Antigen capturing nanoparticle and dendritic cells for in SITU cancer immunization

Abstract

Compositions comprising migratory type 1 conventional dendritic cells in combination with a positively charged antigen capturing nanoparticle comprising a hydrophobic surface and use of the nanoparticle and dendritic cells optionally in combination with an immune checkpoint inhibitor in methods of treating cancer or preventing or reducing the risk of reoccurrence of a cancer in a subject.

Description

UIC0112WO PATENTANTIGEN CAPTURING NANOPARTICLE AND DENDRITIC CELLS FOR IN SITU CANCER IMMUNIZATIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit from U.S. Patent Application Serial No. 63 / 682,533, filed August 13, 2024, the content of which is incorporated herein by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under grant no. R35GM150507 awarded by the National Institutes of Health. The government has certain rights in this invention.BACKGROUND

[0003] Cancer immunotherapy marks a transformative development in cancer treatment. By activating the body's immune system to fight malignant cells, cancer immunotherapy has the potential to induce lasting immunological memories, thereby preventing tumor recurrence. Various types of immunotherapies, including immune checkpoint inhibitors and adoptive cell therapies, have shown promising efficacy in the clinic in extending overall and progression-free survival, offering new hope to patients who did not benefit from conventional interventions like chemotherapy.

[0004] Inducing a strong immune response against tumors hinges on effectively activating a complex series of cellular and molecular interactions in the cancer-immunity cycle (CIC). Dendritic cells (DCs), a key type of antigen presenting cells (APCs), play an essential role in the CIC. DCs capture tumor- associated antigens and present them to T cells, inducing antigen-specific T cells and augmenting T-cell-mediated tumor killing. Due to DC's crucial role in the CIC, DC-based cell therapies, especially DC vaccines that involve the adoptiveUIC0112WO PATENT transfer of antigen-pulsed DCs, have been explored as a promising immunotherapeutic approach (Sabado et al. (2017) Cell Res, 27:74- 95; Perez & De Palma (2019) Nat. Commun. 10:5408; Carreno et al. (2015) Science 348:803-808; Palucka & Banchereau (2012) Nat. Rev. Cancer 12:265-277; Zhang et al. (2023) Nat. Nanotechnol. 18:1364- 1374). In contrast to genetically engineered cell therapies such as chimeric antigen receptor (CAR) T cells and CAR natural killer (NK) cells, which are tailored to target a single tumor antigen, DC-based cell therapies can be used to tackle different tumor antigens, which can more effectively counteract the tumor's antigen heterogeneity and may be personalized with different patients. However, despite extensive studies, ex vivo engineered DC therapies have only shown modest clinical efficacy, largely due to their suboptimal activity induced by the ex vivo preparation process and their inability to effectively counteract the tumor's immunosuppressive microenvironment. A distinguishing challenge is the poor-match between the antigens displayed by the DC therapies and the heterogenous antigens within the patient's tumor, due to the high inter- and intra-patient tumor antigenic heterogeneity as well as antigen loss during the ex vivo process, which collectively compromises the effectiveness of DC-based cell therapies in generating a broad T cell response. Additionally, the effectiveness of ex vivo DC-based cell therapies is further hampered by limited intratumoral infiltration of T cells because of their inability to alter the tumor's immunosuppressive microenvironment. In situ immunization, which leverages native tumor-derived antigens directly within the tumor, holds potential to enhance antigen coverage and stimulate systemic antitumor immunity against heterogenous tumor antigens. However, inducing a robust immune response via in situ immunization is hampered by the lack of effective DC subtypes in the tumor and their suboptimal capability for antigen presentation within the immunosuppressive tumor microenvironment.UIC0112WO PATENT

[0005] Needed is an effective in situ immunization strategy allowing for remodeling of the tumor microenvironment. The present invention addresses this need in the art.SUMMARY OF THE INVENTION

[0006] In one aspect is provided a method of treating cancer or preventing or reducing the risk of reoccurrence of a cancer in a subject comprising administering to the subject an effective amount of (i) an antigen capturing nanoparticle, the antigen capturing nanoparticle comprising a positively charged and hydrophobic surface; and (ii) migratory type 1 conventional dendritic cells, thereby treating cancer or preventing the reoccurrence of cancer in the subject.

[0007] In another aspect, a composition comprising (i) an antigen capturing nanoparticle, the antigen capturing nanoparticle comprising positively charged and hydrophobic surface; (ii) migratory type 1 conventional dendritic cells; and (iii) a pharmaceutically acceptable carrier or aqueous medium is provided.

[0008] In a further aspect, a kit comprising (i) an antigen capturing nanoparticle, the antigen capturing nanoparticle comprising positively charged and hydrophobic surface; (ii) migratory type 1 conventional dendritic cells; and (iii) an immune checkpoint inhibitor is provided.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIG. 1. Tumor protein binding capability of antigencapturing nanoparticles (AC-NPs) (n=3, independent samples). Statistical analysis: two-way ANOVA followed by Dunnett test. P < 0.0001 as compared to NPPEGand NPNe9.

[0010] FIG. 2. Mean fluorescence intensity (MFI) of FITC-tumor lysate in type 1 conventional dendritic cells (cDCls) (n=5, biologically independent samples). Statistical analysis: one-wayUIC0112WO PATENTANOVA followed by Dunnett test. Data are presented as mean values ± SEM.

[0011] FIG. 3. Relative quantity of H-2Kb-Ova peptide expressed on cDCls (n==3, biologically independent samples), Statistical analysis: one-way ANOVA followed by Dunnett test. Data are presented as mean values± SEM. P < 0.0001.

[0012] FIG. 4. Distribution of AC-NPs in different cells in the tumor 6 hours after AC-NPs or Antigen Capturing nanoparticle Transformed Dendritic Cell therapy (ACT-DC) injection (n=4 for AC-NP, n=5 for ACT-DC, biologically independent animals). AC-NPs and CD103+ cDCls were labeled with 1,1'-Dioctadecyl-3,3,31,3'- Tetramethylindo-dicarbocyanine (DiD) and IVISense™ DiR (NIR fluorescent dye), respectively. Data are presented as mean values ± SEM.

[0013] FIG. 5. Quantification of injected CD103+ cDCls migrated to tumor draining lymph nodes (tDLNs) (n=5, biologically independent mice). CD103+ cDCls were labeled with IVISense™ DiR. Statistical analysis: two-tailed unpaired student's t test. Data are presented as mean values ± SEM.

[0014] FIG. 6. Tumor growth curve showing therapeutic efficacy of ACT-DC in early-stage MC38 tumors (n=4, biologically independent animals). Statistical analysis: two-way ANOVA followed by Dunnett test. Data are presented as mean values ± SEM. PC0.0001, ACT-DC compared to DC; P=0.0038, ACT-DC compared to AC-NP.

[0015] FIG. 7. Survival curve of mice treated with ACT-DC or control formulations showing therapeutic efficacy of ACT-DC in early-stage MC38 tumors (n=4, biologically independent animals).

[0016] FIG. 8. Growth curve of primary tumors (n=15 for ACT-DC, n=8 for ACT-DC + anti-PDl antibody 1 (aPD), n=7 for the other groups, biologically independent animals) showing therapeutic efficacy of ACT-DC in an established MC38 tumor model. Statistical analysis: two-way ANOVA followed by Dunnett test.UIC0112WO PATENTData are presented as mean values± SEM. P<0.0001, ACT-DC compared to DC+NPEEG; P=0.0060, ACT-DC compared to DC+NPNEG; P=0.0026, ACT- DC compared to aPDl.

[0017] FIG. 9. Survival curve of animals in FIG. 8 showing therapeutic efficacy of ACT-DC in an established MC38 tumor model. Statistical, analysis: two-sided Mantel-Cox tests. P=0.0009, ACT-DC compared to DC+NPPEG; P-0.0303, ACT-DC compared to DC+NPKEG; PcO.OOOl, ACT-DC compared to AC-NP and aPDl.

[0018] FIG. 10. Tumor growth curve showing efficacy of ACT-DC in controlling the 1stsubcutenous (s.c.) tumor rechallenge (n=5 for ACT-DC and Naive groups, n=6 for ACT-DC+aPDl, biologically independent animals). Rechallenge was conducted on day 86 post primary tumor inoculation. Statistical analysis: two-way ANOVA followed by Dunnett test. Data are presented as mean values ± SEM.

[0019] FIG. 11. Survival curve post the 1strechallenge of animals in FIG. 10. Statistical analysis: two-sided Mantel-Cox tests. P=0.0137, ADT-DC+aPDl compared to ACT-DC; P=0.00009, ADT-DC+aPDl compared to naive.

[0020] FIG. 12. Number of macrophages in the tumor draining lymph node (tDLN) of mice treated with ACT-DC or control therapies (n=5 biologically independent animals per group). Statistical analysis: one-way ANOVA followed by Dunnett test. Data are presented as mean values ± SEM.

[0021] FIG. 13. Number of cDCl in the tDLN of mice treated with ACT-DC or control therapies (n=5 biologically independent animals per group). Statistical analysis: one-way ANOVA followed by Dunnett test. Data are presented as mean values ± SEM.

[0022] FIG. 14. Number of cDC2 in the tDLN of mice treated with ACT-DC or control therapies (n=5 biologically independent animals per group). Statistical analysis: one-way ANOVA followed by Dunnett test. Data are presented as mean values + SEM.UIC0112WO PATENT

[0023] FIG, 15. Number of CD8 and CD4 T cells in the tDLN of mice treated with ACT-DC or control therapies (n=5 biologically independent animals per group). Statistical analysis: one-way ANOVA followed by Dunnett test. Data are presented as mean values ± SEN.

[0024] FIG. 16, Number of Adpgk tetramer-positive CD8 T cells in the tDLN of mice treated with ACT-DC or control therapies (n=5 biologically independent animals per group). Statistical analysis: one-way ANOVA followed by Dunnett test. Data are presented as mean values ± SEM.

[0025] FIG. 17. Number of interferon (IFN)-y expressing CD4 and CD8 T cells in the tDLN of mice treated with ACT-DC or control therapies (n=5 biologically independent animals per group). Statistical analysis: one-way ANOVA followed by Dunnett test. Data are presented as mean values ± SEM.

[0026] FIG. 18. Total number of effector memory (CD44+CD62L-) and central memory (CD44+CD62L+) CDS T cells in the tDLN. Statistical analysis for: one-way ANOVA followed by Dunnett test. Data are presented as mean values ± SEM.

[0027] FIG. 19. cDCl / cDC2 ratio in the tumor (n=4-5 biologically independent mice per group). Statistical analysis was performed using one-way ANOVA.

[0028] FIG. 20. Growth curve of primary tumors in a bilateral B16F10 melanoma tumor model. n=9 for PBS, n=7 for the other groups, biologically independent mice. Statistical analysis: two- way ANOVA with Dunnett test. Data are presented as mean ± SEM. P<0.0001, ACT-DC compared to PBS; P=0.0001 ACT-DC+aPDl compared to aPDl.

[0029] FIG. 21. Growth curve of distant tumors in a bilateral B16F10 melanoma tumor model. n=9 for PBS, n=7 for the other groups, biologically independent mice. Statistical analysis: two- way ANOVA with Dunnett test. Data are presented as mean ± SEM.UIC0112WO PATENTP<0.0001, ACT-DC compared to PBS; P=0.0570 ACT-DC+aPDl compared to aPDl.

[0030] FIG. 22. Survival curves of mice treated with different formulations in the orthotopic CT-2A glioblastoma model. N=6 for the RT+ACT-DC+aPDl group, n=7 for the other groups, biologically independent mice. Statistical analysis was performed using two- sided Mantel-Cox tests. P=0.0261, RT+ACT-DC+aPDl compared to RT+aPDl.DETAILED DESCRIPTION OF THE INVENTION

[0031] Migratory CD103+ cDCls are a crucial DC subtype that most effectively present tumor antigens to CD8 T cells, with other critical functions in regulating cell-cell interactions within the tumor microenvironment. However, these cells are poorly present in the tumor and their antigen capturing ability is significantly restricted by the immunosuppressive tumor microenvironment. The invention described herein is based, in part, on the development of a novel Trojan horse strategy based on the integration of antigen-capturing nanoparticles (AC-NPs) and migratory CD103+ type 1 conventional dendritic cells (cDCls), referred to herein as Antigen Capturing nanoparticle Transformed Dendritic Cell therapy (ACT-DC), for effective in situ immunization and remodeling of the tumor microenvironment. ACT- DC employs the adoptive transfer of migratory CD103* cDCls to increase their overall frequency and alter their spatial distribution within the tumor. This is coupled with the use of a novel AC-NP that directly collects antigens from the tumor and in situ delivers them to these cDCls, facilitating antigen presentation to activate CD8 T cell in the lymph nodes. Moreover, activation of cDCls by AC-NPs also transforms the local tumor microenvironment into an "immune-hot" state, facilitating immune cell infiltration and tumor eradication. The results herein show that ACT-DC, optionally when combined with immune checkpointUIC0112WO PATENT inhibitors, led to the effective eradication of primary tumors across multiple tumor models. Moreover, this strategy resulted in a robust immune memory, leading to the rejection of distant tumors after two separate tumor rechallenges. Accordingly, provided herein are compositions, kits, and methods for treating cancer or preventing or reducing the risk of reoccurrence of a cancer in a subject using (i) an antigen capturing nanoparticle, the antigen capturing nanoparticle comprising a positively charged and hydrophobic surface; and (ii) migratory type 1 conventional dendritic cells.

[0032] In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, "an antigen capturing nanoparticle" may refer to a single nanoparticle or a population of nanoparticles.

[0033] As used interchangeably herein, "subject," "individual," or "patient" may refer to a vertebrate organism, such as a mammal (e.g., human). In some aspects, a "subject" means an individual. Thus, subjects include, for example, domesticated animals, such as cats and dogs, livestock (e.g., cattle, horses, pigs, sheep, and goats), laboratory animals (e.g., mice, rabbits, rats, and guinea pigs) mammals, non-human mammals, primates, non-human primates, rodents, birds, reptiles, amphibians, fish, and any other animal. The subject is preferably a mammal such as a primate or a human.

[0034] As used herein, the terms "treat," "treating" and "treatment" refer generally to obtaining a desired pharmacological and / or physiological effect. The effect may be therapeutic in terms of a partial or complete cure of a disease, condition, symptom or adverse effect attributed to the disease, disorder, or condition. The term "treatment" as used herein mayUIC0112WO PATENT include treatment of a cancer in a subject, particularly a human and may include inhibiting the progression of the disease, i.e., arresting its development, inhibiting worsening of the disease, and / or relieving the disease, i.e., mitigating or ameliorating the disease and / or its symptoms or conditions. Treatment of cancer may include ameliorating one or more signs or symptoms of the particular disease. A subject in need of treatment include those already diagnosed with the disorder as well as those prone or predisposed to contract the disorder or those in whom the disorder is to be prevented. For example, in tumor (e.g. cancer) treatment, a therapeutic agent may directly decrease the pathology of tumor cells, or render the tumor cells more susceptible to treatment by other therapeutic agents or by the subject's own immune system. "Preventing reoccurrence of a cancer" or "reducing the risk of reoccurrence of a cancer" refers to averting, obviating, forestalling, stopping, or hindering the reoccurrence of a cancer in a subject that has already had and been treated for a cancer, e.g., a subject in remission. Thus, a subject in need of treatment may be a subject treated in the past for a cancer.

[0035] Cancers that may be treated using the compositions, kits and methods herein include solid or non-solid cancer diseases selected from the group consisting of gastric carcinoma, adenocarcinoma, melanoma, in particular malignant melanoma, glioma, colon cancer, pancreatic carcinoma, ovarian carcinoma, uterine cancer, breast cancer, in particular metastatic breast cancer, hepatocellular carcinoma, bronchial carcinoma, leukemias, preferably acute or chronic leukemias of myeloid or lymphoid origin, lymphomas, preferably Hodgkin-lymphoma, nonHodgkin lymphoma, e.g., diffuse large B-cell lymphoma, Ewing's sarcoma, fibrosarcoma, leiomyosarcoma, myeloma, thymoma and other soft tissue sarcomas and blastomas. More specifically, blastomas may be selected from the group consisting of hepatoblastoma,UIC0112WO PATENT medulloblastoma, nephroblastoma, neuroblastoma, pancreatoblastoma, pleuropulmonary blastoma, retinoblastoma and glioblastoma. In some aspects, the compositions, kits and methods herein are of use in the treatment of melanoma, glioma, colon cancer, or breast cancer.

[0036] In accordance with the method herein, a subject in need of treatment is administered (e.g., by injection or transplantation) an effective amount of a nanoparticle as described herein and an effective amount of migratory type 1 conventional dendritic cells. As used herein, the term "amount effective," "effective amount" or a "therapeutically effective amount" refers to an amount of a composition, nanoparticle and / or cells as described herein to achieve the desired result of treating a cancer; preventing or reducing the risk of reoccurrence of a cancer; reducing tumor volume by, e.g., 10% to 100% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any value or range therebetween); increase survival, e.g., by at least 1 month (e.g., 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years or more); increasing the total number of CD103+ cDCls within a tumor by at least 2-fold (e.g., 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9 -or 10-fold) increasing distribution of cDCls throughout both the marginal and central areas of a cancer; increased trafficking and delivery of tumor antigens to the tDLNs; increasing the number of innate immune cells in tDLNs including macrophages, cDCls, and cDC2s; increasing central memory and effector memory CD8+ and CD4+ T cells in tDLNs; and preventing cancer metastasis as compared to a subject that has not been treated with the nanoparticles and / or cells. The amount of the composition, nanoparticle and / or cells, which constitutes an "effective amount" or "therapeutically effective amount" may vary depending on the severity of the disease, the condition, weight, or age of the patient to be treated, the frequency of dosing, or the route of administration,UIC0112WO PATENT but may be determined routinely by one of ordinary skill in the art. In some aspects, an effective amount of a composition comprising nanoparticles and / or cells may be administered in a single injection or infusion or divided into multiple administrations delivered at predetermined intervals. A clinician may titer the dosage or route of administration to obtain the optimal therapeutic effect.

[0037] To elicit a robust immune response against a tumor, aspects of this invention provide for the use of an antigen capturing nanoparticle. An "antigen capturing nanoparticle" or "AC-NP" refers to a nanoparticle that binds to or otherwise associates with an antigen directly obtained from a tumor. In some aspects, an AC-NP herein directly collects antigens from a tumor and in situ delivers the tumor antigen to migratory type 1 conventional dendritic cells (cDCl), thereby facilitating antigen presentation to activate CDS T cell in the lymph nodes. Activation of cDCls by AC-NPs also transforms the local tumor microenvironment into an "immune-hot" state, facilitating immune cell infiltration and tumor eradication.

[0038] A "nanoparticle" refers to a particle having a core and shell (a core-shell nanoparticle), which may be generally spherical and have a diameter of less than about 1000 nm, e.g., about 5 nm to about 990 nm, or any value or range therebetween. In some aspects, a nanoparticle is an "empty nanoparticle," i.e., the core does not encapsulate a compound. In some aspects, a nanoparticle is a "loaded nanoparticle," i.e., the shell encapsulates a compound within the core of the nanoparticle. In some aspects, an empty nanoparticle may have a diameter that is smaller than the diameter of a loaded nanoparticle. In some aspects, a loaded nanoparticle may have a diameter of 5 to 200 nm, about 60 to about 200 nm, about 70 to about 150 nm, about 80 to about 160 nm, about 100 to about 200, about 120 to about 300, about 150 to about 400 nm, or about 200 to 1000 nm. TheUIC0112WO PATENT hydrodynamic diameter of a given nanoparticle core may be dependent upon the solvent in which it is suspended. For example, nanoparticle cores that are suspended in water generally have larger hydrodynamic diameters than nanoparticle cores that are suspended in phosphate-buffered saline (PBS).

[0039] Methodology for determining the hydrodynamic diameter of a nanoparticle is well known in the art and described, for example, in US 2007 / 0258907. Hydrodynamic diameter measurements often include a determination of dynamic light scattering (DLS), such as may be achieved with a ZetaPALS dynamic light scattering detector (DLS, Brookhaven Instruments Corporation),

[0040] In some aspects, the surface of a nanoparticle herein may be hydrophobic. "Hydrophobic" is given its ordinary meaning in the art and, as will be understood by those skilled in the art, in many instances herein, is a relative term. The relative hydrophobicity of a material may be determined by measuring the contact angle of a water droplet on a planar surface of the substance to be measured, e.g., using an instrument such as a contact angle goniometer and a packed powder of the core material.

[0041] In some aspects, a nanoparticle comprises a hydrophobic surface by having a shell composed of one or more hydrophobic polymers. Examples of hydrophobic polymers include, but are not limited to, polytetrafluoroethylene (PTFE), poly (lactic acid) (PLA), poly (lactic-co-glycolic acid) (PLGA), poly (e- caprolactone) (PCL), polyurethane (PU), polypropylene carbonate (PPC), polyhydroxybutyrate (PHB), and the like, and combinations thereof.

[0042] In some aspects, a hydrophobic polymer of use in the preparing a nanoparticle herein may include poly (lactic-co- glycolic acid) (PLGA). PLGA is a biocompatible and biodegradable co~polymer of lactic acid and glycolic acid, and various forms of PLGA are characterized by the ratio of lactic acid:glycolic acid. Lactic acid may be L-lactic acid, D-lactic acid, or D,L-UIC0112WO PATENT lactic acid. The degradation rate of PLGA may be adjusted by altering the lactic acid:glycolic acid ratio. In some aspects, a PLGA of use herein may be characterized by a lactic acid:glycolic acid ratio of approximately 85:15, approximately 75:25, approximately 65:35, approximately 50:50, approximately 35:65, approximately 25:75, or approximately 15:85.

[0043] In some aspects, a nanoparticle herein is positively charged, i.e., the nanoparticle is cationic. In some aspects, the surface of a nanoparticle herein may be characterized by having a positive surface charge of greater than about +1 mV, e,g., a surface charge range from about +5 mV to about +80 mV, e,g.fabout +5, +10, +15, +20, +25, +30, +35, +40, +45, +50, +55, +60, +70, or +80 mV or any value or range therebetween.

[0044] In some aspects, a nanoparticle comprises a positive or cationic charge by having a shell composed of one or more cationic polymers and / or lipids. Representative cationic polymers and lipids of use in the preparing a nanoparticle herein may include, but are not limited to, polyethylenimine (PEI), chitosan, poly(HPMA-jb-DMAPMA), poly-(propylenimine) (PPI), DEAE-dextran, poly(amidoamine) (PAMAM), poly-L-(lysine) (PLL), poly[2-(N,N- dimethylamino)ethyl methacrylate] (PDMAEMA), poly(amidoamine) (PAA), 1,2-di-0-octadecenyl-3-trimethylammonium-propane (DOTMA), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 2,3- dioleyloxy-W-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1- propanaminium trifluoroacetate (DOSPA), and / or ethylphosphatidylcholine (ePC).

[0045] In some aspects, the shell of a nanoparticle herein comprises polyethyleneimine. Polyethyleneimine (PEI) is highly basic and positively charged aliphatic polymer, containing primary, secondary and tertiary amino groups in a 1:2:1 ratio. As the polymer is composed of repeating units of ethylamine, PEI is also highly water soluble. Polyethyleneimine is available inUIC0112WO PATENT both linear and branched forms with molecular weights ranging from 700 Da to 1000 kDa.

[0046] In some aspects, an antigen capturing nanoparticle herein has a wall comprising, consisting essentially of or consisting of a hydrophobic polymer and a positively charged polymer or lipid. In some aspects, an antigen capturing nanoparticle herein has a wall comprising, consisting essentially of or consisting of PLGA and a positively charged polymer or lipid. In some aspects, an antigen capturing nanoparticle herein has a wall comprising, consisting essentially of or consisting of a hydrophobic polymer and PEI. In some aspects, an antigen capturing nanoparticle herein has a wall comprising, consisting essentially of or consisting of PLGA and PEI. In some aspects, the ratio of the hydrophobic polymer (e.g., PLGA) and the positively charged polymer or lipid (e.g,, PEI) used in the preparation of a nanoparticle herein ranges from 20:1 to 1:20, e.g., 20:1, 10:1 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:10, or 1:20. In some aspects, the ratio of the hydrophobic polymer (e.g., PLGA) and the positively charged polymer or lipid (e.g., PEI) used in the preparation of a nanoparticle herein is 2:1.

[0047] Methods for producing an antigen capturing nanoparticle include those described in the Examples as well as other suitable methods such as, for example, coacervation-phase separation, single emulsion, double emulsion, melt dispersion, interfacial deposition, in situ polymerization, self-assembly of macromolecules, spray drying and spray-congealing, electrospray, air suspension coating, pan and spray coating, freeze- drying, air drying, vacuum drying, fluidized-bed drying, precipitation, critical fluid extraction, and lithographic approaches.

[0048] Advantageously, a nanoparticle comprising a positively charged and hydrophobic surface as described herein captures aUIC0112WO PATENT significantly higher number and / or amount of tumor proteins compared to negatively charged nanoparticles or nanoparticles with a hydrophilic surface. An antigen, in particular a tumor antigen, is "captured" by a nanoparticle herein when the nanoparticle binds to or otherwise associates with, e.g., via noncovalent interactions, a tumor antigen. Capture of a tumor antigen by a nanoparticle described herein may be determined by, e.g., an increase in the size of the nanoparticle, a shift in the charge of the nanoparticle from positive to neutral or negative, and / or analyzing protein content / identity, e.g., using a BCA assay, polyacrylamide gel electrophoresis, and / or liquid chromatography-mass spectrometry.

[0049] As used herein, the term "tumor antigen" refers to an immunogenic epitope (e.g., protein) expressed by a tumor cell that is capable of being recognized by the immune system and triggering an immune response. The protein may be expressed by non-tumor cells but be immunogenic only when expressed by a tumor cell. Alternatively, the protein may be expressed by tumor cells, but not normal cells. Exemplary tumor antigens include, but are not limited to, carcinoembryonic antigen (CEA), CA-125, MUC-1, epithelial tumor antigen (ETA), CA15-3, tyrosinase, melanoma- associated antigen (MAGE), abnormal products of ras or p53, alphafetoprotein, beta subunit of hCG, prostate specific antigen (PSA), beta 2 microglobulin, CA19-9, chromogranin A, TA90, GP100, MelanA / MARTl, MHC, CTLA-4, mesothelin, PD-L1, TRP1, CD40, EGFP, Her2, TCR alpha, trp2, TCR, cdr2, 4-1BB, CT26, GITR, 0X40, TGF- a, WT1, LMP2, EGFRvIII, HER-2 / neu, NY-ESO-1, PSMA, GD2, Proteinase3 (PR1), bcr-abl, Survivin, hTERT, EphA2, PAP, ML-IAP, EpCAM, NA17, PAX3, ALK, Androgen receptor, Cyclin Bl, Polysialic acid, MYCN, RhoC, TRP-2, GD3, Fucosyl GM1, PSCA, CYP1B1, PLAC1, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGSS, SART3, STn, Carbonic anhydrase IX, PAXS, OY-TES1, Sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, Legumain, Tie 2, Page4,UIC0112WO PATENTVEGFR2, MAD-CT-1, FAP, PDGFR-p, MAD-CT-2, Hnrnpf, Aatf, Copb2, Kpna6, and Fos-related antigen 1.

[0050] In some aspects, an antigen capturing nanoparticle herein also captures one or more damage-associated patterns (DAMPs) proteins. DAMPS are endogenous molecules released from damaged or dying cells that trigger and amplify the innate immune response. DAMPS are recognized by pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) and the receptor for advanced glycation end products (RAGE), on immune cells, leading to an immune response. Examples of DAMPs that may be captured by a nanoparticle herein include, but are not limited to, fibronectin, S100 proteins, heat shock proteins, F-actin, histones, IL-lct, IL-33, and the like.

[0051] In situ capture of tumor antigens (and optionally DAMPs) by a nanoparticle herein facilitates delivery of the tumor antigens to adoptively transferred migratory cDCls thereby enhancing antigen presentation to the lymph nodes and reshaping the tumor microenvironment. Accordingly, the compositions, kits, and methods herein further include the use of migratory type 1 conventional dendritic cells. "Type 1 conventional dendritic cells" or "cDCls" refer to conventional dendritic cells (DCs) that differentiate from pre-cDC precursors under the control of BATF3, ID2 and IRF8 and are specialized in the induction of cytotoxic T cell (CTL) responses. Prominent cDCl markers include CD8A, CLEC9A, ITGAE, ITGAX, THBD (CD141), and XCR1. In addition, human eDCs may be characterized by the cell surface markers of at least CDllc+ MHCII+ CD16" BDCA2- and CD14" and either CD8od or CD103+. In some aspects, a cDCl of use in the compositions, kits, and methods herein is a migratory cDCl characterized by cell surface expression of CD103+and one or more of the abovereferenced markers.

[0052] Migratory cDCl cells may be isolated and / or enriched from, e.g., bone marrow, for use in the compositions, kits, and methodsUIC0112WO PATENT herein using conventional methods such as buoyant density centrifugation, magnetic-activated cell sorting (MACS), and / or fluorescently activated cell sorting (FACS). The term "isolated," as used herein, means having been removed from its natural environment. "Isolated" does not require absolute isolation; rather, it is intended as a relative term. Thus, for example, an isolated cell subset is one in which said cell subset is more pure than the cell subset in its natural environment within a tissue. The term "enriched", as used herein, means having increased the purity of a cell type or cell subset within a cell population. In some aspects, migratory cDCls may be enriched such that the migratory cDCls represent at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% of the total cell content of a cell preparation.

[0053] To facilitate activation of cDCls, the compositions, kits, and methods herein may further include the use of at least one immune stimulating agents encapsulated within the core of the antigen capturing nanoparticle. An "immune stimulating agent" or "immune stimulating adjuvant" refers to a molecule that may enhance the immune response to an antigen. In some aspects, an antigen capturing nanoparticle may encapsulate one, two, three, four, five, six, seven, eight, nine, 10 or more immune stimulating agents. In some aspects, an immune stimulating agent may comprise a toll-like receptor (TLR) ligand, nucleotide-binding oligomerization domain (NOD) ligand, retinoic-acid-inducible protein 1(RIG-1)-like receptor (RLR) ligand, C-type lectin receptor ligand, cytosolic DNA sensing ligand, stimulator of interferon genes (STING) ligand, aryl hydrocarbon receptor (AhR) ligand, and / or alpha protein kinase 1 (ALPKl)-TRAF interacting forkhead-associated protein A (TIFA) inducer. In some aspects, an immune stimulating agent may be a TLR ligand, e.g.fa TLR2 ligand, TLR3 agonist, TLR4 agonist, TLR5 agonist, TLR7 / 8 agonistUIC0112WO PATENT and / or TLR9 agonist. In some aspects, an immune stimulating agent may be a NOD ligand, e.g., NODI agonist and / or NOD2 agonist. In some aspects, an immune stimulating agent may be a RLR ligand, e.g., a RIG-I agonist. In some aspects, an immune stimulating agent may be a C-type lectin receptor ligand, e.g., a Dectin-1 agonist, Dectin-2 agonist and / or Mincle agonist. In some aspects, an immune stimulating agent may be a cytosolic DNA sensing ligand. In some aspects, an immune stimulating agent may be a STING ligand. In some aspects, an immune stimulating agent may be an AhR ligand. In some aspects, an immune stimulating agent may be an ALPK1-TIFA inducer. Examples of immune stimulating agents include, but are not limited to, the immune stimulating agents provided in Table 1.TABLE 1UIC0112WO PATENT

[0054] In one aspect, the immune stimulating agent may be a TLR agonist. A "TLR agonist" is a substance that binds, directly or indirectly, to a TLR to induce TLR signaling. Any detectable difference in TLR signaling can indicate that an agonist stipulates or activates a TLR. Signaling differences can be manifested, for example, as changes in the expression of target genes, in the phosphorylation of signal transduction components, in the intracellular localization of downstream elements such as NK-KB, in the association of certain components (such as IRAK) with other proteins or intracellular structures, or in the biochemical activity of components such as kinases (such as MAPK). In some aspects, the immune stimulating agent may be a TLR3 agonist. In another aspect, the immune stimulating agent may be polyinosinic:polycytidylic acid (PIC).

[0055] Alternatively, or in addition to an immune stimulating agent, an antigen capturing nanoparticle may further encapsulate or be modified, e.g., by surface attachment, to deliver of one or more therapeutic agents (including, for example, anti-cancer agents, anti-angiogenic agents, etc.) to an individual in need of the agent(s). The nanoparticle, in some cases, allows delivery of more than one agent, and such multiple agents may be of the same type of agent (nucleic acid or drug, for example) or not. Thus, in a plurality of nanoparticles, there may be a mixture of nanoparticles with more than one agent but with each separate nanoparticle having only one agent; a therapeutically effective amount of the agent may be provided to the individual. In some aspects, there may be a mixture of nanoparticles with more than one agent but with a particular nanoparticle having more than one agent. In any case, a therapeutically effective amount of the agent may be provided to the individual. In some aspects, aUIC0112WO PATENT therapeutic agent is one or more of a nucleic acid, small molecule, protein, peptide, or mixture thereof.

[0056] As used in accordance with the methods, compositions and kits herein, the antigen capturing nanoparticles and migratory cDCl cells herein may be administered by injection, for example, intratumorally, intravenously, intra-muscularly, intraarterially, intra-bone, intratracheally, and the like. In some aspects, administration involves providing to a subject about 102, 103, 104, 105, 106, 101, 108, 109, 1010, 1012, or more cells. The number of cells administered may be chosen based on the route of administration and / or the severity of the condition for which the cells are administered.

[0057] Compositions, e.g., pharmaceutical or therapeutic compositions containing antigen capturing nanoparticles and / or migratory cDCl may be prepared by combining the cells and / or nanoparticles with a pharmaceutically acceptable carrier or aqueous medium. The phrase "pharmaceutically acceptable" or "pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the cells of the present disclosure, its use in therapeutic compositions is contemplated. Pharmaceutical compositions may be determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format and desired dosage. See, for example, Remington: The Science and Practice of Pharmacy, recent edition.

[0058] Compositions herein may be incorporated in an injectable formulation. The formulation may also include the necessaryUIC0112WO PATENT physiologically acceptable carrier material, excipient, lubricant, buffer, surfactant, antibacterial, bulking agent (such as mannitol), antioxidants (ascorbic acid or sodium bisulfite) and the like.

[0059] Acceptable formulation materials preferably are nontoxic to recipients at the dosages and concentrations employed. A pharmaceutical composition may contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. Suitable formulation materials may include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite); buffers (such as borate, bicarbonate, Tris- HC1, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA; complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; saltforming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as poloxamers, PEG, sorbitan esters, polysorbates such as polysorbate 20 and polysorbate 80, Triton™,UIC0112WO PATENT trimethamine, lecithin, cholesterol, or tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol, or sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants.

[0060] The primary vehicle or carrier in a pharmaceutical composition may be either aqueous or nonaqueous in nature. For example, a suitable aqueous medium or carrier may be water for injection, physiological saline solution or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary media. Pharmaceutical compositions may include Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0- 5.5, which may further include sorbitol or a suitable substitute thereof. Pharmaceutical compositions herein may be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents in the form of a lyophilized cake or an aqueous solution.

[0061] The cells and / or nanoparticle compositions herein may be provided by sustained release systems, by encapsulation or by implantation devices. The cells and / or nanoparticle compositions may be administered by bolus injection or continuously by infusion, or by implantation device. The cells and / or nanoparticle compositions may also be administered locally via implantation of a membrane, sponge or another appropriate material onto which the cell or cells have been absorbed or encapsulated. Where an implantation device is used, the device may be implanted into any suitable tissue or organ. The injections may be given as a one-time treatment, repeated (daily, weekly, monthly, annually etc.) to achieve the desired therapeutic effect.UIC0112WO PATENT

[0062] The cells and / or nanoparticle compositions herein may be delivered parenterally. When parenteral administration is contemplated, the compositions for use herein may be in the form of a pyrogen-free, parenterally acceptable aqueous solution. A particularly suitable vehicle for parenteral injection is sterile distilled water. Preparation can involve the formulation with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads or liposomes, that may provide controlled or sustained release of the cell or cells, which may then be delivered via a depot injection. Formulation with hyaluronic acid has the effect of promoting sustained duration in the circulation. Implantable drug delivery devices may be used to introduce the desired composition.

[0063] These compositions herein may also contain adjuvants such as preservative, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid and the like. It may also be desirable to include isotonic agents such as sugars, sodium chloride and the like.

[0064] The cells and / or nanoparticle compositions herein may include classic pharmaceutical preparations. Administration of these compositions according to the present disclosure may be via any common route so long as the target tissue is available via that route. Such routes include oral, nasal, buccal, rectal, vaginal or topical route. Ideally, administration may be by intratumoral delivery, intratracheal instillation, intratracheal inhalation, intravenous delivery, intramuscular delivery, intraarterial delivery, topical delivery, renal artery injection, portal vein injection, intrabone delivery, intraarticular delivery, intralymphatic delivery, intrathymic delivery, intrarenal delivery, intracorneal delivery, intraportalUIC0112WO PATENT delivery, intrahepatic delivery, or intracardiac injection. Such compositions would normally be administered as pharmaceutically acceptable compositions.

[0065] Compositions comprising the migratory cDCl cells and compositions comprising antigen capturing nanoparticles may be administered sequentially or simultaneously to a subject. In some aspects, a composition comprising antigen capturing nanoparticles may be administered prior to a composition comprising migratory cDCl cells. In other aspects, a composition comprising migratory cDCl cells may be administered prior to a composition comprising antigen capturing nanoparticles. In a further aspect, a composition comprising antigen capturing nanoparticles and migratory cDCl cells may be administered to a subject.

[0066] In some aspects, a composition, kit, or method herein may further include the use of an immune checkpoint inhibitor. As used herein, the term "immune checkpoint inhibitor" refers to any modulator that inhibits the activity of the immune checkpoint molecule. Immune checkpoint inhibitors may include, but are not limited to, immune checkpoint molecule binding proteins, small molecule inhibitors, antibodies, antibody-derivatives (including Fc fusions, Fab fragments and scFvs), antibody-drug conjugates, antisense oligonucleotides, siRNA, aptamers, peptides and peptide mimetics. In some aspects, the immune checkpoint inhibitor may be an inhibitor of cytotoxic T-lymphocyte antigen 4 (CTLA4, also known as CD152), T cell immunoreceptor with Ig and ITIM domains (TIGIT), glucocorticoid-induced TNFR-related protein (GITR, also known as TNFRSF18), inducible T cell costimulatory (ICOS, also known as CD278), CD96, poliovirus receptor-related 2 (PVRL2, also known as CD112R, programmed cell death protein 1 (PD-1, also known as CD279), programmed cell death 1 ligand 1 (PD-L1, also known as B7-H3 and CD274), programmed cell death ligand 2 (PD- L2, also known as B7-DC and CD273), lymphocyte activation gene-3 (LAG-3, also known as CD223), B7-H4, killer immunoglobulinUIC0112WO PATENT receptor (KIR), Tumor Necrosis Factor Receptor superfamily member 4 (TNFRSF4, also known as 0X40 and CD134) and its ligand OX4OL (CD252), indoleamine 2,3-dioxygenase 1 (IDO-1), indoleamine 2,3- dioxygenase 2 (IDO-2), carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), B and T lymphocyte attenuator (BTLA, also known as CD272), T-cell membrane protein 3 (TIM3), the adenosine A2A receptor (A2Ar), and V-domain Ig suppressor of T cell activation (VISTA protein). In some aspects, the immune checkpoint inhibitor is an inhibitor of CTLA4, PD-1, or PD-Ll. In some aspects, the immune checkpoint inhibitor is an antibody is selected from: ipilimumab (targeting CTLA4), pembrolizumab (targeting PD-1), nivolumab (targeting PD-1), atezolizumab (targeting PD-Ll), avelumab (targeting PD-Ll), and durvalumab (targeting PD-Ll).

[0067] In some aspects, a composition comprising an AC-NP, migratory cDCl cells, and optionally an immune checkpoint inhibitor may be administered in combination with a chemotherapeutic agent and / or radioisotope. Chemotherapeutic agents are well known to those of skill in the art. Examples of such chemotherapeutics include alkylating agents, antibiotics, antimetabolitic agents, plant-derived agents, and hormones. Among the suitable alkylating agents are nitrogen mustards, such as cyclophosphamide, aziridines, alkyl alkone sulfonates, nitrosoureas, nonclassic alkylating agents, such as dacarbazine, and platinum compounds, such as carboplatin and cisplatin. Among the suitable antibiotic agents are dactinomycin, bleomycin, mitomycin C, plicamycin, and the anthracyclines, such as doxorubicin (also known as adriamycin) and mitoxantrone. Among the suitable antimetabolic agents are antifols, such as methotrexate, purine analogues, pyrimidine analogues, such as 5- fluorouracil (5-FU) and cytarabine, enzymes, such as the asparaginases, and synthetic agents, such as hydroxyurea. Among the suitable plant-derived agents are vinca alkaloids, such asUIC0112WO PATENT vincristine and vinblastine, taxanes, epipodophyllotoxins, such as etoposide, and camptothecan. Radioactive isotopes include, e.g., iodine-131, iodine-123, technicium-99m, indium-ill, rhenium-188, rhenium-186, gallium-67, copper-67, yttrium-90, iodine-125 or astatine-211). Suitable dosages for the selected chemotherapeutic agent are known to those of skill in the art and the route of administration, the number of doses received, the timing of the doses, and the dosage amount, may be readily adjusted as needed.

[0068] In one aspect, a method of treating cancer or preventing or reducing the risk of reoccurrence of a cancer in a subject is provided that comprises the step of administering to a subject in need of treatment an effective amount of (i) an AC-NP, the AC- NP comprising a positively charged and hydrophobic surface; and (ii) an effective amount of migratory cDCl cells. In another aspect, a method of treating cancer or preventing or reducing the risk of reoccurrence of a cancer in a subject is provided that comprises the step of administering to a subject in need of treatment an effective amount of (i) an AC-NP encapsulating'at least one immune stimulating agent (e.g., a TLR agonist, e.g., a TLR3 agonist such as PIC), the AC-NP comprising a positively charged and hydrophobic surface; and (ii) an effective amount of migratory cDCl cells. In a further aspect, a method of treating cancer or preventing or reducing the risk of reoccurrence of a cancer in a subject is provided that comprises the step of administering to a subject in need of treatment an effective amount of (i) an AC-NP, the AC-NP comprising a positively charged and hydrophobic surface; (ii) an effective amount of migratory cDCl; and (iii) an immune checkpoint inhibitor (e.g., a PD-1 inhibitor). A yet another aspect, a method of treating cancer or preventing or reducing the risk of reoccurrence of a cancer in a subject is provided that comprises the step of administering to a subject in need of treatment an effective amount of (i) an AC-UIC0112WO PATENTNP encapsulating at least one immune stimulating agent (e.g., a TLR agonist, e.g., a TLR3 agonist such as PIC), the AC-NP comprising a positively charged and hydrophobic surface; (ii) an effective amount of migratory cDCl cells; and (iii) an immune checkpoint inhibitor (e.g., a PD-1 inhibitor).

[0069] In one aspect, a composition, e.g., pharmaceutical composition, comprising (i) an AC-NP optionally encapsulating at least one immune stimulating agent as described herein, the AC- NP comprising a positively charged and hydrophobic surface; (ii) migratory cDCl cells; and (iii) a pharmaceutically acceptable carrier or aqueous medium is provided herein. In another aspect, a composition comprising (i) an AC-NP encapsulating at least one immune stimulating agent (e.g., a TLR agonist, e.g., a TLR3 agonist such as PIC), the AC-NP comprising a positively charged and hydrophobic surface; (ii) migratory cDCl cells; and (iii) a pharmaceutically acceptable carrier or aqueous medium is provided herein. In a further aspect, a composition comprising (i) an AC- NP, the AC-NP comprising a positively charged and hydrophobic surface; (ii) migratory cDCl cells; (iii) an immune checkpoint inhibitor (e.g., a PD-1 inhibitor); and (iv) a pharmaceutically acceptable carrier or aqueous medium is provided herein. A yet another aspect, a composition comprising (i) an AC-NP encapsulating at least one immune stimulating agent (e.g., a TLR agonist, e.g., a TLR3 agonist such as PIC), the AC-NP comprising a positively charged and hydrophobic surface; (ii) migratory cDCl cells; and (iii) an immune checkpoint inhibitor (e.g., a PD-1 inhibitor); and (iv) a pharmaceutically acceptable carrier or aqueous medium is provided herein,

[0070] Any of the compositions described herein may be comprised in a kit. In a non-limiting example, an AC-NP, the AC-NP comprising a positively charged and hydrophobic surface; migratory cDCl cells; and an immune checkpoint inhibitor may be comprised in a kit. Such reagents include one or more of cells,UIC0112WO PATENT buffers, media, and so forth. The kits may comprise any of its components in one or more suitable containers. The components of the kits may be packaged either in aqueous media or in lyophilized form. A container of a kit herein may generally include at least one vial, test tube, flask, bottle, syringe or other container, into which a component may be placed, and preferably, suitably aliquoted. Where there are more than one components in the kit, the kit may also generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a single vial. A kit may also include a packaging for containing the components in close confinement for commercial sale. Packaging may include injection or blow-molded plastic containers into which the desired vials are retained. The components of the kit may be provided as dried powder(s). When reagents and / or components are provided as a dry powder, the powder may be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container.

[0071] The following non-limiting examples are provided to further illustrate the present invention.EXAMPLESExample 1: Materials and Methods

[0072] Materials. Poly(lactic-co-glycolic acid) (PLGA, 50:50, Resomer RG503H), linear polyethylenimine (PEI, MW 2500), polyinosinic:polycytidylic acid (PIC), non-essential amino acid solution, and polyvinyl alcohol (PVA) were purchased from Sigma- Aldrich (St. Louis, MO). mPEG-PLGA (MW: 5k / 20k) was purchased from Nanosoft Polymers (Winston-Salem, NC). RPMI-1640 medium, high glucose-Dulbecco's modified eagles' medium (DMEM), and penicillin / streptomycin were obtained from Cytiva (Marlborough, MA). Heat-inactivated fetal bovine serum (FBS) was purchased fromUIC0112WO PATENTCorning (Corning, NY). Recombinant murine FTL3L, GoInVivo™ anti- PD1 antibody, and recombinant murine GM-CSF were obtained from BioLegend (San Diego, CA)• 2-mercaptoethanol, HEPES buffer, ACK buffer, sodium pyruvate, 1,1'-Dioctadecyl-3,3,3',3'- Tetramethylindodicarbocyanine (DiD), 3,3'- Dioctadecyloxacarbocyanine Perchlorate (DiO), 1,11-Dioctadecyl- 3,3,3',3'-Tetramethylindocarbocyanine Perchlorate (Dil), Hoechst 33342, and blasticidin were purchased from Thermo Fisher Scientific (Waltham, MA).

[0073] Antibodies and antibody dilutions. CD45-ALEXA FLUOR® 700 (clone QA17A26, BioLegend) 1:400-1:2000 dilution; B220-APC (clone RA3-6B2, BioLegend) 1:300 dilution; CDllc-APC / Cy7 (clone N418, BioLegend) 1:50 dilution; CDllc-PE (clone N418, BioLegend) 1:1000 dilution; CDllc-BV650 (clone N418, BioLegend) 1:80-1:200 dilution; CD103-ALEXA FLUOR® 700 (clone 2E7, BioLegend) 1:200 dilution; CD103-BV711 (clone 2E7, BioLegend) 1:100 dilution; CD86-APC (clone GL-1, BioLegend) 1:400 dilution; CD86-BV785 (clone GL-1, BioLegend) 1:50-1:200 dilution; MHCII-PB (clone M5 / 114.15.2, BioLegend) 1:1000 dilution; MHCII-BV605 (clone M5 / 114.15.2, BioLegend) 1:50 dilution; CD80-FITC (clone 16-10A1, BioLegend) 1:250 dilution; CD80-BV605 (clone 16-10A1, BioLegend) 1:20-1:40 dilution; CDllb-FITC (clone MI / 70, BioLegend) 1:100- 1:1000 dilution; F4 / 80-BV510 (clone BM8, BioLegend) 1:10-1:200 dilution; CD206-Percp-Cyanine5.5 (clone C068C2, BioLegend) 1:10- 1:200 dilution; Gr-l-Pacific Blue (clone RB6-8C5, BioLegend) 1:50 dilution; CD49b-PE / Dazzle 594 (clone DX5, BioLegend) 1:100-1:500 dilution; CD3-PE / Fire 700 (clone 17A2, BioLegend) 1:50-1:500 dilution; CD4-Spark Blue 550 (clone GK1.5, BioLegend) 1:100-1:400 dilution; CD8a-Spark UV 387 (clone 53-6.7, BioLegend) 1:100- 1:1000 dilution; IFN-y-APC / Fire 750 (clone XMG1.2, BioLegend) 1:100-1:200 dilution; CD62L-PE / Cyanine5 (clone MEL-14, BioLegend) 1:50-1:200 dilution; CD44-BV570 (clone IM7, BioLegend) 1:40 dilution; CD25-PE / Cyanine7 (clone PC61, BioLegend) 1:20-1:40UIC0112WO PATENT dilution; FoxP3-ALEXA FLUOR® 647 (clone MF-14, BioLegend) 1:100- 1:200 dilution; PD-1-BV421 (clone 29F.1A12, BioLegend) 1:100- 1:200 dilution; Adpgk tetramer-PE (Tetramer Core of the National Institutes of Health), 1:100 dilution; OVA tetramer-PE (Tetramer Core of the National Institutes of Health) 1:100 dilution; TRP-2 tetramer-PE (Tetramer Core of the National Institutes of Health) 1:100 dilution; Rpll8 tetramer-BV421 (Tetramer Core of the National Institutes of Health) 1:100 dilution; Granzyme B-PE / Cy7 (clone QA16A02, BioLegend) 1:40 dilution; Lag3-PE / Dazzle594 (clone C9B7W, BioLegend) 1:80 dilution; Tim3-PE / Fire640 (clone RMT3-23, BioLegend) 1:40-1:80 dilution; PDl-PE / Fire810 (clone 29F.1A12, BioLegend) 1:160 dilution; Perforin-APC / Fire750 (clone S16009A, BioLegend) 1:40-1:80 dilution; Ki67-BV711 (clone 11F6, BioLegend) 1:300 dilution; Clec9A-PE (clone 7H11, BioLegend) 1:150 dilution; PE / Cyanine7 anti-mouse H-2Kbbound to OVA agonist peptide (clone 25-D1.16, BioLegend) 1:160 dilution; Zombie NIR (BioLegend) 1:1000 dilution; Zombie UV (BioLegend) 1:1000 dilution.

[0074] Cell lines and animals. The MC38 cell line was purchased from Kerafast (Boston, MA). MC38 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, 1% non-essential amino acid, 0.01 M HEPES, and 50 pg / mL Gentamicin. The CT-2A cells are known in the art and available from, e.g., Millipore Sigma. CT-2A cells were maintained in DMEM with 10% FBS and 1% penicillin / streptomycin. The B16F10 cell line expressing luciferase (B16F10-Luc) and B16F10 cell line were obtained from ATCC (Manassas, VA). The B16F10-OVA cells are known in the art and available from, e.g., Biocytogen. B16F10-Luc cells were cultured in DMEM medium supplemented with 10% FBS, 1% penicillin-streptomycin, and 10 pg / mL blasticidin. B16F10 and B16F10-OVA cells were cultured using a similar method as B16F10-Luc, without adding blasticidin to the culture medium. Male / female C57BL / 6 mice (6-8 weeks ofUIC0112WO PATENT age) and female B6.129S(C)-BatfStmlKmm / J mice (6-8 weeks of age) were purchased from Jackson Laboratory (Bar Harbor, ME). Mice were housed in a facility with controlled conditions, including a 14:10-h light:dark cycle, an ambient temperature maintained at 22±2°C, and a relative humidity of 30-70%.

[0075] Preparation and characterization of AC-NPs. AC-NPs were prepared using a double emulsion method (Zhao et al. (2021) Nat. Biomed. Eng. 5:441-454; Zhao et al. (2019) Sci. Adv. 5:eaax9250). Briefly, 20 mg of PLGA (Resomer RG503H) and 10 mg of PEI (linear, MW 2500) were dissolved in 1 mL chloroform as the organic phase with the assistance of a water-bath sonicator. For the preparation of fluorescently labeled NPs, 10 pL of 5 mg / mL DiD or DiO was dissolved in the organic phase. Subsequently, 150 pL of water containing 3 mg PIC was added to the organic phase followed by a water-bath sonication for 1 minute. The emulsion was then dropwise added to 11 mL of 0.5% polyvinyl alcohol solution. Particle formation was achieved through probesonication for 20 seconds twice with a 20-second break in between, followed by stirring for over 12 hours in a fume hood to completely evaporate the organic solvent. Negatively charged (NpNeg) and PEGylated (NPPEG) NPs were prepared using a similar method without the addition of PEI. NPs were washed three times with deionized water for characterization of their physicochemical properties. The size and surface charge of NPs were measured using dynamic light scattering (DLS) (Malvern Nano- ZS Zetasizer). Additionally, the morphology of NPs was characterized by scanning electron microscopy (SEM) (JEOL JSM- IT500HR). For quantifying the loading of PIC into NPs, NPs were lysed in a buffer containing 100 mM sodium hydroxide and 0.05% sodium dodecyl sulfate with gentle shaking overnight at 37°C. The encapsulated PIC was quantified using Nanodrop 2000 (Thermo Fisher).-Si-UIC0112WO PATENT

[0076] CD103+ cDCl culture. CD103+ cDCls were cultured using a previously reported method with modifications (Mayer et al. (2014) Blood 124:3081-3091). Bone marrow was obtained from the femurs of freshly euthanized C57BL / 6 mice. The harvested bone marrow was seeded to a 150 mm x 20 mm petri dish at a concentration of 1.5*106cells / mL and cultured in RPMI-1640 media supplemented with 10% FBS, 1% Penicillin-Streptomycin, 50 pM 2- mercaptoethanol, 200 ng / mL FTL3L, and 2 ng / mL GM-CSF. The media was refreshed on days 3 and 6. Non-adherent ceils were collected on day 9, counted, and re-plated. Non-adherent cells were harvested on days 12-15. Prior to use, cells were washed 2-3 times with PBS. The obtained CD103+ cDCls were characterized by surface marker expression including CDllc, B220, MHCII, CD103, and Clec9A via antibody staining followed by flow cytometry (CytoFLEX, Beckman).

[0077] Tumor lysate preparation. MC38, B16F10, and CT-2A cells were initially seeded and cultured until reaching 80-90% confluency. Subsequently, the cells were collected and lysed through five freeze-thaw cycles involving rapid freezing at -80°C and thawing at 37°C for 5 minutes each cycle. Following this, the tumor lysates underwent centrifugation to eliminate cell debris, and the resulting supernatant was collected. Protein concentrations in the tumor lysates were quantified using a bicinchoninic acid (BCA) assay. For FITC conjugation to tumor lysates, 1 mg / mL of tumor lysates were combined with 5 mg of fluorescein isothiocyanate (FITC) in a carbonate buffer (0.1 M Na2CO3, 0.1 M NaHCCb, pH 9.5) under stirring for 24 hours. Unconjugated FITC was removed through continuous dialysis for 3 days using a dialysis membrane (12,000-14,000 MW) (Spectrum Labs). The FITC-labeled tumor lysates were stored at -80°C until use.

[0078] Evaluation of the antigen capturing efficiency of NPs. The antigen capturing ability of different NPs was studied usingUIC0112WO PATENT the model antigen ovalbumin (AF647-OVA) and MC38 tumor lysate. The protein content in the tumor lysate was pre- quantified using a BCA assay. In brief, 500 pg of NPs were added to a solution containing different concentrations of AF647-OVA or tumor lysate and incubated at 37°C for 30 minutes. Following incubation, the mixture was centrifuged at 12,000 g, and the pellet containing NPs bound with proteins was collected. The unbound protein in the supernatant was quantified using a BCA assay (for tumor lysates) or a fluorescence-based method using plate reader (for AF647- OVA). The amount of protein bound to the NPs was determined by subtracting the unbound protein from the total added protein. The charge and size of NPs after protein binding were measured using dynamic light scattering (DLS).

[0079] The composition of proteins bound to NPs was evaluated through proteomics analysis. Briefly, 2 mg of NPs were washed three times with ultrapure water before use and then incubated with 100 pg of tumor lysates at 37°C for 30 minutes. The NPs were then washed with 1 mL phosphate-buffered saline (PBS) once and resuspended in 300 pL PBS. The NPs with bound proteins (1-10 pg) were resuspended in 45 pL of 8 M urea / 100 mM Tris buffer (pH8.5). Next, 5 pL of 50 mM tris(2-carboxyethyl)phosphine (TCEP) (Sigma-Aldrich) was added and incubated at 56°C for 1 hour. Then, 5.5 pL of 500 mM 2-chloroacetamide (Alfa Aesar) was added and incubated for 30 minutes in the dark at room temperature. The reaction was diluted with 3 volumes of 100 mM Tris buffer (pH8.5) to reduce the urea concentration to 2 M before trypsin (Sigma-Aldrich) was added to a final protease / protein ratio of 1:50 (w / w) and incubated overnight at 37°C, After that, the sample solution was heated up to 56°C for 5 minutes before being spun down at 18,000 g for 15 minutes to collect the supernatant with peptides. Formic acid was added to a final concentration of 1% and samples were analyzed by liquid chromatography-mass spectrometry (LC / MS). A total of 9 samples (3 types of NPs, 3UIC0112WO PATENT biological replicates for each NP type) were prepared and analyzed. For LC / MS run, up to 1 pg of peptides was loaded onto Evotips and cleaned up following the manufacturer's instructions (Evosep Biosystems, Denmark), Samples were injected into a Thermo Q Exactive HF quadrupole-Orbitrap MS equipped with a nanospray ESI source (Thermo Fisher Scientific) using an Evosep One instrument (Evosep Biosystems). The standard preset method for 60 samples per day (60 SPD) was used for the LC component of the run. The spray voltage was set at 1.9 kV. The mass spectrometer was operated in positive ionization and data-dependent acquisition mode, automatically switching between MSI and MS2 spectra. The MSI scan range was set to 200-2000 m / z with a resolution of 60,000 and an automatic gain control (AGC) target value of 3xl06. Up to 15 peptide precursors were selected for MS2 analysis with an isolation width of 2 m / z at the resolution of 30,000 and AGC target value of lxlO5. The maximum injection time for both MSI and MS2 was 100 ms. The normalized collision energy (NCE) was set at 30% for ion fragmentation by higher-energy collisional dissociation (HCD).

[0080] For data analysis, the RAW files were searched with MSfragger in FragPipe v20.0 against UniProt Mus muculus (mouse) reference database (accessed May 2023) with addition of more entries of frequently mutated proteins reported before (Aurisicchio et al. (2019) J. Exp. Clin. Cane. Res. 38:48; Salvatori et al. (2022) Npj Vaccines 7:15). Minimum peptide length was set to 7. The precursor and fragment mass tolerances were set at 20 ppm. The maximum missed cleavage was set to 2. Carbamidomethylation of cysteine was set as fixed modification and N-terminal acetylation and methionine oxidation were set as variable modification. The false discovery rate was set at 1% at peptide and protein levels. For label-free quantification, maxFLQ and match between runs (MBR) options were enabled. Other parameters were used as default.UIC0112WO PATENT

[0081] Cell viability induced by NP treatment. The viability of CD103+ cDCls, MC38 cells, and doxorubicin (DOX) "pretreated MC38 cells after co-incubation with NPs with different concentration was measured using a cell counting kit-8 (CCK-8, Boster Bio). CD103+ cDCls were seeded at a density of 1.5 *104cells / well, while MC38 were seeded at 5*103cells / well in clear 96-well tissue plates. For DOX-treated MC38 cells, DOX was added to MC38 cell culture with a final concentration of 1 pg / mL. After 24 hours, AC-NPs, NPNe9, and NPPEG(0-1000 pg / mL) were added to each well. The cells were incubated for another 24 hours followed by the CCK-8 assay according to the manufacture's protocol. The absorbance was determined at 450 nm using a plate reader. For each scenario of CD103+ cDCls, MC38, and DOX-pretreated MC38 cells, the control group consisted of cells without NP treatment, with their values normalized to 100%.

[0082] In vitro evaluation of tumor protein uptake by cDCls and their activation. To assess AC-NPs' efficiency in enhancing tumor protein uptake into cDCls, DiD- or DiO-labeled NPs were prepared for fluorescent tracking. CD103+ cDCls were seeded in a 12-well plate (0.5xl06per well) overnight and stained with Hoechst 33342. DiD-labeled NPs were then mixed with FITC-labeled tumor lysates, and the mixture was incubated for 30 minutes. Next, CD103+ DCs were exposed to the mixture at a concentration of 20 pg / mL NPs and 2 pg / mL FITC-labeled tumor lysate. The cells were further incubated with the mixture for 10 minutes or 4 hours. Following the incubation, CD103+ DCs were harvested, washed, and analyzed using flow cytometry (CytoFLEX, Beckman). For imaging purposes, samples were placed to glass-bottom dishes (Thermo Fisher) and imaged using a confocal microscope (ZEISS LSM710). To assess the activation of CD103+ cDCls, the expression of activation markers on CD103+ DCs was evaluated. CD103+ DCs were initially seeded in a 12-well plate (0.5xl06per well) and cultured overnight. Subsequently, 200 pg / mL NPs or 1 pg / mL lipopolysaccharide wereUIC0112WO PATENT added to the cells, along with 20 pg / mL tumor lysates, followed by further incubation for 24 hours. The cells were then harvested, stained with antibodies against MHCII, CD80, CD86, CDllc, CD103 and Zombie NIR (BioLegend), and analyzed by flow cytometry (CytoFLEX, Beckman). To evaluate the level of antigen presented on cDCl, a solution containing 100 pg of AC-NPs and 20 pg of ovalbumin was incubated for 30 minutes. The mixture was then added to cDCl cell culture (IxlO6per well). After a 4-hour incubation, free OVA and AC-NPs were washed out. The surface expression of OVA antigen was subsequently detected by staining with anti-mouse H-2Kb-OVA tetramer (PE-labeled) antibody coupled with flow cytometry analysis after an additional 24-hour culture period.

[0083] Whole cell uptake. To evaluate the internalization of tumor cells by CD103+ cDCls, MC38 cells underwent incubation in culture medium with or without 1 pg / mL of DOX overnight. Afterwards, they were stained with 10 pg / mL DiO for 20 minutes. Following three washes, 300 pg / mL AC-NP, NPSes, and NPPEGwere added to both DOX-treated and non-treated MC38 cells. The mixture was incubated for 30 minutes. CD103+ cDCl were stained with Hoechst 33342 (1:5000 dilution) for 20 minutes and washed three times. Then, the mixture of NPs and MC38 cells was added to CD103+ cDCls and incubated for 10 or 30 hours. The samples were washed three times with PBS and analyzed by Flow cytometry. The percentage of DiO-labeled MC38 cells captured / internalized by Hoechst 33342-labeled CD103+ cDCls was analyzed.

[0084] In vivo trafficking of ACT-DC and cell-level distribution of AC-NPs. The trafficking ability of ACT-DC to tDLNs was evaluated. MC38 cells (0.5*106) were subcutaneously injected into the right flank of C57BL / 6 mice. On day 10, a 30 pL intratumoral injection of a model tumor antigen AF647-OVA (50 pg) was administered. Fifteen minutes later, tumors were injected with 30 pL NPs or PBS, followed by an intratumoral injection of 3><106UICOU 2WO PATENTIVISense™ DiR-labeled CD103+ DCs another 15 minutes later. After 6 or 20 hours, tumors and tDLNs were collected. LagoX images were taken to assess the amount of OVA and injected CD103+ cDCls in the organs. The tumors and tDLNs were then processed into a single cell solution. The cells were stained for CD45, CDllc, CD103, CD86, MHCII, CDllb, F4 / 80, and Zombie NIR, and analyzed by flow cytometry (Aurora).

[0085] The cell-level distribution of AC-NPs in the free or ACT- DC form was also assessed. Briefly, DiD-labeled AC-NPs were prepared as previously described. MC38 cells (0.5><106) were subcutaneously injected into the right flank of C57BL / 6 mice. On day 10, an intratumoral injection of ACT-DC (containing 3*106IVISense™ DiR labeled CD103+ cDCls and 333 pg DiD-labeled AC-NPs) or free DiD-labeled AC-NPs were administered. For ACT-DC formulation injection, AC-NPs were first injected, followed by administration of CD103+ cDCls 15 minutes later. Tumors and tDLNs were dissociated 6 hours after cDCl injection and imaged using LagoX to track the distribution of AC-NPs in the organs. The tumors were then processed into a single cell solution, stained for CD45, CDllc, CD103, CDllb, F4 / 80, CD49b, CD3, CD4, CD8, and Zombie UV, and analyzed using flow cytometry to determined AC- NPs' distribution in different cells.

[0086] Immunostaining and imaging for tumor tissues. To image the resident DCs and ACT-DCs in tumor tissues, MC38 cells (5xl05) were implanted subcutaneously into the right flank of C57BL / 6 mice. ACT-DC in 60 pL PBS was intratumorally injected on day 10 after tumor inoculation. Six hours after injection, tumors were collected, embedded in 2% agarose gel (GeneMate), and sectioned to 400-pm thick slices using a vibratome (VT1200S, Leica). Slices were fixed by 2% paraformaldehyde solution for 15 minutes at room temperature and washed three times in PBS, For immunofluorescence staining, the tumor slices were incubated in a staining buffer (0,5 mL RPMI1640 cell culture media, 1% IgG-free bovine serumUIC0112WO PATENT albumin) containing 0.7 pL of DAPI (5 pg / mL), 5 pL of AF647- labeled anti-mouse CD103 antibody (0.5 mg / mL) and 5 pL of FITC- labeled anti-mouse CDllc antibody (0.5 mg / mL) for 18 hours at 4°C under gentle shaking. Stained tumor slices were washed in PBS three times and then cleared by immersion in 100% D~fructose solution (Sigma-Aldrich) for 30 minutes under gentle shaking. Non-treated control tumors were processed by the same method. The stained tumor slices were imaged by a confocal fluorescence microscope (Caliber ID, RS-G4) using 20x air and 40x oil objectives.

[0087] Preparation of ex-vivo pulsed DC vaccines. To prepare DC vaccines ex vivo loaded with tumor lysate, CD103+ cDCls were seeded at a concentration of 30xl06cells in 10 mL RPMI-1640 media supplemented with 10% FBS, 1% Penicillin-Streptomycin, 50 pM 2- mercaptoethanol, 200 ng / mL FTL3L, and 2 ng / mL GM-CSF. The cells were pulsed with 20 pg / mL PIC and tumor lysates that were collected from 60xl06MC38 or CT-2A cells (tumor cells to DCs ratio of 2) for 4 hours. Another DC vaccine ex vivo loaded with defined antigens were prepared using the same method except that cDCls were pulsed with gpl0025-33 (50 pg / mL) and TRP-2i8o-i88 (50 pg / mL) instead of tumor lysate. Cells were then washed three times and resuspended in PBS for subcutaneous injection. The viability of cells was >95%.

[0088] Therapeutic efficacy studies in the MC38 and B16F10 tumor models. ACT-DC's therapeutic efficacy was evaluated in the MC38 and B16F10 tumor models. For the MC38 models, MC38 cells (3*105for the small tumor model and 5xl05for the large established model) were subcutaneously injected into the right flank of female C57BL / 6 mice (6-8 weeks of age). Tumor volume was calculated using the formula: (length x width2) / 2, with the longest diameter considered as the length and the shortest as the width. When the average tumor volume reached ~35 mm3(for the small tumor model) or -100 mm3(for the large established model),UIC0112WO PATENT an intratumoral injection of 0.1 mg / kg doxorubicin was administered to induce antigen release. Next day, tumors were treated intratumorally with different therapies for the first dose, followed by one or two more doses 4-5 days apart. For the ex vivo MC38 tumor lysate pulsed DC vaccine group, DC vaccines were prepared according to the method described above and subcutaneously administered. For formulations containing anti- PD1 antibody, two doses of 100 pg antibody per dose were intraperitoneally injected on days 1 and 3 following each dose of ACT-DC or other control formulations. For formulations containing NPs plus DCs, 30 pL of NPs were first intratumorally injected, followed by intratumoral administration of DCs after 15 minutes. The respective formulations contained 3*106CD103+ DCs and / or NPs containing 40 pg PIC. Mice were euthanized when tumor size reached 20 mm in any dimension or presented significant ulceration (>3 mm in diameter). For the first rechallenge study, 5xl05MC38 cells were subcutaneously injected into the left flank of tumor-free mice on day 86. For the second rechallenge study, 5*105MC38 cells were injected to the right flank of mice survived from the first rechallenge on day 167. To analyze the immune cells in the blood, blood was collected from mice 15 days after the second rechallenge and lysed using ACK buffer to obtain peripheral blood mononuclear cells (PBMCs). The cells were stained with antibodies (CD45, CDllc, CD103, CD86, MHCII, CDllb, Grl, CD49b, CD3, CD4, CD8, IFN-y, CD62L, CD44, Adpgk tetramer, CD25, FoxP3, and Zombie NIR) and analyzed by flow cytometry (Aurora).

[0089] For the B16F10 model, B16F10-Luc cells (5*105) were injected into the right flank of female C57BL / 6 mice (6-8 weeks of age). The mice were treated following a similar schedule and dosing regimen as in the MC38 model. For the ex vivo DC vaccine loaded with gpl0025-33and TRP-2i8o-ias / the vaccine was prepared as described above and subcutaneously administered. In the B16F10UIC0112WO PATENT model, for the first rechallenge, 5xl05B16F10-Luc cells were subcutaneously injected to the left flank of tumor-free mice on day 79. For the second rechallenge, tumor-free mice survived from the first rechallenge received an intravenous injection of IxlO5B16F10-Luc cells on day 159. Blood collection and immunostaining analysis were performed 14-15 days after the first and second rechallenges to assess the immune cells in the blood. For the bilateral B16F10 tumor model, IxlO6B16F10-OVA cells were inoculated to the right flank of female C57BL / 6 mice (6-8 weeks of age) on day 0, and 5xl05B16F10 cells were inoculated to the left flank of the same mice on day 3. An intratumoral injection of 0.1 mg / kg doxorubicin was administered to the B16F10-OVA tumors on day 6 to induce antigen release. ACT-DC was intratumorally administered to the B16F10-OVA tumors on days 7 and 12. For treatments involving anti-PDl antibody, anti-PDl antibody (100 pg per dose) was intraperitoneally injected on days 8, 11, 13, and 15. Mice were euthanized on day 21 to collect lymph nodes for immune cell analysis.

[0090] To investigate the impact of doxorubicin pre-treatment and administration route on the therapeutic efficacy of ACT-DC, B16F10 cells (5x10s) were inoculated into the right flank of female C57BL / 6 mice (6-8 weeks of age) on day 0, On day 6, mice received one of three treatments: an intratumoral doxorubicin injection (0.1 mg / kg, 50 pL), an intravenous doxorubicin injection (3 mg / kg, 100 pL), or no doxorubicin treatment. ACT-DC was administered intratumorally on days 7 and 12. To evaluate the effects of T cell depletion and egress inhibition on ACT-DC efficacy in the B16F10 tumor model, mice were injected intratumorally with doxorubicin (0.1 mg / kg) on day 6, followed by two doses of ACT-DC on days 7 and 12. For CD4 or CD8 T cell depletion, anti-CD4 or anti-CD8 antibodies (200 pg per dose) were administered intraperitoneally on days 7, 10, 13, and 16. For TUIC0112WO PATENT cell egress inhibition, FTY720 (3 mg / kg) was administered intraperitoneally on days 7, 9, 11, 13, 15, 17, and 19.

[0091] Orthotopic mouse glioma model, cannula implantation, and treatments. Male / female C57BL / 6 mice (6-8 weeks of age) were intracranially implanted with cannulas (Plastics One), and CT-2A glioma cells at 5xl04cells per mouse were implanted following the procedures as described before (Zhang et al. (2023) Nat. Commun. 14:1610; Zhang et al. (2019) Proc. Natl. Acad. Sci. USA 116:23714-23723). All the mice were randomly assigned into different treatment groups. Brain-focused radiotherapy was given using a Gammacell 40 Exactor (Best Theratronics) at a 3 Gy daily dose for three consecutive days starting on day 6 after tumor implantation. Some groups of mice received anti-PDl antibody (100 pg per dose) treatment intraperitoneally on days 9, 11, 13, 15 post-tumor implantation. In some groups, mice also received conventional ex vivo tumor lysate pulsed DC vaccine (3 million cells per injection) through subcutaneous injection or ACT-DC (3 million cells per injection) intracranially through the implanted cannulas on days 8 and 12 post-tumor implantation. Supportive care of mice post-tumor implantation and treatments was provided in full compliance with the approved animal protocols. Long-term survivor (LTS) mice were euthanized 100 days after initial tumor implantation, and brains were collected for immunophenotypic analysis by flow cytometry. Non-tumor-bearing mice or CT-2A- bearing mice (21 days post-tumor inoculation) were used as controls.

[0092] Immune cell profiling in the tumor and peripheral immune organs. To assess the capability of ACT-DC to generate a systemic immune response and modulate tumor microenvironment, the large- established MC38 tumors were established and treated according to a similar schedule and dosing regimen from the efficacy studies. On day 17, tumor, spleen, and tDLNs of mice were collected, weighed, and processed to single cells. The totalUIC0112WO PATENT number of cells obtained from each whole tumor / spleen / tDLN were counted and recorded. The cells were washed with PBS, and 1.5 million cells were stained with antibodies with titrated concentrations. Antibodies were obtained from BioLegend including CD45, CDllc, CD103, CD86, MHCII, CDllb, F4 / 80, CD206, Grl, CD49b, CD3, CD4, CDS, IFN-y, CD62L, CD44, CD25, Foxp3, Zombie NIR, Zombie UV, and aPDl. PE-conjugated Adpgk tetramer and BV421-conjuagetd Rpll8 tetramer were obtained from the Tetramer Core of the National Institutes of Health (NIH). Stained samples were subsequently measured by flow cytometry (Aurora). The total number of a specific cell population (e.g., effector memory CD8 T cell) in the whole spleen or tDLN was calculated using the following method. First, the percentage of the specific cell population within live single cells (denoted as A) was determined by analyzing flow cytometry data using FlowJo. The total number of the specific cell population per whole organ was then calculated as the product of A and the total number of live cells in that organ. The total number of a specific cell population in 100 mg tumor was calculated using a similar method.

[0093] Statistical analysis. All experiments were repeated at least two times. All statistical analyses were carried out using Graphpad Prism (version 10). All flow cytometry data were analyzed using Flowjo (version 10). Confocal imaging data were analyzed using Zen Software (Version 3.9) and Image J. All data are presented as mean ± SEM. Two-sided unpaired Student's t test, one-way ANOVA with Dunnett analysis, two-way ANOVA with Dunnett analysis, or Mann-Whitney test were used to determine significance. For the analysis of Kaplan-Meier survival curves, two-sided Mantel-Cox test was used. All statistical analyses were performed on Graphpad Prism (version 10).UIC0112WO PATENTExample 2: Engineering AC-NPs for efficient capture of tumor antigens

[0094] A crucial element of the Antigen Capturing nanoparticle Transformed Dendritic Cell therapy (ACT-DC) approach described herein is the use of antigen-capturing nanoparticles (AC-NPs), designed to capture tumor antigens in situ, transport them to adoptively transferred cDCls, and activate these DCs. Given that many tumor antigens possess negative charges and / or hydrophobic sequences, it was postulated that a hydrophobic nanoparticle (NP) with a positively charged surface would efficiently capture such antigens through both electrostatic and hydrophobic interactions. To implement this concept, a polymer-based composite AC-NP was synthesized using acid-ended poly(lactic-co-glycolic) acid (PLGA) and polyetherimide (PEI). In this design, PLGA imparts hydrophobicity to the NP, while PEI contributes a positive surface charge. As controls, two additional NPs, NPNes and NPPEG, were created. NPHe9, derived from acid-ended PLGA, features a negatively charged hydrophobic surface, while NPPEG, constructed from PEGylated PLGA, has a hydrophilic surface. To effectively activate cDCls, polyinosinic:polycytidylic acid (PIC), a tolllike receptor 3 (TLR3) agonist, was encapsulated in all three types of NPs.

[0095] AC-NPs exhibited a uniform spherical shape with an average diameter of 160 nm and a zeta potential of +41.2 mV. The energy- dispersive X-ray spectroscopy (EDS) analysis revealed that blank AC-NPs showed a characteristic nitrogen peak derived from PEI in its elemental spectrum, further indicating the successful incorporation of PEI into AC-NPs. NPNea and NPPEGshared a similar size (approximately 153 nm) with AC-NPs but carried a negative charge (approximately -30 mV). Encapsulation efficiency and loading efficiency of PIC in nanoparticles is provided in Table 2 . PIC were encapsulated into AC-NPs with a loading capacity of 128 pg / mg.UIC0112WO PATENTTABLE 2

[0096] The release of PIC from AC-NPs followed a sustained pattern, with a cumulative 45.5% of drug released over 72 hours. Subsequently, AC-NPs' ability to capture tumor proteins from tumor lysates was assessed. Under all tested conditions, AC-NPs indeed captured a significantly higher amount of tumor proteins compared to NPHe9 and NPPEG(FIG. 1). This is further evidenced by AC-NPs' substantial increase in NP size and shift of surface charge from positive to neutral upon incubation with increasing amounts of tumor lysate (0 to 500 pg). Similar results were observed using ovalbumin (OVA) as a model tumor antigen, reaffirming AC-NPs' robust protein-capturing capability. To further determine the composition of the tumor proteins captured by different NPs, proteomic analysis was conducted. While NPNea and NPPEGonly captured around 640 and 500 proteins, respectively, AC-NPs captured a broader range of proteins (approximately 800 proteins) as compared to NPNe9 and NPPEG, which captured approximately 650 and 500 proteins. Notably, compared to NPNe^ and NPPEG, AC-NPs captured several frequently mutated proteins in MC38 tumor cells, including Hnrnpf, Aatf, Copb2, and Kpna6. These proteins have the potential to generate neoantigens in vivo, contributing to the development of a tumor-specific immune response. Additionally, AC-NPs also captured a higher number and / or quantity of damage-associated patterns (DAMPs), e.g., h4cl, H3c2, Hsp90abl, Hl-2, Hl-4, Hl-5, Hl-0, Hl-3, Hsp90aal, Calr, H2ax, and Hspa4, compared to NPNea and NPPEG. As endogenous danger signals, the captured DAMPs have the potential to bind to pattern recognition receptors and activate cDCls. Overall, these data demonstrate that the engineered AC-NPs had a robust capability to concentrate and capture tumor-derived proteins. ItUIC0112WO PATENT is important to note that AC-NPs do not selectively capture only frequently mutated proteins or DAMPs; instead, they capture a broad spectrum of tumor proteins, which include some frequently mutated proteins and DAMPs.Example 3:AC-NPs boost the delivery of antigen to, and activation of, CD103+ cDCls

[0097] CD103+ cDCls, a distinctive subset of DCs, are notable for their robust migratory capacity to lymph nodes, facilitating effective antigen presentation. Previous research has established the pivotal role of cDCl infiltration in fostering a potent antitumor immune response (Bottcher & Sousa (2018) Trends Cancer 4:784-792; Salmon et al. (2016) Immunity 44:924-938). However, their presence within tumors is typically limited, and their activity is hindered by the immunosuppressive tumor microenvironment (Del Prete et al. (2023) Cell Mol. Immunol. 20:432-447). The ACT-DC approach combines immunoactive AC-NPs with the adoptive transfer of CD103+ cDCls. This cascade antigen relay strategy modulates the tumor microenvironment, initiating a sequential process that amplifies antigen presentation and enhances cDCl activity, ultimately enabling potent in situ immunization.

[0098] A robust method for generating CD103+ cDCls from bone marrow was adopted, which involves the use of granulocyte macrophage colony-stimulating factor (GM-CSF) and FMS-like tyrosine kinase 3 ligand (FLT3L)(Mayer et al. (2014) Blood 124:3081-3091). cDCls obtained using this method exhibited the characteristic dendritic morphology upon activation by PIC. They also displayed higher expression levels of CD103 and Clec9A, distinctive surface markers of cDCl(Mayer et al. (2014) Blood 124:3081-3091), than conventional bone marrow derived DCs (BMDCs). Specifically, 92.6% of the obtained cells were CD103- positive, indicating that CD103+ cDCls constitute the majority,UIC0112WO PATENT while other DCs, such as cDC2s and monocyte-derived DCs, represented only a small fraction.

[0099] AC-NPs at a concentration up to 1 mg / mL, did not show obvious toxicity to the CD103+ cDCls. Thus, the efficacy of AC- NPs in enhancing the delivery of tumor antigens to CD103+ cDCls was assessed. Flow cytometry results revealed that AC-NPs significantly increased the uptake of tumor proteins by cDCls at both 30 minutes and 4 hours post-incubation, while the control NPs (NPMe9 and NPPEG) showed limited efficacy (FIG. 2). Further confocal fluorescence (CLSM) imaging revealed substantial colocalization of tumor lysate proteins with AC-NPs inside cDCls after a 4-hour AC-NP treatment, suggesting that the enhanced protein uptake is likely attributed to AC-NPs1ability to capture tumor proteins. Increased accumulation of tumor antigens within cDCls is crucial for subsequent antigenic peptide presentation on DC surfaces, a key step for DC-T cell crosstalk and the activation of antigen-specific T cells. Indeed, using ovalbumin (OVA) as a model tumor antigen, it was observed that AC-NPs resulted in a significantly higher level of H-2Kb-OVA peptide presented on cDCl surface compared to free OVA (with or without free PIC) or free OVA plus control NPs (FIG. 3). Additionally, AC-NPs caused increased cell death in doxorubicin-treated MC38 tumor cells, likely due to their vulnerability to NP-binding, which could induce cell membrane destabilization or rupture. While healthy MC38 cells appeared resistant to these effects, doxorubicin-treated cells were more sensitive. Notably, NPPEGcaused less cell death than AC-NPs or NPHe^, likely because PEGylation reduces NP interaction and binding with cells, thereby minimizing binding-induced membrane destabilization. The enhanced cell death induced by AC-NPs may increase antigen release and facilitate the internalization of MC38 cells to cocultured cDCls. Moreover, when co-incubated with a mixture ofUIC0112WO PATENT cells which were dissociated from an MC38 tumor, AC-NPs were more efficiently taken up by cDCls than by cDC2s.

[0100] The ability of AC-NPs to activate cDCls was subsequently assessed. AC-NPs induced approximately a 6-fold increase in the percentage of CD80+CD86+ double-positive activated cDCls, along with significantly higher expression of these individual activation markers, compared to NPNes and NPPEG. The robust efficiency of AC-NPs in activating DCs may be partly attributed to the stronger ability to capture DAMPS. Overall, the data demonstrated that, owing to its efficient capture of tumor proteins, AC-NPs enhanced tumor antigen delivery to and presentation on cDCls while promoting efficient cDCl activation.Example 4: ACT-DCs migrate to tumor draining lymph nodes (tDLNs) and activate cDCls

[0101] While CD103+ cDCls play a crucial role in initiating and enhancing antitumor immune response, their presence within tumors has been shown to be notably limited, partly due to the immunosuppressive characteristics of the tumor microenvironment (Del Prete et al. (2023) Cell Mol. Immunol. 20:432-447). Therefore, the abundance of endogenous CD103+ cDCls within the tumor microenvironment was measured in the MC38 tumor model. As evident from confocal fluorescence microscopic images of MC38 tumors at the margin and center regions, CDllc+ DCs were distributed both in the marginal and central regions of the tumor, whereas CD103+ cDCls were primarily localized at the tumor periphery. Quantitative analysis revealed that endogenous CD103+ cDCls only constituted <1.5% of total CD45+ cells within the MC38 tumor. These findings provided a rationale for incorporating CD103+ cDCls into the ACT-DC approach. Indeed, ACT-DC resulted in a 5.8-fold increase in the total number of CD103+ cDCls within the tumor 6 hours after intratumoral administration. Additionally, ACT-DC also induced a shift in the spatialUIC0112WO PATENT distribution of cDCls within the tumor. Unlike untreated tumors where cDCls predominantly accumulated at the tumor margin, in ACT-DC treated tumors, cDCls showed a more broad distribution throughout both the marginal and central areas. This altered spatial distribution, coupled with the increased abundance of cDCls facilitated by ACT-DC, may reshape the tumor microenvironment into a "hot" state, leveraging native tumor antigens with the assistance of AC-NPs. Ultimately, this approach enables in situ immunization, triggering a robust antitumor immune response. Notably, in all studies involving the injection of ACT-DC, AC-NPs were administered first, followed by cDCls, with a 15-minute interval between the two injections. The ACT-DC approach hinges on the incorporation of AC-NPs, facilitating their hitchhiking onto adoptively transferred CD103+ cDCls. This process triggers the subsequent transport and presentation of in situ captured tumor antigens to the tDLNs. To demonstrate this mechanism, the cellular-level distribution of AC-NPs was determined upon intratumoral administration, either in their free form or as part of an ACT-DC formulation. Six hours after intratumoral injection, AC-NPs in the free form were mainly distributed in tumor cells (non-CD45+ cells) and tumor-resident myeloid cells (CDllb+CDllc- cells)(FIG. 4). In contrast, when integrated into the ACT-DC approach, most of the AC-NPs were associated with the adoptively transferred cDCls, though they were also present in tumor cells and tumor-resident myeloid cells at a lower frequency. Moreover, in a separate study, where a model tumor antigen (AF488-OVA), AC-NPs, and cDCls were sequentially administered intratumorally, a markedly higher uptake of AF488-OVA by the adoptively injected cDCls was observed compared to other resident cells in the tumor. These findings indicate that AC-NPs efficiently target the adoptively injected cDCls in the ACT-DC approach, crucial for the cascade antigen reUIC0112WO PATENT relay mechanism facilitating in situ immunization enabled by ACT- DC.

[0102] Subsequently, the efficiency of ACT-DC in trafficking and delivering tumor antigens to the tDLNs was evaluated. The comparison between 20 hours and 6 hours after intratumoral administration of ACT-DC revealed an increase in the quantity of injected cDCls in the tDLNs and a decrease in the tumor (FIG. 5). This result indicates that ACT-DC efficiently traffics from the tumor to the tDLNs. Notably, a substantial number of injected cDCls remained in the tumor 20 hours post-administration. In addition, ACT-DC demonstrated a trend toward higher accumulation in tDLNs compared to BMDCs plus AC-NPs, indicating the stronger migratory capability of CD103+ cDCls.

[0103] The intratumoral retention of doxorubicin (a chemotherapeutic drug to enhance antigens release), AC-NPs, and cDCl was also measured following serial intratumor administration. Upon injection, doxorubicin rapidly diffused from the injection site, and by 24 hours post-injection (just prior to ACT-DC administration), its signal was almost undetectable. In contrast, cDCl and AC-NPs exhibited prolonged retention at the injection site, with their signals gradually declining over >7 days and becoming undetectable by day 9. Additionally, further imaging of tDLNs revealed that the intratumorally injected cDCls remained detectable in tDLNs even 9 days after ACT-DC administration.

[0104] The ability of AC-NPs to enhance antigen delivery to the adoptively transferred cDCls and their activation status in the tDLNs was also determined. In this study, AF647-labeled OVA was intratumorally injected as a model tumor antigen to fluorescently track its delivery to cDCls and lymph nodes. At 15 and 30 minutes post tumor antigen injection, AC-NPs and DCls were respectively intratumorally injected. Notably, AF647-OVA is not an endogenous tumor antigen; however, its use as a model antigen is well-UIC0112WO PATENT established and it provided a method for monitoring antigen delivery. ACT-DC led to a 2.1-3.3-fold higher uptake of OVA by the adoptively transferred cDCls compared to cDCl alone or cDCls combined with control NPs (NPHegand NPPEG). Moreover, ACT-DC resulted in a 2.3- to 4.7-fold increase in the expression level of CD86 on the injected cDCls, compared to cDCls alone or with control NPs (NPNe<!and NPPEG). Furthermore, data from a separate study showed that ACT-DC led to a 2.7- to 3.8-fold higher number of OVA and NP double-positive injected cDCls in the tDLNs, compared to cDCls with control NPs (NP,,e'J and NPPEG). Additionally, CLSM imaging confirmed the co-localization of OVA and AC-NPs within the injected cDCls in the tDLNs. Collectively, these data demonstrate that ACT-DC captures AC-NPs within the tumor, efficiently traffics to tDLNs, and results in the presence of activated, antigen-carrying cDCls in the tDLNs.Example 5: ACT-DC eradicates small tumors and inhibits established tumors in a MC38 model

[0105] The therapeutic efficacy of ACT-DC in an early-stage subcutaneous MC38 tumor model was assessed. Two doses of ACT-DC (10 and 14 days post-MC38 (0.3M) tumor inoculation) resulted in complete regression of primary tumors (FIG. 6), leading to survival to day 80 without any detectable tumor recurrence (FIG. 7). Treatment with CD103+ cDCl alone did not induce tumor size reduction or prolonged survival. Administration of AC-NP alone led to partial tumor regression in only 50% of the mice.

[0106] The therapeutic efficacy of ACT-DC in controlling larger tumors in the subcutaneous MC38 tumor model was also assessed, with an average tumor volume of approximately 100 mm3at the initiation of ACT-DC treatment. In this experiment, MC38 cells (0.5 M) were injected, followed by doxorubicin at day 8 and three doses of ACT-DC at days 9, 13 and 18. Compared to cDCl+NPNe9 and cDCl+NPPEG, ACT-DC showed significantly better efficacy inUIC0112WO PATENT inhibiting tumor growth and extending animal survival (FIG. 8 and FIG. 9). On day 25 post-primary tumor inoculation, ACT-DC exhibited a 6.9-fold and 9,1-fold greater efficacy in reducing tumor size compared to cDCl+NPt,e9 and cDCl+NPPEG, respectively. The therapeutic efficacy of ACT-DC was also compared to two clinically approved or investigated immunotherapies—immune checkpoint blockade (anti-PDl antibody, aPDl) and ex vivo pulsed / activated DC vaccine. ACT-DC displayed better efficacy in inhibiting tumor growth and extending survival than the conventional DC vaccine pulsed with tumor lysate and activated with PIC ex vivo. ACT-DC also demonstrated better therapeutic efficacy than aPDl (FIG. 8 and FIG. 9). Moreover, the combination of ACT-DC and aPDl further enhanced ACT-DC's efficacy (FIG. 8 and FIG. 9). Notably, 75% of mice treated with ACT-DC plus aPDl achieved complete tumor eradication and survived without detectable tumor recurrence on day 80 post-primary tumor inoculation, while the complete remission rate in the ACT-DC group was 33%. This synergy between ACT-DC and aPDl likely stems from their complementary mechanisms of action; ACT-DC enhances the induction and infiltration of cytotoxic CD8 T cells, while the aPDl improves the activity of infiltrated CDS T cells through immune checkpoint blockade. The similar average tumor volume curves observed between the ACT-DC and ACT-DC+aPDl groups during the first 32 days (FIG. 8) were attributed to two non-responder mice in the ACT-DC+aPDl group that developed large tumors. Additionally, more mice (15) were used in the ACT-DC group than the other treatment groups (7-8) to ensure an adequate number of surviving mice for subsequent re-challenge studies.

[0107] Subsequently, the surviving tumor-free mice were rechallenged subcutaneously on day 86 to evaluate the immune memory generated by ACT-DC. ACT-DC, either alone or in combination with aPDl, demonstrated significantly superior efficacy in inhibiting the growth of the rechallenged tumorsUIC0112WO PATENT compared to age-matched naive mice (FIG. 10 and FIG. 11). Notably, 100% of the mice treated with ACT-DC plus aPDl, which had survived the primary tumors, completely rejected the rechallenged tumors, and remained tumor-free for 165 days. Mice that had survived the first rechallenge were subsequently subjected to a second subcutaneous rechallenge at day 167 post-primary tumor inoculation to further evaluate the long-term immune memory induced by ACT-DC. All mice treated with ACT-DC or ACT-DC plus aPDl, which had previously thwarted the first rechallenge, exhibited complete rejection of the second tumor rechallenge. The immune cell profiles in the blood of these animals was measured 15 days after the second rechallenge. The ACT-DC group was not included in this analysis because only two mice survived, which was not sufficient for statistical analysis. In comparison to age-matched naive mice, mice treated with ACT-DC plus aPDl showed elevated levels of antigen-specific CDS T cells (approximately 0.5% vs, 1.0% of CD45+ cells; P<0.0001), memory CD8 T cells (approximately 5% vs. 10% of CD8 T cells (P=0.0024) for TEH and approximately 20% vs. 30% of CD8 T cells (P=0.0007) for TCM), antigen-specific memory CD8 T cells, memory CD4 T cells, along with a reduced number of immunosuppressive cells, including regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs). ACT-DC plus aPDl also increased the CD8 / CD4 T cell ratio in the blood. These data provide additional mechanistic support for the enduring immune memory effect induced by ACT-DC. In a separate study, the therapeutic efficacy of ACT-DC was compared to BMDC combined with AC-NPs. ACT-DC, which includes CD103+ cDCls and AC-NPs, demonstrated significantly greater efficacy in eradicating MC38 tumors. This enhanced performance was likely due to the superior migratory capacity of CD103+ cDCls toward tDLNs and their stronger antigen-presenting capabilities.UIC0112WO PATENTExample 6:ACT-DC induces a systemic immune response and reshapes tumor microenvironment

[0108] ACT-DC is designed to transport captured tumor antigens in situ to tDLNs and in turn, initiate a cascading systemic antitumor immune response. To evaluate this mechanism, mice were inoculated with MC38 cells, treated with doxorubicin at day 9, injected with two doses of ACT-DC (days 10 and 14 post-tumor cell injection, and immune cell profiles in the tDLNs of the mice were measured at day 17 post-tumor cell injection. As shown in FIGS. 12-14, compared to PBS, ACT-DC resulted in a significant increase in the number of innate immune cells in tDLNs including macrophages, cDCls, and cDC2s. A 5.7-fold higher number of cDCls was detected in the tDLNs of mice treated with ACT-DC than in those treated with PBS. In addition, the combination of ACT-DC and aPDl further elevated the number of all these tested innate immune cells in the tDLNs. Additionally, compared to PBS treatment, ACT-DC, either alone or in combination with aPDl, increased the expression of DC activation marker CD86 on cDCls in the tDLN by 1.8- to 1.9-fold. ACT-DC also led to an increase in CD86 expression on cDC2s compared to PBS, although this difference was not statistically significant. Furthermore, ACT- DC also resulted in an enhanced adaptive immune response, as evidenced by the increased number of T cells (FIG. 15), antigenspecific CD8 T cells (FIG. 16), IFN-y+ CDS T cells and Thl cells (FIG. 17), Granzyme B+ and perforin! CD8 T cells, TCF-1+ CDS T cells, and proliferating Ki67+ CD8 T cells. A 3.4-4.1-fold higher number of antigen-specific CDS T cells against two different epitopes (Adpgk and Rpll8) were observed in the ACT-DC group compared to the PBS group (FIG. 16). Notably, AC-NP alone, cDCl alone, and cDCl plus free PIC also led to increased numbers of innate and adaptive immune cells in the tDLNs, however, their efficacy was not as potent as ACT-DC (FIGS. 12-17), again highlighting the importance of both the AC-NP and cDCl componentsUIC0112WO PATENT in the ACT-DC approach. Moreover, the capability of ACT-DC to induce immune memory, which is crucial for preventing tumor relapse, was also evaluated. ACT-DC, either alone or in combination with aPDl, significantly elevated the number of both central memory and effector memory CD8 T cells in the tDLNs compared to PBS or other control formulations (FIG. 18). ACT-DC also resulted in more central memory CD8 T cells in the spleen, while its combination with aPDl further increased the frequency of both central memory and effector memory CD8 T cells in the spleen. Overall, these tDLN and spleen immune cell profiling data indicate that ACT-DC induced a potent systemic anti-tumor response with robust memory.

[0109] The immune cell profiles within tumors were analyzed to evaluate ACT-DC's capability to modulate the tumor immune microenvironment. ACT-DC significantly increased the infiltration of CD4 and CD8 T cells by approximately 5-fold, including Thl cells, effector CD8 T cells (IFN-y+ CD8 T cells, Granzyme B+ CD8 T cells, and perforin+ CD8 T cells), TCF-1+ CDS T cells, proliferating Ki67+ CD8 T cells, and antigen-specific CD8 T cells against two different epitopes (Adpgk and Rpll8), compared to the PBS treatment. Specifically, in comparison to PBS and other control formulations, ACT-DC led to a 1.8-4.2-fold increase in effector CD8 T cells and a 5.3-12.4-fold increase in Adpgk tetramer-positive CDS T cells. ACT-DC also changed the profiles of DCs within the tumor microenvironment. ACT-DC significantly increased the number of cDCls while decreasing the number of cDC2s in the tumor compared to PBS (FIG. 19). cDC2s were the dominant DC subtype within the tumors of PBS-treated mice, however, ACT-DC treatment shifted this predominance toward cDCls. ACT-DC's effect on the cDCl / cDC2 ratio may not be merely due to injecting cDCls directly into the tumor, as the treatments with cDCls alone or with cDCls plus free PIC did not significantly alter this ratio. Moreover, ACT-DC also significantly changed theUIC0112WO PATENT frequency of immunosuppressive cells including Tregs and macrophages within the tumor. ACT-DC led to a 36.5-72.7% reduction in Tregs and a 58.5-63.1% reduction in macrophages compared to PBS and other control formulations. Furthermore, data from a separate study indicated that ACT did not significantly increase the expression of T cell exhaustion markers (LAG-3, TIM- 3, and PD-1) on intratumoral CD4 T cells. However, ACT-DC led to a significant increase in TIM-3 expression on intratumoral CD8 T cells, without affecting LAG-3 or PD-1 levels. Combining ACT-DC with TIM-3 blockade provides a promising strategy to further enhance the therapeutic efficacy of ACT-DC. Overall, these data show that ACT-DC reshapes the innate and adaptive immune cell profiles within the tumor microenvironment, transforming it into a more "immune-hot" state and facilitating tumor elimination.Example 7: ACT-DC eliminates primary tumors and rejects tumor rechallenge in other tumor models

[0110] To evaluate the broad applicability of ACT-DC, its therapeutic efficacy was examined in two additional tumor models with lower immunogenicity, specifically the B16F10 melanoma model and the CT-2A glioma model. In the B16F10 model, mice were initially inoculated with a primary tumor, followed by a dose of doxorubicin at day 6 and three doses of ACT-DC at days 7, 11 and 16 post-tumor inoculation. ACT-DC demonstrated significantly better efficacy in tumor eradication compared to AC-NPs, cDCl, and the combination of cDCl with free PIC. Notably, 80% of mice treated with ACT-DC survived without detectable tumors on day 78 post-primary tumor inoculation. The combination of ACT-DC and aPDl further enhanced ACT-DC s efficacy, resulting in 100% tumor- free survival on day 78 post-primary tumor inoculation. Surviving tumor-free mice were subcutaneously rechallenged on day 79. ACT- DC and ACT-DC plus aPDl led to tumor rejection in 75% and 80% of the rechallenged mice, respectively. The blood immune cellUIC0112WO PATENT profiles 14 days after the first rechallenge revealed that ACT- DC, either alone or in combination with aPDl, significantly increased effector memory CD4 T cells (2.5-3.4-fold enhancement) and effector memory CD8 T cells (1.6-2.1-fold enhancement) compared to naive mice. Moreover, ACT-DC elevated the CD8 / CD4 T cell ratio and CD8 / Treg ratio while reducing the number of MDSCs in the blood. A further intravenous rechallenge of the mice that survived the first rechallenge was carried out at day 159 postprimary tumor inoculation to assess ACT-DC's ability to induce long-term immune memory. Despite all age-matched naive mice developing lung metastasis, mice treated with ACT-DC or ACT-DC plus aPDl continued to survive without detectable tumors for over 260 days. Blood immune cell profiling further revealed an increased presence of effector and central memory CD4 and CD8 T cells and increased ratios of CD8 / CD4 T cells and CD8 / Tregs in ACT-DC and ACT-DC plus aPDl treated mice, supporting ACT-DC's efficacy in rejecting rechallenged tumors. In a separate study, ACT-DC was directly compared with a conventional ex vivo DC vaccine loaded with two defined antigens (gpl0025-33 and TRP-2ieo- 188)• ACT-DC demonstrated significantly better therapeutic efficacy, further indicating the advantages of the in situ immunization approach enabled by ACT-DC.100111]To further assess the systemic and multivalent immune response triggered by ACT-DC, a bilateral tumor model was used. In this model, B16F10-OVA cells and B16F10 cells were inoculated into the right and left flanks, respectively, with only the B16F10-OVA tumor receiving ACT-DC treatment. Specifically, B16F10-OVA cells were inoculated into the right flank and, 3 days later, B16F10 cells were inoculated into the left flank, followed by the administration of doxorubicin at day 6 and two doses of ACT-DC to the primary tumor at days 7 and 12 post-primary tumor inoculation. Remarkably, ACT-DC, both alone and in combination with aPDl, significantly inhibited not only the treated primaryUIC0112WO PATENTB16F10-OVA tumors but also the untreated distant B16F10 tumors, compared to PBS or aPDl treatments (FIG. 20 and FIG. 21). On day 21, the distant tumors became undetectable in 42.8% of mice in the ACT-DC group and in 71.4% of mice treated with ACT-DC plus aPDl. This inhibition of distant B16F10 tumors shows that ACT-DC induced a robust systemic immune response targeting B16F10 tumor antigens. Additionally, significantly higher numbers of both OVA- specific and TRP-2-specific CD8+ T cells (TRP-2 being a B16F10 neoantigen) were observed in the draining lymph nodes of the primary B16F10-OVA tumor, supporting the induction of a multivalent antigen-specific immune response.

[0112] To investigate the roles of CD4 and CDS T cells, as well as their egress from lymph nodes, on ACT-DC's therapeutic efficacy, mice were treated with ACT-DC followed by the administration of anti-CD4 antibody, anti-CD8 antibody, or the T cell egress inhibitor FTY720 (an S1PR inhibitor). Specifically, mice were inoculated with B16F10 cells, administered a dose of doxorubicin at day 6 followed by a dose of ACT-DC, anti-CD4 or anti-CD8 antibody (i.p.), and FTY720 (i.p.) at day 7 post-primary tumor inoculation. At days 9, 11, 13, 15, 17, and 19 post-primary tumor inoculation additional doses of FTY720 (i.p.) were administered; at days 10, 13, and 16 post-primary tumor inoculation additional doses of anti-CD4 or anti-CD8 antibody (i.p.) were administered; and at day 11 post-primary tumor inoculation a second dose of ACT-DC was administered. Depletion of either CD4 or CDS T cells significantly reduced the efficacy of ACT-DC, indicating the essential role of both T cell subsets in the ACT-DC approach. Notably, CD8 T cell depletion resulted in a more dramatic reduction in therapeutic efficacy compared to CD4 T cell depletion, indicating a potentially more critical function of CD8 T cells in ACT-DC's effectiveness. FTY720 treatment also significantly impaired ACT-DC's efficacy, indicating that T cell trafficking and egress from lymph nodesUIC0112WO PATENT are essential for ACT-DC's therapeutic effectiveness. To evaluate the contribution of endogenous cDCls to ACT-DC's efficacy, therapeutic studies were conducted inmice, which lack endogenous cDCls. While ACT-DC treatment in J3atf3~ / ~ mice significantly delayed tumor growth and improved survival rates, its therapeutic efficacy was markedly reduced compared to that in the wide-type mice. These data indicate that in addition to the adoptively transferred cDCls, endogenous cDCls are also critical to the success of the ACT-DC approach.

[0113] Notably, in both the B16F10 and MC38 models, tumors were pre-treated with intratumoral doxorubicin before ACT-DC therapy to promote tumor antigen release. Accordingly, a comparative study was carried out to assess the impact of doxorubicin pretreatment and its administration route on ACT-DC's therapeutic efficacy. Even without doxorubicin pre-treatment, ACT-DC significantly delayed tumor growth and achieved tumor-free survival in 42.9% of treated mice, although its efficacy was less potent than in doxorubicin pre-treated mice. Moreover, the route of doxorubicin administration did not significantly influence ACT-DC's efficacy. Pre-treatment with either intravenous or intratumoral doxorubicin before ACT-DC therapy resulted in comparable tumor growth inhibition and an 85.7% tumor-free survival rate.

[0114] The therapeutic efficacy of ACT-DC was also demonstrated in an orthotopic CT-2A glioma model. Glioma is one of the deadliest tumors, known for its immunologically cold microenvironment, characterized by limited lymphocyte infiltration, resistance to both conventional and immune-based therapies, and high recurrence rate. Radiation therapy (RT) is a standard-of-care treatment for glioma and is known to trigger the release of tumor antigens. In this model, animals were inoculated with CT-2A tumor cells, followed by three doses of RT (3 Gy) at days 6, 7, and 8 post-tumor cell inoculation. At days 8 and 12UIC0112WO PATENT post-tumor cell inoculation, the animals received doses of ACT- DC and at days 9, 11, 13, and 15 post-tumor cell inoculation animals received treatment with aPDl. The results of the analysis herein indicated that RT alone or its combination with aPDl did not improve animal survival, confirming the therapy resistance feature of this model. Encouragingly, although ACT-DC did not greatly improve the RT effects as compared to RT alone, a combination of RT, ACT-DC, and aPDl led to tumor regression in 50% of the treated mice (FIG. 22). Notably, ACT-DC significantly outperformed the conventional ex vivo tumor lysate-pulsed DC vaccine in improving glioma response to RT and aPDl therapy. The immune cell composition in the brains of long-term survivor (LTS) mice, defined as mice surviving 100 days post-tumor inoculation, was evaluated. Compared to mice with existing primary CT-2A tumors (the "Tumor" group), LTS mice exhibited a significantly higher ratio of tumor-infiltrating lymphocytes (TILs) to tumor- associated myeloid cells (TAMCs) in the brain (ratio of 1:1 vs. approximately 3:1; P<0.0001). Additionally, LTS mice had 1.7-fold and 4.4-fold higher abundance of CD8 and CD4 T cells in their brain, respectively, compared to the "Tumor" mice. Further analysis of CD4 T cell populations indicated that over 95% of CD4 T cells in LTS brains were CD4 T helper cells, and there was an 86,2% reduction in Tregs as compared to "Tumor" mice. It is also worth noting that, compared to healthy control mice without tumor implantation (the "non-tumor" group), LTS mice, which were also tumor-free, demonstrated a significantly higher abundance of lymphocytes, both CD8 and CD4 T cells, in the brain, indicting an immune surveillance or memory induced by ACT-DC therapy. These immune cell profiling results support the robust therapeutic efficacy of ACT-DC in the CT-2A model.

[0115] The therapeutic efficacy of ACT-DC in the 4T1 triple negative breast cancer model, known for its immunologically cold nature characterized by limited immune cell infiltration, wasUIC0112WO PATENT also evaluated. This analysis indicated that the combination of ACT-DC with aPDl not only significantly delayed tumor growth but also prolonged animal survival, with an approximately 25% survival rate at 200 days post primary tumor inoculation. Importantly, this combination exhibited better efficacy compared to aPDl combined with AC-NP, cDCl, or cDCl plus free PIC. These data collectively demonstrated that ACT-DC is effective in treating solid tumors, inducing immune memory, and displaying applicability across multiple tumor models.

Claims

UIC0112WO PATENTWhat is claimed is:

1. A method of treating cancer or preventing or reducing the risk of reoccurrence of a cancer in a subject comprising administering to the subject an effective amount of(i) an antigen capturing nanoparticle, the antigen capturing nanoparticle comprising a positively charged and hydrophobic surface; and(ii) migratory type 1 conventional dendritic cells, thereby treating cancer or preventing the reoccurrence of cancer in the subject.

2. The method of claim 1, wherein the antigen capturing nanoparticle comprises poly(lactic-co-glycolic) acid (PLGA) and a positively charged polymer or lipid.

3. The method of claim 2, wherein the positively charged polymer or lipid comprises polyethylenimine, chitosan, poly(HPMA~ jb-DMAPMA), poly- (propylenimine) (PPI), DEAE-dextran, poly (amidoamine) (PAMAM), poly-L-(lysine) (PLL), poly[2-(N,N- dimethylamino)ethyl methacrylate] (PDMAEMA), poly(amidoamine) (PAA), 1,2-di-0-octadecenyl-3-trimethylammonium-propane (DOTMA), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 2,3- dioleyloxy-W-(2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1- propanaminium trifluoroacetate (DOSPA), ethylphosphatidylcholine (ePC), or a combination thereof.

4. The method of claim 2, wherein the positively charged polymer or lipid comprises polyethyleneimine.

5. The method of claim 1, wherein the antigen capturing nanoparticle encapsulates at least one immune stimulating agent.UIC0112WO PATENT6. The method of claim 5, wherein the at least one immune stimulating agent comprises a toll-like receptor (TLR) ligand, nucleotide-binding oligomerization domain (NOD) ligand, retinoic-acid-inducible protein 1(RIG-1)-like receptor (RLR) ligand, C-type lectin receptor ligand, cytosolic DNA sensing ligand, stimulator of interferon genes (STING) ligand, aryl hydrocarbon receptor ligand, and / or alpha protein kinase 1 (ALPKl)-TRAF interacting forkhead-associated protein A (TIFA) inducer.

7. The method of claim 6, wherein the TLR ligand comprises a toll-like receptor 3 (TLR3) agonist.

8. The method of claim 7, wherein the TLR3 agonist comprises polyinosinic:polycytidylic acid (PIC).

9. The method of claim 1, wherein the antigen capturing nanoparticle and the migratory type 1 conventional dendritic cells are administered by injection or infusion.

10. The method of claim 1, further comprising administering to the subject an immune checkpoint inhibitor.

11. A composition comprising(i) an antigen capturing nanoparticle, the antigen capturing nanoparticle comprising positively charged and hydrophobic surface;(ii) migratory type 1 conventional dendritic cells; and(iii) a pharmaceutically acceptable carrier or agueous medium.UIC0112WO PATENT12. The composition of claim 11, wherein the antigen capturing nanoparticle comprises poly(lactic-co-glycolic) acid (PLGA) and a positively charged polymer or lipid.

13. The composition of claim 12, wherein the positively charged polymer or lipid comprises polyethylenimine, chitosan, poly(HPMA-b-DMAPMA), poly-(propylenimine) (PPI), DEAE-dextran, poly(amidoamine) (PAMAM), poly-L-(lysine) (PLL), poly[2-(N,N- dimethylamino)ethyl methacrylate] (PDMAEMA), poly(amidoamine) (PAA), 1,2-di-0-octadecenyl-3-trimethylammonium-propane (DOTMA), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 2,3- dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1- propanaminium trifluoroacetate (DOSPA), ethylphosphatidylcholine (ePC), or a combination thereof.

14. The composition of claim 11, wherein the antigen capturing nanoparticle encapsulates at least one immune stimulating agent.

15. The composition of claim 14, wherein the at least one immune stimulating agent comprises a toll-like receptor (TLR) ligand, nucleotide-binding oligomerization domain (NOD) ligand, retinoic-acid-inducible protein 1(RIG-1)-like receptor (RLR) ligand, C-type lectin receptor ligand, cytosolic DNA sensing ligand, stimulator of interferon genes (STING) ligand, aryl hydrocarbon receptor ligand, and / or alpha protein kinase 1 (ALPKl)-TRAF interacting forkhead-associated protein A (TIFA) inducer.

16. The composition of claim 15, wherein the TLR ligand comprises a toll-like receptor 3 (TLR3) agonist.

17. The composition of claim 16, wherein the TLR3 agonist comprises polyinosinic:polycytidylic acid (PIC).UIC0112WO PATENT18. The composition of claim 11, further comprising an immune checkpoint inhibitor.

19. A kit comprising(i) an antigen capturing nanoparticle, the antigen capturing nanoparticle comprising positively charged and hydrophobic surface;(ii) migratory type 1 conventional dendritic cells; and(iii) an immune checkpoint inhibitor.

20. The kit of claim 19, wherein the antigen capturing nanoparticle encapsulates at least one immune stimulating agent.

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