thickening agents such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified cellulose, or modified mineral materials may also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like for direct application to the skin of a subject.

[0097] As used herein, the terms "therapeutically effective", "therapeutically effective dose", or "therapeutically effective amount" (unless otherwise specifically stated) refer to an amount of a nanoconstruct and / or compound (e.g., a therapeutic agent) that, when administered once or over the course of a treatment cycle, affects the health, well-being, or mortality of a subject (for example and without limitation, delays the onset and / or reduces the severity of one or more symptoms associated with cancer). In certain embodiments, a therapeutically effective amount may provide a prophylactic effect. Useful dosages of nanoconstructs may be determined by comparing their in vitro activity with their in vivo activity in animal models. Methods for extrapolating effective dosages in mice and other animals to human subjects are known in the art. In practice, dosages of nanoconstructs may vary widely depending on the condition of the host subject, the type of cancer being treated, how advanced the condition is, the route of administration and tissue distribution of the compound, and the possible co-use of other therapeutic treatments (such as radiation therapy or additional drugs in combination therapy). The amount of a composition required for use in therapy (e.g., a therapeutically effective amount or dose) will vary with the particular application, as well as with the selected salt (if applicable) and subject characteristics (e.g., age, medical condition, sex, subject's body surface area and / or weight, tolerance to drugs, etc.), and will ultimately be at the discretion of the attending physician, clinician, or otherwise. Therapeutically effective amounts or doses can range, for example, from about 0.05 mg / kg of patient weight to about 30.0 mg / kg of patient weight, or from about 0.01 mg / kg of patient weight to about 5.0 mg / kg of patient weight, including but not limited to 0.01 mg / kg, 0.02 mg / kg, 0.03 mg / kg, 0.04 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 1.5 mg / kg, 2.0 mg / kg, 2.5 mg / kg, 3.0 mg / kg, 3.5 mg / kg, 4.0 mg / kg, 4.5 mg / kg, and 5.0 mg / kg, all of which are in kg of patient weight. The total therapeutically effective amount of the nanoconstructs can be administered in single or divided doses and can, at the discretion of the practitioner, fall outside the typical ranges given herein.

[0098] In certain embodiments, the nanostructure carries PTX and is formulated into a pharmaceutical composition for intravenous administration, and the therapeutically effective amount is about 15 mg / kg to about 30 mg / kg (e.g., 15 mg / kg to about 30 mg / kg or about 15 mg / kg to 30 mg / kg). In certain embodiments, the nanostructure carries PTX and is formulated into a pharmaceutical composition for intrathecal administration, and the therapeutically effective amount is about 2 mg / kg to about 10 mg / kg (e.g., 2 mg / kg to about 10 mg / kg or about 2 mg / kg to 10 mg / kg). In certain embodiments, the nanostructure carries PTX and the therapeutically effective amount is 20 mg / kg for intravenous administration and 5 mg / kg for intrathecal administration.

[0099] In another embodiment, the nanoconstruct comprises from about 0.5 g / m to about 500 mg / m 2 , about 0.5g / m 2 ~about 300mg / m 2 , or about 100 g / m 2 ~about 200mg / m 2 In other embodiments, the amount is about 0.5 mg / m 2 ~about 500mg / m 2 , about 0.5mg / m 2 ~about 300mg / m 2 , about 0.5mg / m 2 ~about 200mg / m 2 , about 0.5mg / m 2 ~about 100mg / m 2 , about 0.5mg / m 2 ~about 50mg / m 2 , about 0.5mg / m 2 ~about 600mg / m 2 , about 0.5mg / m 2 ~about 6.0mg / m 2 , about 0.5mg / m 2 ~about 4.0mg / m 2 , or about 0.5 mg / m 2 ~about 2.0mg / m 2 The total amount may be administered in single or divided doses and, at the physician's discretion, may fall outside the typical ranges given herein. These amounts are based on meters of body surface area.

[0100] These and other effective unit dosages may be administered in the form of a single dose or multiple doses, for example, twice a week for a three-week cycle, daily, weekly, or monthly. In additional embodiments, dosages may be administered in any suitable dosage regimen in coordination with other treatment regimens, depending on clinical and patient-specific factors. The amount, timing, sequence, and mode of delivery of the composition comprising an effective amount (e.g., therapeutically effective amount) of the nanoconstruct to treat a disease, whether the administration is prophylactic or therapeutic, will be routinely adjusted on an individual basis depending on factors such as the individual's mass, age, sex, and disease state, acuteness of the disease, and / or associated symptoms, as well as based on drug delivery, absorption, pharmacokinetics including half-life, and other factors known to affect efficacy.

[0101] Methods of Treatment and Use Methods are provided for using nanoconstructs to deliver therapeutic agents to a site in a subject. In certain embodiments, the methods include administering a therapeutically effective amount of any of the embodiments of the nanoconstruct or compositions comprising the same to a subject under conditions that deliver the nanoconstruct to a target site, where the nanoconstruct is internalized by cells when administered under the conditions. The target site may be a solid tumor, a cancer, and / or a cancerous cell or tissue. In certain embodiments, the nanoconstruct elicits an enhanced anti-tumor specific immune response at the target site. For example, the enhanced anti-tumor specific immune response may be the recruitment of APCs, NK cells, and T cells to the target site.

[0102] In certain embodiments, the nanoconstruct enhances immune cell activation at the target site. For example, ATP conjugated to the surface of the nanoconstruct can recruit tumor-specific cells to the target site and create an inflammatory T cell-infiltrated tumor microenvironment (TME). In other words, the nanoconstruct, composition, and method can be useful for sensitizing anti-cancer responses and overcoming immune suppression by inducing the infiltration of NK cells, dendritic cells, and effector T cells into the TME.

[0103] In some embodiments, accumulation of dendritic and other effector immune cells in the TME leads to the uptake and presentation of tumor antigens by antigen-presenting cells (APCs), such as dendritic cells. Tumor antigen-laden dendritic cells then migrate to tumor-draining lymph nodes (TDLNs), where they continue to present tumor antigens and induce effector T cell activation, proliferation, and development.

[0104] In certain embodiments, administering a therapeutically effective amount of a nanoconstruct induces ICD at the target site or accelerates ICD at the target site (compared to the rate of ICD without administration of the nanoconstruct).

[0105] Where the target site is a solid tumor, administration of a therapeutically effective amount of the nanoconstruct may result in a reduction in the volume / size of the solid tumor. In certain embodiments, administration may result in the regression of the tumor (e.g., complete regression of the cancer).

[0106] Also provided are methods for treating cancer in a subject. In certain embodiments, the methods for treating cancer in a subject include administering to the subject a therapeutically effective amount of a nanoconstruct or a composition comprising the same. In certain embodiments, administration is performed subcutaneously, intravenously, intramuscularly, intraperitoneally, intratumorally, or locally.

[0107] The nanoconstruct and / or composition comprising same may be delivered to the subject in conjunction with anti-cancer therapy and / or other means for sensitizing the tumor to cancer treatment. In certain embodiments, the method further comprises administering an immunotherapy (such as immune checkpoint inhibitor therapy) to the subject. Such immunotherapy may be administered subsequent to or in conjunction with administration of a therapeutically effective amount of the nanoconstruct or composition comprising same. The immunotherapy may be immune checkpoint inhibitor therapy.

[0108] Generally, immune checkpoint inhibitor therapy uses antibodies that block T cell negative regulatory molecules such as CTLA-4 and PD-1. However, due to the complex network of immunosuppressive pathways present in advanced tumors and the TME, such therapies face challenges when used alone. The use of nanoconstructs in conjunction with immune checkpoint inhibitor therapy contributes to enhancing the antitumor efficacy of immune checkpoint inhibitor therapy administered after at least one nanoconstruct dosage. Indeed, a single priming dose of nanoconstructs can activate NK cells, T cells, and recruit APCs and dendritic cells to the target site. Furthermore, these activated cells release cytokines to expand and further recruit additional immune cells to the target site, which leads to increased infiltration of cytotoxic T lymphocytes and other in the TME, and then synergizes with the immune checkpoint inhibitor therapy or other anticancer therapy administered later.

[0109] Thus, in certain embodiments, a combination method for treating cancer in a subject is provided. The method includes administering to the subject a priming dose comprising a therapeutically effective amount of a nanoconstruct or a composition comprising the same; and a second therapy selected from the group consisting of an immune checkpoint inhibitor, a tumor-targeting antibody, and a cancer vaccine, where the priming dose induces or enhances an anti-tumor immune response in the subject at the target site. When the second therapy includes an immune checkpoint inhibitor therapy, the immune checkpoint inhibitor can be an antibody or antibody fragment targeting PD-1 (e.g., nivolumab or pembrolizumab) or PD-L1 (e.g., atezolizumab, avelumab, or durvalumab), CTLA-4 (e.g., tremelimumab or ipilimumab), an anti-CD25 antibody (e.g., basiliximab), or decitabine (e.g., a demethylating agent that controls T cell exhaustion). In certain embodiments, the priming dose of the combination immunotherapy induces the recruitment of NK cells and DC cells to TDLN.

[0110] Methods are provided for enhancing an anti-cancer immune response in a subject, which in certain embodiments comprise administering to the subject a therapeutically effective amount of a nanoconstruct or composition.

[0111] Methods of enhancing infiltration of immune cells to a target site are also provided. Such methods may include, for example, delivering one or more therapeutic agents to a target site in a subject by administering a therapeutically effective amount of a nanoconstruct or a composition comprising the same under conditions that deliver the nanoconstruct to the target site. The target site may be, for example, a solid tumor, and the nanoconstruct may enhance an immune response (e.g., an anti-tumor specific immune response) at the target site (e.g., by activating and recruiting immune cells to the site). In certain embodiments, administering a therapeutically effective amount of the nanoconstruct or composition induces ICD at the target site. Administration may be performed subcutaneously, intravenously, intramuscularly, intraperitoneally, intratumorally, locally, or otherwise as desired and / or required.

[0112] Administration of a therapeutically effective amount of a nanoconstruct or composition (e.g., in any of the methods) can result in a reduction in the volume of a cancer (e.g., a solid tumor). In certain embodiments, administration of a therapeutically effective amount of a nanoconstruct or composition results in the regression of the tumor.

[0113] A specific definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art of chemistry and biology. Additionally, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, if a compound / composition is substituted with "an" alkyl or aryl, the compound / composition is optionally substituted with at least one alkyl and / or at least one aryl. Additionally, unless specifically stated otherwise, the term "about" refers to a value plus or minus 10% range for percentages and plus or minus 1.0 units for unit values, e.g., about 1.0 refers to a value range of 0.9 to 1.1.

[0114] As used herein, the terms "salt" and "pharmaceutically acceptable salt" refer to derivatives of the disclosed compounds in which the parent compound is modified by making acid or base salts thereof. "Pharmaceutically" and "therapeutically" are used interchangeably herein. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic groups such as amines; and alkali or organic salts of acidic groups such as carboxylic acids. Pharmaceutically acceptable salts include the conventional non-toxic salts or quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, and nitric acids; as well as salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, and isethionic acid.

[0115] Pharmaceutically acceptable salts can be synthesized from parent compounds containing basic or acidic moieties by conventional chemical methods.In some cases, such salts can be prepared by reacting the free acid or basic form of these compounds with stoichiometric amounts of appropriate base or acid in water or organic solvent, or in a mixture of the two; generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.Lists of suitable salts can be found in Remington's Pharmaceutical Sciences, 21st ed., Lippincott Williams & Wilkins, 2006, for example, Chapter 38, the disclosure of which is hereby incorporated by reference.

[0116] As used herein, the term "treating" includes therapeutic treatment (e.g., a subject having signs and symptoms of a disease state being treated) and / or prophylactic treatment. Prophylactic treatment includes preventing and inhibiting or slowing the progression of a disease state. EXAMPLES

[0117] The following examples serve to illustrate the present disclosure and are not intended to limit its scope in any way.

[0118] material Adenosine triphosphate (ATP) determination kit, Cell Tracker Green and Cell Tracker Deep Red, Transwell® (8 um pore size), ATP, dopamine HCL, and apyrase were all purchased from Thermo Fisher Scientific Inc. (Waltham, MA).

[0119] Poly(lactic-co-glycolic acid) (PLGA) (Lactel B6006-1, LA:GA=85:15, ester terminated, MW 97 kDA) was purchased from Sigma Aldrich (St. Louis, MO). All other PLGA used was purchased from Akina Inc. (West Lafayette, IN).

[0120] (3-(4,5-Dimenthylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Thermo Fisher Scientific Inc. (Waltham, MA). Paclitaxel (PTX) was a gift from Samyang Biopharm Corporation (Seoul, Korea). Carfilzomib (CFZ) was purchased from Shenzhen Chemical Co. Ltd. (Shanghai, China). Oxaliplatin (OXA) was purchased from Thermo Fisher Scientific Inc. (Waltham, MA). Gemcitabine (GEM) was purchased from LC Laboratories (Woburn, MA).

[0121] CT26, B16F10, 4T1, JAWSII, and THP-1 cells were purchased from the American Type Culture Collection (ATCC) (Manassas, VA). Dulbecco's Modified Eagle Medium (DMEM) and Roswell Park Memorial Institute (RPMI) were purchased from Thermo Fisher Scientific Inc. (Waltham, MA). Mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) was purchased from Shenandoah Biotechnology, Inc. (Warminster, PA).

[0122] Calreticulin (CRT) antibody conjugated to fluorescein isothiocyanate (FITC) was purchased from Abcam plc (Cambridge, UK). High mobility group B1 (HMGB1) antibody was purchased from Novus Biologicals (Littleton, CO). LEGEND MAX™ Mouse IFN-γ ELISA kit was purchased from BioLegend (San Diego, CA).

[0123] Purified rat anti-mouse CD16 / CD32 (Fc block) was purchased from BioLegend (San Diego, CA). Collagenase type 4, deoxyribonuclease I, and hyaluronidase were purchased from Worthington Biochemical Corporation (Lakewood, NJ). Anti-mouse CD16 / 32, CD45, CD11c, CD86, F4 / 80, CD206, NKp46, CD3, CD4, CD8, FOXp3, CD11b, and GR1 antibodies were purchased from BioLegend (San Diego, CA). Anti-mouse programmed cell death protein 1 (PD-1) antibody (CD279) was purchased from Bio X Cell (Lebanon, NH, USA). Female Balb / c mice (5-6 weeks old), male C57BL / 6 mice (5-6 weeks old), and female athymic nude (Foxn1nu) mice (5-6 weeks old) were purchased from Envigo (Indianapolis, IN). Cy7 amine was purchased from Lumiprobe (Cockeysville, MD).

[0124] All animal procedures conformed to the National Institutes of Health (NIH) guidelines for the care and use of laboratory animals and were approved by the Purdue Animal Care and Use Committee.

[0125] All other materials were purchased from Thermo Fisher Scientific Inc. (Waltham, Mass.).

[0126] Example 1 In vitro evaluation of the immunogenic cell death (ICD) potential of PTX and CFZ ICD inducers can be identified by detecting the release of immunostimulatory damage-associated molecular patterns (DAMPs), such as (a) surface exposure of CRT, (b) passively released chromatin-bound HMGB1, and (c) extracellularly secreted ATP. These molecules act as "danger signals" to the immune system, inducing antigen-presenting cells (APCs) to recognize and process antigens and elicit adaptive immunity.

[0127] Therapeutic agents (Carfilzomib (CFZ) and Paclitaxel (PTX)) were tested against CT26 mouse carcinoma along with a positive control (Oxaliplatin (OXA), a known ICD inducer) and a negative control (Gemcitabine (GEM), a known non-ICD inducer). The release of DAMPs from treated cells was then analyzed (e.g., CRT exposure was measured by flow cytometry, HMGB1 was measured by quantifying Western blots, and ATP was measured by an ATP determination kit).

[0128] CRT exposure: CT26 mouse carcinoma, B16F10 mouse carcinoma, and 4T1 breast cancer cells were treated with PBS (control), IC 50 (half-maximal inhibitory concentration of related substances) of either gemcitabine (GEM), oxaliplatin (OXA), paclitaxel (PTX), or carfilzomib (CFZ) for 6 hours. After 6 hours, cells were washed with PBS and fixed with 4% perfluoroalkoxyalkane (PFA). Fixed cells were incubated with FITC-labeled CRT antibody. Cells were analyzed by Accuri C6 flow cytometer to determine the population of CRT-positive cells.

[0129] GEM did not induce CRT exposure. Compared to the control group, both PTX and CFZ induced CRT exposure (see Figures 1A, 2A, and 3A).

[0130] HMGB1 release: CT26 mouse carcinoma, B16F10 mouse carcinoma, and 4T1 breast cancer cells were treated with IC 50 The cells were treated with GEM, OXA, PTX, or CFZ at concentrations of 0.01 mg / mL for 24 hours. After 24 hours, supernatants were collected and Western blot immunoassays were performed to determine the amount of HMGB1 present.

[0131] With regard to HMGB1, similar results were obtained as with CRT exposure, with OXA inducing HMGB1 release and GEM not inducing HMGB1 secretion. Both PTX and CFZ induced HMGB1 release (see Figures 1C, 2C, and 3C).

[0132] ATP secretion: CT26 mouse carcinoma, B16F10 mouse carcinoma, and 4T1 breast cancer cells were treated with IC 50 The cells were treated with GEM, OXA, PTX, or CFZ at a concentration of 0.01 mg / mL for 24 hours. After 24 hours, the supernatant was collected and the amount of ATP in the supernatant was measured with an ATP determination kit. Both PTX and CFZ induced the secretion of ATP (see Figures 1B, 2B, and 3B).

[0133] Overall, the DAMP screen supports that PTX and CFZ are ICD inducers, with both CFZ and PTX inducing comparable DAMP production. Various cell lines were used in this study to ensure that ICD induction occurs in a variety of cell lines.

[0134] Example 2 PTX and CFZ enhance phagocytosis Phagocytosis assays were performed to observe whether cells killed by ICD inducers would be taken up by dendritic cells (DCs). As potential ICD inducers, PTX and CFZ could enhance the uptake of dying tumor cells by dendritic cells.

[0135] CT26 cells were stained with Cell Tracker Green (0.5 μM) for 30 min and washed once with chilled serum-free DMEM. CT26 cells were incubated overnight in 10% FBS medium to inactivate any unbound dye. CT26 cells were harvested and seeded (150,000 cells / well) in 6-well plates for 24 h. JAWSII cells were harvested and stained with Cell Tracker Deep Red (0.5 μM) for 30 min and washed once with chilled serum-free RPMI. JAWSII cells were incubated overnight in 10% FBS medium to inactivate any unbound dye. After 24 h of seeding, CT26 cells were treated with IC50 of PTX (250 nM) or CFZ (15 nM) for 0 or 6 h. After treatment, the 6-well plates containing CT26 were centrifuged at 500 g×8 min. The supernatant was aspirated to remove any remaining drug. JAWSII cells (150,000 cells / well) were added to CT26 and the cells were co-cultured for 24 hours. The co-cultured cells were harvested, resuspended in staining buffer, and analyzed by an Accuri C6 flow cytometer.

[0136] Treatment with either PTX or CFZ significantly increased the uptake of CT26 into dendritic cells (see FIG. 4), supporting that treatment with PTX and CFZ, when used in vivo, can enhance phagocytosis and lead to a strong anti-tumor immune response.

[0137] Example 3 In vivo tumor vaccination studies Vaccination studies, the standard test for verifying bona fide ICD, were performed in vivo with PTX and CFZ (e.g., candidate drugs). The vaccination studies involved generating tumor antigens by exposing tumor cells to a lethal dose of an ICD inducer. Cells treated with a potential ICD inducer (i.e., primary challenge) were subcutaneously injected into immunocompetent mice. One week later, live tumor cells were injected into the contralateral side (i.e., rechallenge), and tumor growth was monitored to determine whether the drug-killed tumor cells (i.e., primary challenge) could act as a vaccine and prevent the growth of the live cell challenge.

[0138] Here, CT26 cells were cultured in a T150 flask to 30% confluency and treated with 100× IC 50 Ex vivo vaccines were generated by treating the cells with OXA, GEM, PTX, or CFZ for 24 hours. Dead cells were harvested and washed twice with PBS to remove excess drug.

[0139] Three million dead cells were injected into the right flank of 6-week-old immune-competent female Balb / c mice, syngeneic hosts of CT26 cells (i.e., primary dosing). Nine days later, 500,000 live CT26 cells (live tumor cells) were subcutaneously inoculated into the opposite side of the mice (i.e., rechallenge), and tumor growth was monitored to determine the development of antitumor immunity. Tumors grew in all animals that received PBS or GEM-treated cells (Figure 5). In contrast, tumors did not grow in animals vaccinated with OXA, PTX, and CFZ-treated cells, supporting that the drug-treated tumor cells (i.e., primary dosing) served as a tumor vaccine to establish antitumor immunity.

[0140] To test whether anti-tumor immunity was tumor specific, splenocytes obtained from vaccinated mice at the end of the study in Example 2 were loaded with AH1 peptide, a CT26 immunodominant MHC class I-restricted antigen, and assessed for interferon gamma (IFN-γ) production (Figure 6).

[0141] Briefly, spleens were harvested, minced, and passed through a 100 μm filter followed by a 40 μm filter. Single cell suspensions were treated with ammonium chloride-potassium (ACK) lysis buffer and then washed. Splenocytes were seeded in 96-well plates (300,000 cells per well) and stimulated with 10 μg / mL AH1 peptide in the presence of 20 ng / mL GM-CSF. Cells were incubated for 72 hours and then centrifuged down to collect the supernatant. The concentration of IFN-γ in the harvested supernatant was measured using an ELISA kit.

[0142] Vaccination studies support that PTX and CFZ are ICD inducers.

[0143] Example 4 ATP-coated PLGA nanoparticles (NP-pD-ATP) characterization PLGA, a biodegradable polymer that can be used to enhance the delivery of hydrophobic molecules by surface functionalization of the vehicle, was selected as a vehicle for the ICD inducer. PLGA nanoparticles can be coated with polydopamine (pD), e.g., functionalized, to increase the recruitment of DCs. ATP was selected as a potential candidate for a chemokine to recruit DCs, since it contains amine groups that can be conjugated to the surface of pD-coated nanoparticles.

[0144] PLGA nanoparticles were incubated with dopamine under oxidizing conditions to yield pD-coated PLGA nanoparticles (nanoparticle-pD). NP-pD was further incubated with ATP to yield ATP-coated nanoparticles (NP-pD-ATP) (see Figures 7A and 7C). The amount of conjugated ATP on NP-pD-ATP was measured by ATP bioluminescence assay, and ATP conjugation was 10.5-11.2 wt%. The maximum amount of conjugation (12.5% ​​w / w) was achieved at an ATP to nanoparticle w / w ratio of 50:1 (Figure 7B).

[0145] Example 5 Immunoreactivity of NP-pD-ATP It is important that ATP maintains its chemoattractant activity after conjugation to nanoparticles. The chemotactic ability of ATP is related to the triphosphate group; therefore, conjugating ATP via amine groups is not expected to affect the chemotactic function of ATP. The chemotactic ability of ATP or NP-pD-ATP (i.e., the ability of ATP or ATP-decorated nanoparticles to recruit DCs) was assessed using THP-1 human monocytes or JAWSII mouse DCs in Transwell. The two cell lines are cell populations involved in initiating immune responses; monocytes serve as precursors of DCs, which present antigens to T cells.

[0146] Monocytes (2 × 10 in 100 μL) suspended in medium were placed in the upper well of a Transwell (8 μm pore size). 5 THP-1 cells) or DCs (2 x 10 in 100 µL 5 JAWSII cells (immune cells of an immortalized immature DC line) were added. 600 μL of medium containing 10 μM ATP or ATP-conjugated nanoparticles (NP-pD-ATP) was added to the lower well. The cells were incubated for 4 hours at 37° C. After 4 hours of incubation, the lower medium was collected and the number of monocytes or DCs that migrated from the upper well to the lower well was counted using an Accuri C6 flow cytometer.

[0147] Based on the Transwell assay, NP-pD-ATP maintained the activity of ATP, and the chemoattractant activity was comparable to that of free ATP (see FIG. 9A). Based on the Transwell assay, NP-pD-ATP maintained the activity of ATP, and the chemoattractant activity was comparable to that of free ATP (see FIG. 9B and FIG. 9C).

[0148] Example 6 Stability assessment of NP-pD-ATP The stability of ATP bound to NP was assessed by incubating NP-pD-ATP (i) in serum-containing medium or (ii) in the presence of the ATP-degrading enzyme apyrase.

[0149] NP-pD-ATP is stable in serum-containing medium NP-pD-ATP equivalent to 1 mg ATP was incubated in 1 mL 10% fetal bovine serum (FBS) and 90% DMEM for up to 72 hours. At various time points during incubation (e.g., 6 hours, 12 hours, 24 hours, 48 ​​hours, and 72 hours), the nanoparticles were separated from the supernatant by centrifugation at 25000rcf×15 minutes, and the nanoparticles and supernatant were collected. The incubated nanoparticles and serum-containing medium (used to contain NP-pD-ATP) were used in Transwell assays with JAWSII DCs.

[0150] Briefly, nanoparticles were resuspended in 1 mL PBS. NP-pD-ATP equivalent to 10 μM ATP of the resulting supernatant from the incubated medium was added to the lower well of the Transwell. NP-pD-ATP still maintained ATP activity after 72 h of incubation, while particle-conditioned supernatant did not induce migration of JAWSII DCs. This supports that ATP was stably bound to nanoparticles even in the presence of serum (Figure 8D).

[0151] FIG. 8E shows the stability of PLGA-pD-ATP as estimated by the percentage of JAWSII cells that migrated across the Transwell in response to each treatment, including nanoparticles preincubated in 10% FBS-containing medium for 24 hours. Specifically, nanoparticles were preincubated in 10% FBS-containing medium for 24 hours and separated into supernatant and pellet. The lack of JAWSII cell migration in response to the supernatant indicates that ATP was not released into the supernatant. Furthermore, nanoparticle pellets induced JAWSII migration to a similar extent as fresh nanoparticles, indicating that the majority of ATP remained bound to the nanoparticles.

[0152] NP-pD-ATP is stable in apyrase Although ATP may play a role as a chemokine to stimulate the immune system, its degradation products (i.e., adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine) play opposing roles, inducing an immunosuppressive environment. This mechanism is utilized in our body to prevent unnecessary chronic inflammation. Thus, if bound ATP is hydrolyzed to ADP, AMP, or adenosine, DC recruitment will be hindered, creating an immunosuppressive environment that promotes tumor growth. ATP-degrading enzymes such as CD39 and CD73 are commonly expressed on the surface of tumors; therefore, it is critical to understand how ATP behaves in the presence of the enzymes.

[0153] To screen the effect of degradative enzymes on ATP binding to nanoparticles, apyrase (CD39 equivalent) was co-incubated with ATP or NP-pD-ATP, and DC migration assays were performed in Transwell (Figure 9A). For the migration assay, 100 μL of 2 × 10 5 JAWSII cells were added to the top of a Transwell (8 μm pore size). 600 μL of medium containing 10 μM ATP-conjugated nanoparticles was added to the bottom well (with or without 0.5 units of apyrase). The cells were incubated at 37° C. for 4 hours. The bottom medium was collected and the number of migrated cells was analyzed by flow cytometry.

[0154] To further confirm the activity of apyrase on ATP, 10 μM ATP was incubated with or without 5 units / mL apyrase for 10 min at 37° C. The ATP levels after incubation were measured by ATP bioluminescence assay.

[0155] In the Transwell migration assay, apyrase completely abolished the chemotactic activity of free ATP, as shown by the significantly reduced migration of DCs (Figure 9B). However, NP-pD-ATP resisted the degradation of apyrase and induced the migration of DCs to the lower well. Surface-bound ATP may induce steric hindrance for the apyrase enzyme, preventing the degradation of ATP.

[0156] In addition, ATP was undetectable after free ATP incubation with apyrase, supporting the activity of apyrase to catalyze the sequential hydrolysis of ATP to ADP, AMP, and finally adenosine, releasing inorganic phosphate (Figure 9C).

[0157] Overall, the results of the stability experiments support that ATP was stably conjugated to the surface of the nanoparticles and that such conjugation increased the stability of ATP against its degrading enzymes.

[0158] Example 7 Encapsulation of ICD-inducing agents within nanoparticles Following the development of NP-pD-ATP, ICD inducers were encapsulated. PTX or CFZ were encapsulated in PLGA nanoparticles by single emulsion method with a target loading efficiency of 5 wt%. PLGA nanoparticles were selected for investigation due to their compatibility with hydrophobic drugs and their high surface area to mass ratio that facilitates the conjugation of functional ligands.

[0159] 100 mg of PLGA (MW 97 kDa, LA:GA=85:15) and 10 mg of PTX or CFZ were dissolved in 1 mL of dichloromethane. The solution was added to 4 mL of 4% polyvinyl alcohol (PVA). The mixture was emulsified by sonication for 2 minutes (4 seconds on, 2 seconds off, 40% amplitude). 20 mL of DI water was added to the emulsion. The solution was stirred for 3 hours and then further evaporated using a rotavapor for an additional 30 minutes.

[0160] Nanoparticles loaded with PTX cargo or nanoparticles loaded with CFZ cargo were collected by centrifugation (45k rcf×20 min) and coated with a layer of pD to enable subsequent conjugation of ATP to pD via Michael addition and Schiff base reaction (FIG. 11). The collected particles were washed twice with deionized (DI) water. PTX-loaded PLGA nanoparticles (PTX@NP) were incubated in dopamine HCl solution in Tris buffer (10 mM, pH 8.5) with a dopamine HCl to NP mass ratio of 0.5 / 1 for 2 h. When the nanoparticles showed the dark color of polymerized dopamine, they were collected by centrifugation (20 min×25000 rpm) and washed twice with DI water to remove excess dopamine and pD.

[0161] PTX-loaded NP-pD (PTX@NP-pD) was incubated with ATP in Tris buffer (10 mM, pH 8.5) at an ATP to nanoparticle mass ratio of 20:1 for 1 h. Nanoparticles were collected by centrifugation at 45k rcf for 20 min at 4°C and washed twice with DI water. Size was measured by NS90 Zetasizer, zeta potential was measured by NS90 Zetasizer, loading efficiency was measured by high performance liquid chromatography (HPLC), and ATP content was measured using an ATP determination kit. Dipalmitoylphosphatidylcholine (DPPC) and cholesterol were purchased from Avanti Polar Lipids, Inc. (Birmingham, AL).

[0162] The size and surface charge of the drug-encapsulated nanoparticles were measured by a Malvern NS90 Zetasizer. The loading efficiency was defined as drug per nanoparticle (quantified by HPLC). PTX and CFZ were encapsulated in PLGA nanoparticles with loading efficiencies of 3.4 and 4.4%, respectively. The drug-encapsulated nanoparticles had a suitable particle size for systemic injection (Table 1).

[0163] [Table 1]

[0164] Example 8 Release rate from NP-pD-ATP The release rates of PTX-loaded PLGA-pD-ATP and CFZ-loaded PLGA-pD-ATP were measured in 0.2% Tween 80 / PBS under sink conditions. The saturated solubilities of PTX and CFZ in 0.2% Tween 80 / PBS were determined to be 13 μg / mL and 9 μg / mL, respectively. The supernatant was collected and the amount of drug was measured by HPLC at each time point indicated in Figure 11A and Figure 11B.

[0165] As shown in Figure 11A and Figure 11B, PTX@NP-pD-ATP released 82.5% of the total loaded drug over 96 h, and CFZ@PLGA-pD-ATP released 46%. An initial burst release was observed to different extents for both drugs, likely due to leakage of drug located near the surface of the particles and poorly encapsulated drug that diffuses easily. The delayed and incomplete release of CFZ from CFZ@PLGA-pD-ATP is likely due to the poor solubility of the compound in FBS-containing medium as previously observed.

[0166] Example 9 Enhancement of release of encapsulated therapeutic agents Various investigations were performed to assess the effect of variables on the release rate of ATP-decorated nanoparticles. First, CFZ encapsulated within PLGA did not provide a percentage release of more than 50% of the loaded cargo, so further investigations were performed to provide complete release.

[0167] Varying the lactic acid to glycolic acid (LA:GA) ratio or molecular weight (MW) First, the LA:GA ratio or the MW of the PLGA polymer was changed to provide either a hydrophobic or hydrophilic environment for CFZ. Since CFZ is hydrophobic and can be molecularly dispersed in the PLGA matrix, increasing the hydrophobicity has the potential to improve the release rate. As a result of molecular dispersion, CFZ cannot crystallize, thereby increasing the release. On the other hand, decreasing the hydrophobicity by changing the LA:GA ratio can help release CFZ by increasing water infiltration. Various LA:GA ratios and MW were used to produce nanoparticles (Table 2).

[0168] [Table 2]

[0169] The release of CFZ in the produced (uncoated) nanoparticles (CFZ@NP), prepared at a concentration equivalent to 3 μg / mL of CFZ, was measured in 15 mL of 0.2% Tween80 / PBS under sink conditions (maximum solubility of CFZ in sink conditions - 0.2% PBS solution with low concentration detergent solution (PBST): 9 μg / mL) and the concentration of CFZ in the release medium was measured at different time points (Figure 12A). 200 μL samples were collected at each time point indicated in Figure 12A and then centrifuged at 16000 rcf for 20 min. 150 μL of the supernatant was collected and then analyzed. 150 μL of 0.2% PBST was added to the collected sample and then added back to the original sample. No significant changes in the release were observed regardless of the formulation.

[0170] Changes in PLGA composition: Another approach is to improve the release by utilizing polyethylene glycol (PEG)-conjugated PLGA, which can increase the porosity of the polymer by forming water channels. The wettability of PEG can facilitate the diffusion and release of the drug. Previous studies have shown that the incorporation of PEG into PLGA polymers improves the release of doxetaxel (see Rafiei and Haddadi, Docetaxel-loaded PLGA and PLGA-PEG nanoparticles for intravenous application: pharmacokinetics and biodistribution profile, Int J Nanomedicine 12:935-947 (2017)). Therefore, PEG-conjugated PLGA was assessed in an attempt to improve the release of encapsulated CFZ by replacing PLGA with PEG-conjugated PLGA to various degrees.

[0171] Briefly, various ratios of PLGA to PLGA-methoxy-polyethylene glycol (mPEG) were mixed to encapsulate CFZ. More specifically, PLGA (LA:GA=85:15, 97 kDA) was mixed with PLGA-mPEG (MW approx. 5,000:55,000 Da LA:GA=50:50) in 1 mL dichloromethane (DCM) to replace 10%, 50%, or 100% of PLGA. 5 mg CFZ was added (at 10% mass ratio) to 1 mL of PLGA / PLGA-mPEG dissolved in DCM.

[0172] The CFZ+PLGA / PLGA-mPEG solution was added to 5 mL of 4% PVA. The mixture was emulsified by sonication for 2 minutes (2 seconds on, 2 seconds off, 40% amplitude). The emulsion was added to 25 mL of DI water. The solution was stirred for 3 hours and then rotavaped at 20 mbar for an additional 30 minutes. The CFZ-loaded PLGA / PLGA-mPEG-NPs were collected using centrifugation at 43400rcf for 20 minutes. The CFZ-loaded PLGA / PLGA-mPEG-NPs were then washed with DI water, sonicated (10 seconds, 20% amplitude), and centrifuged twice at 43400rcf for 15 minutes. The pellet was collected and stored at 4°C.

[0173] The substitution of PEG did not affect the physical characteristics of the particles (Table 3).

[0174] [Table 3]

[0175] As described above, the release of CFZ in the nanoparticles produced was measured in 0.2% Tween 80 / PBS under sink conditions. Samples were taken at various time points and the amount of CFZ release was measured (Figure 12B). As expected, the more PLGA was replaced with PEG-conjugated PLGA, the more CFZ release was observed.

[0176] Alternative Delivery Vehicles: An alternative approach to enhance the release of CFZ was also explored by encapsulating CFZ in liposomes instead of PLGA. Liposomes have been shown to provide sustained release of PTX (see, e.g., Liu et al., Mixed Liposome Approach for Ratiometric and Sequential Delivery of Paclitaxel and Gemcitabine, AAPS PharmSciTech, 19:693-699 (2018)). Therefore, liposomes were screened for delivery of CFZ.

[0177] Liposomes containing CFZ were produced using a high-pressure homogenizer at 5,000 psi or 20,000 psi (Table 4).

[0178] [Table 4]

[0179] Briefly, DPPC was dissolved in a mixture of 3 mL chloroform. CFZ was added to this solution at 2.1 wt% of lipid, and the CFZ / lipid solution was transferred to a round-bottom flask and spun on a rotary evaporator under vacuum at 45 °C for 30 min to remove the solvent and form a thin lipid film. The liposome suspension was sonicated briefly using an ultrasonic probe. Liposomes were extruded through a high-pressure homogenizer at 5,000-20,000 psi. Liposomes were collected by centrifugation at 175,000 rcf for 20 min. CFZ-loaded liposomes were prepared in 15 mL 0.2% Tween 80 / PBS at a concentration equivalent to 3 μg / mL of CFZ (sink condition - maximum solubility of CFZ in 0.2% PBST: 9 μg / mL). 200 μL samples were collected at each time point displayed in Figure 12C and centrifuged at 175,000 rcf for 20 min. 150 μL of the supernatant was collected and then analyzed. 150 μL of 0.2% PBST was added to the collected sample and then added back to the original sample.

[0180] Regardless of the pressure used to produce the nanoparticles, the nanoparticles showed similar size and drug loading. The release of CFZ was tested in a similar manner as in other studies. The liposomal formulations showed similar release profiles over time regardless of the pressure used for production (Figure 12C), with 70% of the loaded CFZ being released over 144 hours.

[0181] FIG. 12D shows the liposomally encapsulated CFZ release rate mass balance, indicating that the majority of the encapsulated CFZ was released within 6 days (e.g., released linearly over time).

[0182] Example 10 Cytotoxicity of PTX or CFZ encapsulated PLGA nanoparticles The cytotoxicity of PTX and CFZ was tested against CT26 murine carcinoma and JAWSII DC. Because the nanoparticles herein can be used to deliver ICD-inducing agents to create tumor antigens that need to be taken up by APCs, in certain embodiments, it is important that the formulation kills tumor cells while sparing host immune cells.

[0183] Cytotoxicity was assessed by MTT assay. CT26 cells were seeded in 96-well plates at 3,000 cells / well and cultured to 30% confluency. Cells were treated with various concentrations of drug, drug@NP, drug@NP-pD, drug@NP-pD-ATP for 24 hours. After 24 hours, the medium was replaced and cells were incubated for an additional 24 hours, and an MTT assay was performed to measure the number of viable cells present (see Figure 13A and Figure 14A).

[0184] For JAWSII cells, JAWSII cells were seeded in 96-well plates at 20,000 cells / well. Cells were treated with various concentrations of PTX for 96 hours, and an MTT assay was performed to measure the number of viable cells present (see Figures 13B and 14B).

[0185] PTX nanoparticle formulations showed cytotoxicity against CT26 tumor cells with minimal toxicity against JAWSII DCs (Figures 13A-B). PTX, a cytostatic chemotherapy, does not affect cells that do not proliferate as rapidly as tumor cells, such as DCs, and therefore PTX is generally better tolerated by immune cells than cancer cells. We suspect that the sudden decrease in DC viability above 30 μM may be related to the crystallization of free PTX, which may induce physical damage to the cell membrane (cancer cells were not exposed above 30 μM due to their high sensitivity).

[0186] CFZ nanoparticle formulations were toxic to both CT26 tumor cells and JAWSII dendritic cells (Figures 14A-B). Such differences are likely due to different mechanisms of action: CFZ is a proteasome inhibitor that can block the degradation of proapoptotic factors such as p53 protein, allowing activation of programmed cell death regardless of cell type. Given these findings, PTX was selected as a lead candidate for future investigations to minimize the potential for immunotoxicity.

[0187] Example 11 Tumor Processing for Immune Cell Profiling The following protocol was used for processing tumors for the in vivo studies described herein.

[0188] Tumors were harvested, minced into small pieces, and digested for 2 h at 37 °C with medium containing 1.5 mg / mL collagenase type 4, 0.2 mg / mL DNase I, and 0.2 mg / mL hyaluronidase. The cell suspension was filtered through a 100 μm followed by a 40 μm cell strainer. The cells were centrifuged at 500 g for 8 min. The pellet was resuspended in 5 mL of ACK lysis buffer and then preincubated for 5 min. Lysis was stopped by adding 50 mL PBS and centrifuged at 500 g for 8 min. The cells were resuspended in staining buffer, a buffered saline solution containing FBS and sodium azide (0.09%) as a preservative. The cells were stained with anti-mouse CD16 / 32 antibody for 10 min at 4 °C to block nonspecific binding of immunoglobulins to Fc receptors, followed by staining antibody for 20-30 min at 4 °C and centrifugation at 500 g for 8 min.

[0189] The cells were fixed with 1 mL of 4% PFA at room temperature for 20 min, then centrifuged at 500 g for 8 min. The cells were harvested and suspended in 300 μL of PBS at 4° C. for flow cytometry.

[0190] Example 12 NP-pD-ATP recruits DCs to tumors APCs can initiate antitumor immune responses by presenting tumor antigens produced by ICD inducers to T cells. To test whether NP-pD-ATP can attract APCs as suggested by the in vitro Transwell study above, PTX@NP-pD-ATP was intratumorally injected into CT26 tumor-bearing mice, and the number of DCs in the tumor microenvironment (TME) and tumor growth were analyzed 3 days after injection (Figures 15A and 15B).

[0191] More specifically, female BALB / c mice (4-6 weeks old) were inoculated with 3 × 10 5 CT26 cells were inoculated subcutaneously. 3 At the time when the tumors had grown to 100 μL, mice were randomly assigned to three groups (n=3 per group) and treated with intratumoral injection of 100 μL of PBS, PTX@NP-pD, or PTX@NP+ATP mixture. Tumor growth was monitored, and 72 hours after injection, tumors were harvested to evaluate the number of dendritic cells in the tumor. Tumors were prepared into single cell suspensions (as described in Example 11) and stained with antibodies for immunophenotyping. DCs (CD45 + CD86 + CD11c + ) was measured by flow cytometry.

[0192] Tumor volume was best suppressed with PTX@NP-pD-ATP at 3 days compared to PBS, PTX@NP-pD, or a mixture of PTX@NP-pD and soluble ATP. PTX@NP-pD-ATP significantly increased the number of DCs in the tumor when compared to other groups. The addition of soluble ATP did not increase DC infiltration into the tumor, but rather reduced it compared to PTX@NP-pD-ATP. Such effects may be driven by the instability of ATP due to the presence of degradative enzymes (CD73 and CD39) commonly expressed on the surface of tumor cells. Such degradative enzymes may convert ATP to AMP or adenosine, which may induce an immunosuppressive environment that may hinder DC recruitment.

[0193] Unlike the mixture of PTX@NP-pD and free ATP, PTX@NP-pD-ATP, when locally injected in the TME, helped recruit DCs to the TME (Fig. 15B) and inhibited tumor growth for the first 3 days (Fig. 15A), supporting that NP-pD-ATP may help maintain the stability of ATP and increase the infiltration of DCs into tumors, thereby inducing antitumor immunity.

[0194] Example 13 Nanoparticle formulation enhances nanoparticle retention in tumors To test whether ATP-conjugated nanoparticles prolong the retention of ATP in tumors, tumor-bearing mice were treated with fluorescent dye-conjugated nanoparticles and imaged over time. Although it is possible to simply measure the amount of ATP in tumors after injection of ATP or NP-pD-ATP, it would be difficult to distinguish between exogenous ATP (e.g., from nanoparticles) and endogenous ATP (e.g., from cells) since ATP is a common energy source in the body. Furthermore, ATP from injection can be degraded to ADP or AMP. Therefore, an amine-terminated dye was used instead of ATP to simulate the behavior of soluble ATP and nanoparticle-bound ATP. More specifically, an amine-conjugated Cy7 dye was used because its water solubility is similar to that of ATP and the florescent spectrum of the amine-conjugated Cy7 dye does not overlap with the autofluorescence from the mouse body.

[0195] Cy7-coated nanoparticles (NP-pD-Cy7) were produced in the same manner as NP-pD-ATP, except for wt%. PLGA nanoparticles were incubated in dopamine HCl solution in bicine buffer (10 mM, pH 8.5) with a dopamine HCl to NP mass ratio of 0.5 / 1 for 1 h. When the nanoparticles showed the dark color of polymerized dopamine, they were collected by centrifugation (20 min x 25000 rpm) and washed twice with DI water to remove excess dopamine and pD.

[0196] 20 mg PLGA-pD was incubated with 50 μg Cy7-amine (0.25% Cy7-amine to nanoparticle mass ratio) in 5 mL bicine buffer (10 mM, pH 8.5) for 1 h. Nanoparticles were collected by centrifugation at 25000 rpm for 20 min at 4 C and washed twice with DI water.

[0197] The resulting NP-pD-Cy7 was injected intratumorally into CT26-bearing mice, and the fluorescence intensity of Cy7 was measured over time with an AMI imager (Figure 16). In contrast, the Cy7 signal from free Cy7 faded rapidly and began to become undetectable 3 days after injection (Figures 16 and 17). On the other hand, the signal of Cy7-conjugated nanoparticles persisted even after 8.2 days (Figures 16 and 18), suggesting that ATP stably bound to nanoparticles may remain longer in tumors compared to soluble ATP.

[0198] Example 14 Antitumor efficacy of PTX-encapsulated NP-pD-ATP via intravenous delivery The systemic antitumor efficacy of PTX@NP-pD-ATP was assessed using different models of tumor-bearing mice (CT26 syngeneic model and B16F10 syngeneic model). 3 Once tumor volume reached 100 μg / kg, mice were treated intravenously every 3 days for 4 doses with the equivalent of 20 mg / kg PTX. Tumor growth was monitored daily.

[0199] CT26 related model Female BALB / c mice (4–6 weeks old) were injected with 3 × 10 5 CT26 cells were subcutaneously inoculated (Figure 19A). 3 When tumors grew to 100x, mice were randomly assigned to three groups and treated with a total of four intravenous injections of 150 μL of PBS, PTX@NP-pD, or PTX@NP+ATP mixture, and PTX@NP-pD-ATP every three days. The PTX dose was 20 mg / kg equivalent for all groups receiving PTX. Tumor growth was monitored.

[0200] Compared with treatment with PBS or mixture group, PTX@NP-pD-ATP showed better antitumor efficacy, resulting in complete regression in one of five mice (Figure 19B and Figure 29C). However, tumors still grew in most of the mice. Although tumors were initially suppressed during treatment, tumors began to regrow when treatment was discontinued after four times.

[0201] B16F10 related model Male C57BL / 6 mice (4–6 weeks old) were inoculated with 5 × 10 5 B16F10 cells were subcutaneously inoculated (Figure 20A). 3 At the time of tumor growth, mice were randomly assigned to three groups and treated with a total of four intravenous injections of 150 μL of PBS, PTX@NP-pD, PTX@NP+ATP mixture, and PTX@NP-pD-ATP every three days. The PTX dose was 20 mg / kg equivalent for all groups receiving PTX. Tumor growth was monitored. Figure 20B shows a graph of tumor size after intravenous injection for each group. Figure 20C shows a graph of the percent survival of mice after treatment (or lack thereof).

[0202] Consistent with previous observations in the CT26 syngeneic model, the PTX@NP-pD-ATP group showed superior antitumor efficacy compared with the PBS and PTX@NP-pD+ATP groups. However, the tumor growth of B16F10 tumors was faster than that of CT26 tumors, possibly due to the lower number of immune cells in the tumors and more rapid doubling time.

[0203] Nonetheless, the efficacy of the PTX@NP-pD-ATP formulation was consistent across various tumor models, supporting the enhanced potency of ATP when conjugated to the surface of nanoparticles. This enhanced potency may result from increased stability and retention of ATP from the coating and nanoparticle formulation, leading to recruitment of nearby immune cells to elicit the desired immune response.

[0204] Example 15 Antitumor efficacy of PTX-encapsulated NP-pD-ATP in CT26 model via intratumoral delivery As for intravenous injection, it is possible that different amounts of PTX may be delivered to the tumor compared to the injected amount. To test whether ATP-conjugated nanoparticles increase the antitumor effect of fixing PTX delivery to the tumor, another set of animal studies was performed in which mice were treated intratumorally. Unlike intravenous injection, intratumoral injection was performed only once directly into the tumor and tumor growth was monitored (Figure 21A). Since the treatment was delivered intratumorally, the amount of PTX delivered to the tumor was expected to be the same between groups. An additional control group was also included in the study, where Abxtal, a PTX carrier-free nanocrystal formulation, was used instead of Abraxane (the current standard for PTX therapy) because Abxtal is as effective as Abraxane. (See Park and Yeo, Albumin-coated nanocrystals for carrier-free delivery of paclitaxel, J. Controlled Release 263:90-101 (2017); see also Park et al., A Comparative In Vivo Study of Albumin-Coated Paclitaxel Nanocrystals and Abraxane, Small 14:e1703670 (2018)).

[0205] Female BALB / c mice (4–6 weeks old) were injected with 3 × 10 5 CT26 cells were inoculated subcutaneously. Tumor growth was monitored and tumors were monitored until they reached approximately 50–100 mm 3 At the time of tumor growth, mice were randomly assigned to five groups and treated with intratumoral injection of 100 μL of PBS, PBS, PTX(Abxtal), PTX(Abxtal)+ATP, PTX@NP-pD, PTX@PLGA-pD+ATP, and PTX@PLGA-pD-ATP. The PTX dose was 5 mg / kg equivalent for all groups receiving PTX. Tumor growth was monitored again.

[0206] Among the treatment groups, PTX@NP-pD-ATP showed the best antitumor efficacy, resulting in complete remission in 2 out of 5 mice (Figures 21B and 21C). As demonstrated by tumor growth and survival curves, additional delivery of soluble ATP together with either Abxtal or PTX@NP-pD did not significantly suppress tumors (Figures 21B and 21C). This supports that NP-pD-ATP may enhance ATP retention, thereby increasing immune cell infiltration into tumors, as previously seen in Cy7 studies (Example 13).

[0207] (Example 16) Immune cell profiling of CT26 tumors after intratumoral delivery of PTX-loaded NPs-pD-ATP To better understand immune cell changes in the TME, immune cell analysis was performed 7 days after intratumoral injection (Figure 22). Seven days was determined to be an appropriate time point to investigate immune cell numbers, since both the innate and adaptive immune systems would be active by this time point.

[0208] Female BALB / c mice (4–6 weeks old) were injected with 3 × 10 5 CT26 cells were inoculated subcutaneously. 3 When the mice grew to 100 μL, they were randomly assigned to three groups and treated intratumorally with injections of 100 μL of PBS, PTX@PLGA-pD+ATP, or PTX@PLGA-pD-ATP. The PTX dose was 5 mg / kg equivalent for all groups that received PTX. Seven or 20 days after injection, tumors, spleens, and tumor-draining lymph nodes were harvested, and the number of immune cells was measured by flow cytometry (Figures 23-25). Abxtal, Abxtal+ATP, PTX@NP-pD, and PTX@NP-pD+ATP showed similar antitumor efficacy to each other (see Example 15), so PTX@NP-pD+ATP was selected as a representative control. Therefore, only three treatment groups were compared (PBS, PTX@NP-pD+ATP, and PTX@NP-pD-ATP).

[0209] In the TME (Figure 23), the number of DCs in mice treated with PTX@NP-pD-ATP was significantly higher than that in the PTX@NP-pD+ATP (mixture) treatment group or the PBS control group. The number of M1 macrophages (antitumor-like macrophages) was increased in both the PTX@NP-pD-ATP and mixture groups when compared to PBS (control group); however, the mixture group also had an increase in M2 macrophages (tumor-promoting like macrophages). The M1 / M2 ratio in the tumor was only increased in the PTX@NP-pD-ATP treatment group. Natural killer (NK) cells were increased in both the PTX@NP-pD-ATP and mixture treatment groups compared to PBS (control group). Overall, the innate immune cell population was significantly increased with the treatment of PTX; however, the addition of soluble ATP did not further increase the immune cell population, whereas the use of ATP conjugated to nanoparticles did.

[0210] PTX@NP-pD-ATP significantly increased the amount of CD8+ T cells in the tumor compared to the mixture or PBS. No significant increase in CD4+ T cells was observed. However, PTX@NP-pD-ATP treatment increased the amount of Treg cells in the tumor compared to the PBS (control) group. Such an effect is likely due to the continuous inflammation caused by DCs and CD8+ T cells in the tumor, which promotes the recruitment of Treg cells to counter the activity of dendritic cells and CD8+ T cells. Both PTX@NP-pD-ATP and mixture treatment groups exhibited an apparent increase in myeloid-derived suppressor cell (MDSC) population, but only the mixture treatment group showed a significant difference compared to the control group.

[0211] Tumor-draining lymph nodes (TDLN), immune organs adjacent to tumors, were also analyzed in a similar manner as tumors. As shown in FIG. 24, significant DC and M1 macrophage populations were observed in the mixture and PTX@NP-pD-ATP treatment groups (greater increases were observed in the PTX@NP-pD-ATP treatment group). Overall, the innate immune cell trends in TDLN mirrored those of tumors. However, T cells showed no significant trends. Additionally, the population of Treg cells in TDLN was decreased with respect to treatment groups (PTX@NP-pD-ATP or mixture). Both PTX@NP-pD-ATP and mixture treatment groups showed significant increases in MDSCs in TDLN; however, the PTX@NP-pD-ATP treatment group had a significantly lower number of MDSCs in TDLN compared to the mixture treatment.

[0212] The population of immune cells in the spleen was also analyzed (Figure 25). The spleen contains various types of immune cells (1.5% DC, 5% macrophages, 23% T cells, 1.2% Treg, 64% B cells) and regulates the development of innate and adaptive immunity. Both DC and M1 macrophage populations were increased in the PTX@NP-pD-ATP or mixture treatment groups compared to the PBS (control) group, and the degree of increase was significantly higher in the PTX@NP-pD-ATP treatment group compared to the mixture treatment group. A significant increase in CD8+ T cells, along with a slight increase in CD4+ T cells, was observed only in the PTX@NP-pD-ATP treatment group. The Treg cell population could not be compared. The MDSC population was significantly decreased with PTX@NP-pD-ATP treatment, in contrast to the observations in the tumor.

[0213] Overall, regardless of the sampling location (i.e., tumor, TDLN, spleen), DC populations showed a consistent increase with PTX@NP-pD-ATP or mixture treatment. However, the degree of increase observed was higher for PTX@NP-pD-ATP treatment than mixture treatment. Also, M1 population and M1 / M2 macrophage ratio were significantly higher in the PTX@NP-pD-ATP treatment group than in the PBS control group or mixture treatment group. This supports that ATP conjugation of nanoparticles enhanced the recruitment of DCs and macrophages, and such enhancement of innate immune cell recruitment led to stronger activation of adaptive immune cells, as demonstrated by higher CD8+ T cell populations in tumor and spleen. The increase in Tregs and MDSCs observed in tumor and TDLN could potentially explain the tumor growth at later stages.

[0214] (Example 17) Changes in immune cell populations in CT26 tumors relative to tumor size after intratumoral injection Although PTX@NP-pD-ATP inhibited tumor growth better than the mixture treatment group (PTX@NP-pD+ATP), the tumors grew after various degrees of initial inhibition. To investigate the potential role of immune cells in the subsequent recurrence of tumors, CT26 tumors were intratumorally treated with PTX@NP-pD-ATP, followed by analysis of immune cells from tumors (Figure 26A), lymph nodes (Figure 26B), and spleens (Figure 26C) 25 days after intratumoral injection (performed as previously described in the aforementioned study).

[0215] The number of anti-tumor immune cells was highly correlated with the size of the tumor. As the tumor grew, the Treg population increased along with the MDSC population, and the number of anti-tumor immune cells (e.g., DC, macrophages, and T cells) decreased. Although the spleen and TDLN did not necessarily show the same results as the tumor (e.g., the M1 macrophage population decreased in the tumor but increased in the spleen and TDLN), the overall trend supports that the lack of anti-tumor immune cells is involved in the late recurrence of tumors.

[0216] (Example 18) Combination therapy with anti-PD-1 antibody and PTX@NP-pD-ATP The increase in immunosuppressive cells in tumors observed in Example 17 was expected because tumors have feedback mechanisms that counteract the activity of T cells. Although an increase in the number of T cells in tumors was observed in previous studies, IFN-gamma released from T cells can induce upregulation of PD-L1 expression in tumors, which can lead to exhaustion of CD8 T cells and recruitment of Tregs. See, for example, Jorgovanovic et al., Roles of IFN-gamma in tumor progression and regression: a review, Biomark Res 8:49 (2020). Recruited Tregs can downregulate the activation of CD8 T cells and DCs in tumors, further reducing the activation of adaptive immunity.

[0217] Because the presence of Tregs is highly correlated with poor outcomes of cancer immunotherapy, therapies have been developed to block Treg proliferation or interfere with Treg interactions with other immune cells. See Han et al., Turning the Tide Against Regulatory T Cells, Front Oncol 9:279 (2019). Although the mechanisms of action for each strategy differ from one another, three strategies can be used to block or deplete Treg cell activation: (i) anticancer drugs, (ii) Treg cell depleting agents, and / or (iii) conversion of Treg cells into immune stimulatory cells. Han (2019), supra.

[0218] Among possible therapeutic options for downregulating Treg cells, we identified anti-PD-1 antibodies as an additional therapeutic agent for further investigation because they can relieve the suppression of cytotoxic CD8 T cells and downregulate Tregs while blocking T cell interactions with other immunosuppressive cells. See Yoshida et al., Anti-PD-1 antibody decreases tumour-infiltrating regulatory T cells, BMC Cancer 20:25 (2020), and Ravelli et al., Immune-related strategies driving immunotherapy in breast cancer treatment: a real clinical opportunity, Expert Rev Anticancer Ther 15:689-702 (2015).

[0219] The systemic antitumor efficacy of PTX@NP-pD-ATP administered in combination with an anti-PD-1 antibody was assessed using CT26 tumor-bearing mice. 3 Starting when the tumor volume reached 100 mg / kg, mice were treated intravenously every 3 days with the equivalent of 20 mg / kg PTX for 4 doses. Mice additionally received sequential injections of anti-PD-1 antibody intraperitoneally every 3 days for 4 doses (Figure 27).

[0220] More specifically, female BALB / c mice (4-6 weeks old) were inoculated with 3 × 10 5 CT26 cells were inoculated subcutaneously. 3 When mice reached adulthood, they were randomly assigned to four groups and treated with PBS, PBS + anti-PD-1 antibody, PTX@NP-pD-ATP, or PTX@NP-pD-ATP + anti-PD-1 antibody. PBS or PTX@NP-pD-ATP (PTX equivalent 20 mg / kg) was given intravenously every 3 days up to four times.

[0221] Since the study was designed to downregulate immunosuppressive Treg cells in tumors, the antibody was injected 7 days after the first injection of PTX, at the time when T cells are activated after antigen presentation. Indeed, providing sequential injections has shown higher antitumor efficacy in other combination therapies. See, for example, Messenheimer et al., Timing of PD-1 Blockade Is Critical to Effective Combination Immunotherapy with Anti-OX40, Clin Cancer Res 23:6165-6177 (2017), and Kim et al., Sequential and Timely Combination of a Cancer Nanovaccine with Immune Checkpoint Blockade Effectively Inhibits Tumor Growth and Relapse, Angew Chem Int Ed Engl 59:14628-14638 (2020). Therefore, starting 7 days after the first injection of PBS or PTX@NP-pD-ATP, anti-PD-1 antibody (150 μg per dose) was administered intraperitoneally every 3 days up to 4 times. Tumor growth was monitored.

[0222] The combination of PTX@PLGA-pD-ATP and anti-PD-1 antibody significantly improved the outcome of tumor-bearing mice, resulting in complete tumor regression in 75% (6 / 8) of mice and slowing tumor growth in the other two mice (Figures 28A and 28B).

[0223] Anti-PD-1 antibody was used to treat tumors up to 500 mm 3 Because the tumors were given 7 days after PBS treatment, when tumors had already reached a volume larger than 10 mm, it is possible that the anti-PD-1 antibody may not be effective because large tumors may already exhibit a strong immunosuppressive TME. 3 To observe how the effect of CT26 tumors on the IL-18 expression of IL-18 in mice was observed, additional animals were inoculated with CT26 tumors as described above, with tumor volumes ranging from 50 to 100 mm in volume. 3Anti-PD-1 antibodies were given first at some point before or after the treatment, and although the anti-PD-1 antibodies suppressed tumor growth compared to the control group (Figures 29A and 20B), they were not as effective as the combination therapy and did not have complete tumor regression.

[0224] To examine whether mice that survived the combination therapy had developed adaptive immunity against CT26 tumors, mice that had achieved complete remission were rechallenged with CT26 cells (per previous protocol) on the opposite flank and observed for tumor growth (Figure 30). Tumors did not grow in surviving mice; however, tumors began to grow in age-matched naive mice already 7 days after inoculation, supporting that tumor-free mice from the combination treatment group had developed anti-tumor immunity against CT26.

[0225] (Example 19) Antitumor efficacy against CT26 in immunodeficient mice To confirm that the enhanced antitumor response was dependent on adaptive immunity, nude mice, which lack mature T cell populations, were also tested with PTX@NP-pD-ATP.

[0226] First, female nude mice (4-6 weeks old) were inoculated with 3 × 10 5 CT26 cells were inoculated subcutaneously. 3 Upon reaching a tumor volume of 100 μL, mice were randomly assigned to three groups and treated with a total of four intravenous injections of 150 μL of PBS, PTX@NP-pD-ATP, or PTX@NP+ATP mixture every three days. The PTX dose was 20 mg / kg equivalent for all groups receiving PTX. Tumor growth was monitored.

[0227] Unlike the immune-competent model, PTX@NP-pD-ATP showed no difference in tumor growth (specific tumor growth rate) compared to the mixture group (Figures 31A-C), supporting that the activity of ATP-coated nanoparticles is dependent on T cell activation.

[0228] (Example 20) ATP does not affect PTX accumulation in tumors Based on the overexpression of P2X7 receptors on cancer cells, ATP has previously been utilized as a tumor-targeting ligand to enhance delivery of cargo. See, e.g., Rajabnia and Meshkini, Fabrication of adenosine 5'-triphosphate-capped silver nanoparticles: Enhanced cytotoxicity efficacy and targeting effect against tumor cells, Process Biochemistry 65:186-196 (2018). Although studies in nude mice (Example 19) suggest activation of antitumor immunity by ATP-decorated nanoparticles, ATP conjugated to PLGA nanoparticles could also enhance delivery of PTX to CT26 or B16F10 tumors by targeting the tumor (compared to non-targeted, uncoated nanoparticles).

[0229] To screen whether ATP-decorated nanoparticles enhance nanoparticle uptake via P2X7 receptor interaction, nanoparticles were formed using rhodamine B-labeled PLGA, and the surface of rhodamine B-labeled PLGA nanoparticles was left uncoated (i.e., undecorated), coated with polyethyleneimine (PEI), or coated with ATP. PEI was used as a positive control due to its known ability to nonspecifically promote cellular uptake. Nanoparticle uptake by tumor cells was analyzed by confocal microscopy and flow cytometry.

[0230] Confocal microscopy showed that ATP-coated nanoparticles were not actively taken up by CT26 or B16F10 cells, whereas PEI-coated nanoparticles were taken up by tumor cells (Figure 32A and Figure 32B). Flow cytometry results showed a consistent trend (Figure 33). The in vitro results support that ATP conjugation cannot enhance nanoparticle uptake into tumors.

[0231] To evaluate the targeting effect in vivo, CT26 tumor-bearing mice were treated with either the PTX@NP-pD+ATP mixture (i.e., mixture or mixture treatment group) or PTX@NP-pD-ATP, and the amount of PTX accumulation in the tumor was measured 24 hours after the first treatment. The 24-hour time point was chosen based on literature findings suggesting that the highest accumulation of nanoparticles in tumors would occur 24 hours after injection. See Hyun et al., Surface modification of polymer nanoparticles with native albumin for enhancing drug delivery to solid tumors, Biomaterials 180:206-224 (2018), and Xu et al., Quinic Acid-Conjugated Nanoparticles Enhance Drug Delivery to Solid Tumors via Interactions with Endothelial Selectins, Small 14:e1803601 (2018).

[0232] Female BALB / c mice (4–6 weeks old) were injected with 3 × 10 5 CT26 cells were inoculated subcutaneously. 3 When the tumors grew to 100 mm Hg, the mice were randomly assigned to three groups and treated once with PBS, PTX@NP-pD+ATP, or PTX@NP-pD-ATP (PTX equivalent: 20 mg / kg). After 24 h, the tumors were harvested and the amount of PTX in the tumors was measured after extraction.

[0233] The amount of PTX in the tumor was measured by HPLC using carbamezapine as an internal standard. The accumulation of PTX in the tumor did not differ between the mixture and PTX@NP-pD-ATP groups (Figure 34). This result indicates that ATP-coated nanoparticles did not enhance the accumulation of PTX in the CT26 tumor model. Therefore, the improved antitumor activity of PTX@NP-pD-ATP compared with PTX@NP-pD or the PTX@NP-pD+ATP mixture was not due to altered delivery of PTX to the tumor, but instead was due to enhanced antitumor immunity via surface-bound ATP, which recruited DCs and then activated T cells.

Claims

1. Nanoparticles having an outer surface; One or more therapeutic agents encapsulated within the nanoparticles; and An immune adjuvant-modified polyphenol compound bound to the outer surface of the nanoparticles A nanoconstruct comprising.

2. The polyphenol compound is selected from the group consisting of polymerized dopamine (pD), tannic acid, tannic acid-iron complex, gallic acid, ellagic acid, hydroxyhydroquinone, epigallocatechin, epicatechin gallate, epigallocatechin gallate, and pyrogallol, the nanoconstruct according to claim 1.

3. The immune adjuvant is selected from the group consisting of adenosine triphosphate, calreticulin, high mobility group box 1, deoxyribonucleic acid, annexin A1, type I interferon, heat shock protein 70, and heat shock protein 90, the nanoconstruct according to claim 1.

4. The nanoparticles are selected from the group consisting of poly(lactic-co-glycolic acid) (PLGA), polycaprolactone, D-α-tocopherol polyethylene glycol 1000 succinate-PLGA conjugate, polylactic acid, PLGA-methoxy-polyethylene glycol, ethylene vinyl acetate, mesoporous silica, liposomes, nanocrystals, and polyphenol aggregates, the nanoconstruct according to any one of claims 1, 2, or 3.

5. The one or more therapeutic agents are one or more chemotherapeutic agents, the nanoconstruct according to any one of claims 1, 2, or 3.

6. The one or more therapeutic agents are carfilzomib or paclitaxel, the nanoconstruct according to any one of claims 1, 2, or 3.

7. Nanoparticles having an outer surface, One or more therapeutic agents encapsulated within the nanoparticles, and An immune adjuvant-modified polyphenol compound bound to the outer surface of the nanoparticles A priming dose comprising a therapeutically effective amount of the nanoconstruct containing ; and Administering to the subject an immune checkpoint inhibitor, a tumor-targeting antibody, or a cancer vaccine A method for treating cancer in a subject, comprising the step of, wherein the priming dose induces or enhances an anti-tumor immune response in the subject at the target site.

8. The immunoadjuvant is selected from the group consisting of adenosine triphosphate, calreticulin, high mobility group box 1, deoxyribonucleic acid, annexin A1, type I interferon, heat shock protein 70, and heat shock protein 90, in the method according to claim 7.

9. The one or more therapeutic agents are selected from the group consisting of oxaliplatin, carfilzomib, paclitaxel, mitoxantrone, bleomycin, doxorubicin, epirubicin, idarubicin, cyclophosphamide, and cardiac glycosides, in the method according to claim 7.

10. The nanoparticles are selected from the group consisting of poly(lactic-co-glycolic acid) (PLGA), polycaprolactone, D-α-tocopherol polyethylene glycol 1000 succinate-PLGA conjugate, polylactic acid, PLGA-methoxy-polyethylene glycol, ethylene vinyl acetate, mesoporous silica, liposomes, nanocrystals, and polyphenol aggregates, in the method according to claim 7.

11. The one or more therapeutic agents are chemotherapeutic agents, in the method according to claim 7, 8, 9, or 10.

12. The polyphenol compound is selected from the group consisting of polymerized dopamine (pD), tannic acid, tannic acid-iron complex, gallic acid, ellagic acid, hydroxyhydroquinone, epigallocatechin, epicatechin gallate, epigallocatechin gallate, and pyrogallol, in the method according to claim 7, 8, 9, or 10.

13. The priming dose is administered at least 4 days before the immune checkpoint inhibitor, whereby cancer in the subject is treated, in the method according to claim 7.

14. The immune checkpoint inhibitor is an antibody or antibody fragment targeting PD-1 (e.g., nivolumab or pembrolizumab) or PD-L1 (e.g., atezolizumab, avelumab, or durvalumab), CTLA-4 (e.g., tremelimumab or ipilimumab), an anti-CD25 antibody (e.g., basiliximab), or decitabine (e.g., a demethylating agent that controls T cell exhaustion), in the method according to claim 13.

15. The target site is cancerous tissue or cells, in the method according to claim 13.

16. A method for enhancing an anti-cancer immune response in a subject, comprising the step of administering to the subject a therapeutically effective amount of the nano-construct according to claim 1, 2 or 3.

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