Combination therapies and methods and uses thereof for treating cancer

Abstract

Described herein are combination treatments and related compositions, kits, and methods. In aspects, the combination comprises targeted hyperthermia therapy (THT) and immunotherapy, wherein the combination is for synergistically treating a cancer. In additional or alternative aspects, the combination comprises targeted hyperthermia therapy (THT) and immunotherapy, wherein the combination causes an abscopal effect.

Description

[0001] COMBINATION THERAPIES AND METHODS AND USES THEREOF FOR TREATING CANCER

[0002] FIELD

[0003] The present disclosure relates generally to combination therapies, and methods and uses thereof for treatment of cancer.

[0004] BACKGROUND

[0005] There are many types of cancer treatment such as, and without being limited thereto, biomarker testing for cancer treatment, chemotherapy, hormone therapy, hyperthermia, immunotherapy, photodynamic therapy (PDT), photothermal therapy (PTT), radiation therapy, and others. PTT, for example, is a minimally invasive, local treatment that relies on an optical absorbing agent (i.e. photosensitizer), which can absorb energy and convert it into heat upon stimulating by an electromagnetic radiation (EMR) such as radiofrequency, microwaves, near infrared irradiation, or visible light. PTT is a therapy that induces hyperthermia, including sub-ablative hyperthermia, or ablation in tumour cells. It may be difficult to specifically target cell populations for destruction using PTT. Metal nanoparticles such as gold nanorods (GNRs) can be used as the photosensitizer in PTT.

[0006] There is a need for the development of improved compounds, compositions, uses and / or methods for treatment of cancer.

[0007] The background disclosed herein is included solely to explain the context of the application. This is not to be taken as an admission that any of the material referred to herein was published, known, or part of the common general knowledge as of the priority date.

[0008] SUMMARY

[0009] In accordance with an aspect, there is provided a combination comprising targeted hyperthermia therapy (THT) and immunotherapy, wherein the combination is for synergistically treating a cancer.

[0010] In accordance with an aspect, there is provided a combination comprising targeted hyperthermia therapy (THT) and immunotherapy, wherein the combination causes an abscopal effect.

[0011] In an aspect, the combination is synergistic for treating a cancer.

[0012] In an aspect, the combination is additive for treating a cancer.

[0013] In an aspect, the THT comprises administration of a metal nanoparticle (MNP) and application of infrared light to heat the MNP.

[0014] In an aspect, the MNP is a tube, rod, shell, cage, sphere, fiber, wire, stars, plate, sea archon, or a combination thereof.

[0015] In an aspect, the MNP has an average particle size that is less than about 1 m in size, and typically from about 1 nm to about 900 nm in size and various ranges therebetween.

[0016] In an aspect, the average particle size of the MNP is about 1 nm to about 100 nm.

[0017] In an aspect, the MNP has one or more dimensions of the order of 100 nm or less.

[0018] In an aspect, the MNP is a metal nanorod (MNR).

[0019] In an aspect, the MNP comprises a transition metal, a precious metal, or a combination thereof.

[0020] In an aspect, the MNP comprises gold, nickel, palladium, platinum, copper, silver, zinc, cadmium, or any combination thereof.

[0021] In an aspect, the MNP is a gold nanoparticle. In an aspect, the MNP is a gold nanorod (GNR).

[0022] In an aspect, the MNP is capped with a capping agent, such as carboxylic acid, citrate, a positively charged ligand, or any combination thereof.

[0023] In an aspect, the metal nanoparticles have a surfactant monolayer, or a surfactant bilayer wrapped in a polymer.

[0024] In an aspect, the polymer comprises proteins, gelatin, bovine serum albumin, polystyrene sulfonate, polyethylene oxides, thiolated polyethylene oxides, thiolated polyethyene oxides with terminating carboxylic acid functionalities, thiolated polyethyene oxides with terminating amine acid functionalities, or any combination thereof.

[0025] In an aspect, the polymer forms covalent or non-covalent bonds with at least one of a protein, a polypeptide, an antibody, an antibody fragment, an IgG class of antibody, a polyclonal antibody, a monoclonal antibody, or any combination thereof.

[0026] In an aspect, the metal nanoparticles comprise pegylated GNRs.

[0027] In an aspect, the metal nanoparticles are conjugated to the immunotherapy.

[0028] In an aspect, the metal nanoparticles are not conjugated to the immunotherapy.

[0029] In an aspect, the immunotherapy comprises administration of a tumor-specific antigen (TSA), a tumor-associated antigen (TAA), an antibody, a modified immune cell, a cytokine, a cytokine agonist, a chemokine, a chemokine agonist, a toll-like receptor (TLR) agonist, an immune checkpoint blockade molecule, a virus, a nucleic acid, or any combination thereof.

[0030] In an aspect, the tumor-specific antigen (TSA) or tumor-associated antigen (TAA) comprises MAG-Tn3, MAGE-A3, New York esophageal squamous cell carcinoma antigen (NY-ESO-1), HER-2 / neu, p53, melanoma-associated antigen recognized by T cells 1 (MART-1 ), glycoprotein (gp) 100, alphafetoprotein (AFP), EGFRvlll-specific 14-amino acid peptide, PEP-3 chemically conjugated to keyhole limpet hemocyanin (KLH), CA-125, MUC-1 , carcinoembryonic antigen (CEA), epithelial tumor antigen (ETA), tyrosinase, prostatic acid phosphatase (PAP), prostate-specific antigen (PSA), Sialyl-Tn, prostate specific membrane antigen (PSMA), non-catalytic hTERT, or any combination thereof.

[0031] In an aspect, the antibody is a monoclonal antibody.

[0032] In an aspect, the antibody targets CD52, EGFR, VEGF, HER-2, CD20, CD16, 0X40, CD137, CD27, GITR, CD40, CD19, CD272, CD279, CD274, PAP, CD38, CD47, GD2, or any combination thereof.

[0033] In an aspect, the modified immune cell comprises a dendritic cell that expresses a PAP antigen.

[0034] In an aspect, the modified immune cell expresses a chimeric antigen receptor (CAR).

[0035] In an aspect, the CAR is directed against CD19, melanoma-associated antigen recognized by T cells 1 (MART-1 ), glycoprotein (gp) 100, carcinoembryonic antigen (CEA), p53, MAGE-A3, New York esophageal squamous cell carcinoma antigen (NY-ESO-1 ), or any combination thereof.

[0036] In an aspect, the cytokine comprises an interferon, an interleukin, or a combination thereof.

[0037] In an aspect, the cytokine comprises IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12 IL-15, IL-17, IL-23, IL- 1 p, TNF-a, IFN-a, IFN-p, IFN-y, an agonist of any thereof, or any combination thereof.

[0038] In an aspect, the chemokine comprises CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11 , CXCL12, CXCL14, CCL2, CCL5, an agonist of any thereof, or any combination thereof. In an aspect, the TLR agonist comprises a TLR-4 agonist, a TLR-7 agonist, a TLR-8 agonist, a TLR-9 agonist, a TLR-12 agonist, or a combination thereof.

[0039] In an aspect, the immune checkpoint blockade molecule comprises a monoclonal antibody.

[0040] In an aspect, the antibody targets CTLA-4, PD-L1 , PD-1 , or any combination thereof.

[0041] In an aspect, the virus expresses a 5T4 tumor-associated antigen.

[0042] In an aspect, the combination comprises THT, IL-2, and a checkpoint inhibitor.

[0043] In an aspect, the checkpoint inhibitor comprises an anti-PD-1 antibody.

[0044] In an aspect, the combination further comprises an adjuvant, an immune modulator, or a combination thereof.

[0045] In an aspect, the adjuvant or immune modulator comprises GM-CSF, KLH, liposomal AS15, BCG, freeze dried BCG, MONTANIDE, IL-2, or any combination thereof.

[0046] In an aspect, the cancer is brain cancer, nerve sheath cancer, breast cancer, colon cancer, colorectal cancer, skin cancer, lung cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, renal cell carcinoma, carcinoma, melanoma, lymphoma, head and neck cancer, brain cancer, and prostate cancer, or any combination thereof.

[0047] In an aspect, the cancer is breast cancer, colon cancer, or melanoma.

[0048] In an aspect, the breast cancer is triple-negative breast cancer.

[0049] In an aspect, the cancer is metastatic.

[0050] In an aspect, the THT and the immunotherapy are independently administered intratumourally or systemically.

[0051] In an aspect, the THT is administered intratumourally and treats a distant tumour.

[0052] In an aspect, the THT is administered a single time or a plurality of times, wherein each THT administration is separated by a period of time such as from about 1 day to about 3 months, such as about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, with or without further administration of MNPs.

[0053] In an aspect, the immunotherapy is administered a single time or a plurality of times.

[0054] In an aspect, the THT is administered a single time and the immunotherapy is administered a plurality of times, before, during, and / or after the THT, such as at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times, wherein each immunotherapy administration is separated by a period of time such as from about 1 hour to about 1 month, such as about 1 , 2, 3, 4, 5, 6, or 7 days.

[0055] In an aspect, the immunotherapy is for administration before, after, or concurrently with the THT.

[0056] In accordance with an aspect, there is provided a pharmaceutical composition or kit comprising the combination described herein.

[0057] In accordance with an aspect, there is provided a method for sensitizing a cancer to cancer immunotherapy, the method comprising administering the combination, composition, or kit described herein.

[0058] In accordance with an aspect, there is provided a method for exposing cancer antigens for immunotherapy, the method comprising administering the combination, composition, or kit described herein.

[0059] In accordance with an aspect, there is provided a method for treating cancer, the method comprising administering the combination, composition, or kit described herein. In accordance with an aspect, there is provided a method for increasing CD8+ T cell infiltration into a tumour, increasing CD8+ memory T cell subsets, and / or increasing PD-1 expression on CD+ T cells, the method comprising administering the combination, composition, or kit described herein.

[0060] In an aspect, the cancer is selected from brain tumour, nerve sheath tumour, breast cancer, colon cancer, colorectal cancer, skin cancer, lung cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, renal cell carcinoma, carcinoma, melanoma, lymphoma, head and neck cancer, brain cancer, and / or prostate cancer, including but not limited to androgen-dependent prostate cancer and androgen-independent prostate cancer.

[0061] In an aspect, the cancer is selected from breast cancer, colon cancer, and melanoma.

[0062] In an aspect, the cancer is metastatic.

[0063] In an aspect, wherein treatment of a primary tumour further treats one or more metastatic lesions.

[0064] In an aspect, wherein the mammal is a human.

[0065] In an aspect, wherein administering comprises parenteral administration (e.g. formulated for injection via the intravenous, intramuscular, sub-cutaneous, intralesional, and / or intraperitoneal routes).

[0066] In accordance with an aspect, there is provided a use of the combination, composition, or kit described herein for sensitizing a cancer to cancer immunotherapy.

[0067] In accordance with an aspect, there is provided a use of the combination, composition, or kit described herein for exposing cancer antigens for immunotherapy.

[0068] In accordance with an aspect, there is provided a use of the combination, composition, or kit described herein for treating cancer.

[0069] In accordance with an aspect, there is provided a use of the combination, composition, or kit described herein for increasing CD8+ T cell infiltration into a tumour, increasing CD8+ memory T cell subsets, and / or increasing PD-1 expression on CD+ T cells.

[0070] In an aspect, the cancer is selected from brain tumour, nerve sheath tumour, breast cancer, colon cancer, colorectal cancer, skin cancer, lung cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, renal cell carcinoma, carcinoma, melanoma, lymphoma, head and neck cancer, brain cancer, and / or prostate cancer, including but not limited to androgen-dependent prostate cancer and androgen-independent prostate cancer.

[0071] In an aspect, the cancer is selected from breast cancer, colon cancer, and melanoma.

[0072] In an aspect, the cancer is metastatic.

[0073] In an aspect, treatment of a primary tumour further treats one or more metastatic lesions.

[0074] In an aspect, the mammal is a human.

[0075] In an aspect, administering comprises parenteral administration (e.g. formulated for injection via the intravenous, intramuscular, sub-cutaneous, intralesional, and / or intraperitoneal routes).

[0076] In accordance with an aspect, there is provided a use of MNP-mediated THT to stimulate an immune response.

[0077] In accordance with an aspect, there is provided a method for stimulating an immune response, the method comprising administering MNP-mediated THT to a subject in need thereof. In accordance with an aspect, there is provided a use of MNP-mediated THT to treat a tumour, whererin the THT is for administration at least twice, wherein each THT administration is separated by a period of time such as from about 1 day to about 3 months, such as about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, or 14 days, with or without further administration of MNPs.

[0078] In accordance with an aspect, there is provided a method of treating a tumour using MNP- mediated THT, the method comprising administering the THT at least twice, wherein each THT administration is separated by a period of time such as from about 1 day to about 3 months, such as about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, with or without further administration of MNPs.

[0079] It is understood that one or more of the aspects disclosed herein may be combined in any suitable manner. The novel features will become apparent to those of skill in the art upon examination of the following detailed description. It should be understood, however, that the detailed description and the specific examples presented, while indicating certain embodiments of the present invention, are provided for illustration purposes only because various changes and modifications within the spirit and scope of the invention will become apparent to those of skill in the art from the detailed description of the invention and claims that follow.

[0080] BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Reference will now be made, by way of example, to the accompanying drawings and by which the present disclosure can be further understood from the following description with reference to the Figure(s):

[0082] Figure 1 shows tumor growth in animals over time. The combined therapy of THT and IL-2 showed enhanced cancer treatment efficacy in mice models of colorectal cancer, breast cancer, and melanoma. The treated tumors showed significant reduction in volume compared to controls, with evidence of systemic immune responses indicated by shrinkage in distant untreated tumors.

[0083] Figure 2 shows FACS analysis revealing increased infiltration of T-cells, including CD8+ cytotoxic T-cells and memory T-cells, in both treated and untreated tumors, suggesting a systemic abscopal immune response. High levels of PD-1 expression on T-cells in treated tumors indicate potential for additional benefit from PD-1 inhibitors.

[0084] Figure 3 shows the upregulation of inflammatory gene expression in the STING / cGAS / TLR pathway following GNR-induced hyperthermia in both 4T1 tumours and B16 tumours.

[0085] Figure 4 shows that GNR-induced hyperthermia upregulated innate immune cells in 4T 1 and B16 tumours.

[0086] Figure 5 shows enhanced tumour reduction by the combination of IL-2 and GNR / NIR therapies as compared to controls.

[0087] Figure 6 shows enhanced tumour reduction with double NIR treatment in a B16 model.

[0088] Figure 7 shows enhanced infiltration of CD8+ T cells into tumours treated with a combination of GNR and IL-2.

[0089] Figure 8 shows abscopal effects of the therapy combining GNR and IL-2, where increased CD8+ T cells were observed in a contralateral tumour following the combination therapy.

[0090] Figure 9 shows GNR-Enhanced Laser Irradiation Induces T umor THT, Absent in Laser-Only Controls. (A) Schematic representation of the experimental setup, showing a BALB / c mouse bearing a 4T 1 tumor, with two internal temperature probes used to monitor the effects of gold nanorod (GNR)- enhanced laser irradiation versus laser-only controls. (B) Temperature profiles of 4T1 tumors in BALB / c mice during laser irradiation, comparing the temperature changes in GNR-injected tumors to those in laser-only controls. (C-D) Quantitative analysis of surface temperatures in BALB / c mice with 4T 1 tumors (n=18 control, 19 THT) and C57BL / 6 mice with B16-F10 tumors (n=11 control, 14 THT), illustrating the differential heating effects observed between GNR-treated tumors and controls across both models.

[0091] Figure 10 shows GNR-Mediated THT Induces Tumor Shrinkage, Cell Death, and Immune Activation in 4T1 Models and Growth Suppression in B16-F10 Models Within 48 Hours Post-Treatment. (A) Measurement of tumor volumes in 4T 1 tumor models following GNR-mediated THT treatment (n=24 control, n=28 THT). (B) Representative images of a control mouse and a THT-treated mouse 48 hours post-irradiation, alongside a comparison of tumor volumes between GNR-treated and control groups. (C) Flow cytometry analysis of cell viability in 4T 1 tumors at 24 and 48 hours post-THT, highlighting the proportion of non-viable cells (n=5). (D) Levels of extracellular calreticulin in 4T 1 tumors at 24 and 48 hours post-THT (n=5). (E) Flow cytometry data showing the percentage of CD45+immune cells in 4T 1 tumors at 24 and 48 hours post-treatment (n=5). (F) Analysis of M2 macrophage levels in 4T1 tumors at 24 and 48 hours post-THT (n=5). (G) Tumor volume measurements in the B16-F10 model within 48 hours post-laser treatment (n=19 control, n=23 THT).

[0092] Figure 11 shows GNR-lnduced THT Results in Tumor Regrowth After Initial Reduction, Despite Upregulation of STING Pathway Genes and Increased Innate Immune Cell Levels. (A) Tumor volume measurements in 4T1 models showing initial reduction following THT treatment (n=16 control, n=15 THT). (B) Tumor volume analysis in B16-F10 models (n= 9 control, n=11 THT). (C-D) Gene expression analysis 8 days post-laser treatment showing upregulation of STING pathway genes in both 4T 1 (n= 8 control, 7 THT) and B16-F10 tumors (n = 4 control, 7 THT) subjected to THT, indicating enhanced tumor antigen recognition and immune signaling activation. (E-H) Flow cytometry analysis of 7dendritic cells, M1 macrophages, macrophages and NK cells in 4T1 tumors post-THT, (n=6 control, n= 7 THT). (I-L) Flow cytometry analysis of 7dendritic cells, M1 macrophages, macrophages and NK cells in B16 tumors post-THT, (n=3 control, n= 5 THT).

[0093] Figure 12 shows Intratumoral IL-2 Treatments Prevent Tumor Regrowth Following GNR-lnduced THT. (A) Tumor volume measurements over a 14-day period post-laser treatment for each group: THT alone, IL-2 alone, THT combined with IL-2, and control in the 4T 1 model (n=16 control, 14 IL2, 15 THT, 12 THT+IL2). (B) Final tumor weights for the 4T 1 model across the different treatment groups (n=16 control, 10 IL2, 13 THT, ). (C) Tumor volume measurements up to day 8 post-laser treatment in the B16- F10 model, showing the comparison between the same groups (n=9 control, 7 IL2, 10 THT, 10 THT+IL2). (D) Final tumor weights for the B16-F10 model across the different treatment groups. The figure demonstrates the experimental design aimed at testing the hypothesis that IL-2 can enhance the therapeutic effects of THT and prevent tumor regrowth. *p<0.05 vs. control, +p<0.05 vs. IL2, #p<0.05 vs. THT all anova, a p<0.05 t.test.

[0094] Figure 13 shows IL-2 Treatment Enhances CD8+ T Cell Infiltration, Central Memory Differentiation, and PD-1 Expression in Tumors Following GNR-lnduced THT. Panel (A) shows the quantification of CD8+ T cells (gated on CD3+CD8+) in 4T 1 tumors across different treatment groups (n= 6 Control, 3 IL2, 7 THT, 6 THT+IL2). Panel (B) illustrates the expression of PD-1+ on CD8+ T cells (gated on PD-1 +CD8+CD3+) in 4T 1 tumors. Panel (C) displays the frequency of CD8+ central memory (CM) T cells (gated on CD62L+CD44+CD8+CD3+) in 4T1 tumors. Panel (D) presents the quantification of M2 macrophages (gated on CD45+ / CD11b+F480+CD206+CD273+) in 4T1 tumors. In the B16-F10 model, panel (E) quantifies CD8+ T cells (gated on CD3+CD8+) across different treatment groups (n= 4 Control, 4 IL2, 5 THT, 7 THT+IL2). Panel (F) shows the expression of PD-1+ on CD8+ T cells (gated on PD-1+CD8+CD3+) in B16-F10 tumors. Panel (G) illustrates the frequency of CD8+ central memory (CM) T cells (gated on CD62L+CD44+CD8+CD3+) in B16-F10 tumors, while panel (H) quantifies M2 macrophages (gated on CD45+ / CD11 b+F480+CD206+CD273+) in B16-F10 tumors. (I) Tumor volume measurements over a 14-day period post-laser treatment for each group: THT alone, IL-2 alone, THT combined with IL-2, and control in the 4T 1 model (n=16 control, 14 IL2, 15 THT, 12 THT+IL2, 2 PD1, 4 PD1+THT). * represents p<0.05.

[0095] Figure 14 shows GNR-lnduced THT Combined with IL-2 Reduces Contralateral 4T1 Tumor Size and Enhances CD8+ T Cell Infiltration. Panel (A) shows the tumor volume of contralateral (untreated) 4T1 tumors in mice with bilateral tumors. Panel (B) quantifies CD8+ T cells (gated on CD3+CD8+) in contralateral tumors. *p<0.05 vs. control, #p<0.05 vs. THT.

[0096] Figure 15 shows enhanced tumour reduction with double NIR treatment in a B16 model.

[0097] DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS / ASPECTS

[0098] Definitions

[0099] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those disclosed herein can be used in the practice for testing of the present invention, the typical materials and methods are disclosed herein.

[0100] In addition, in describing and claiming the present invention, the common terminology generally used is disclosed herein below. If a term is used in this disclosure but is not specifically disclosed herein, the definition from the IUPAC Compendium of Chemical Terminology can be applied, as long as that definition does not conflict with any other disclosure or description applied herein, or render indefinite or non-enabled any claim to which that definition is applied. All references herein to elements or metals belonging to a certain Group refer to the Periodic T able of the Elements and Hawley's Condensed Chemical Dictionary, 13th Edition. Also, any references to the Group or Groups shall be to the Group or Groups as reflected in the Periodic Table of Elements using the CAS system for numbering groups. To the extent that any definition, description or usage provided by any document incorporated herein by reference conflicts with the description or usage provided herein, the description or usage provided herein controls. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting.

[0101] The term “metal nanoparticle” or “MNP” can refer to an average particle size that is less than about 1 m in size, and typically from about 1 nm to about 900 nm in size and various ranges therebetween. An MNP can take a variety of shapes, including but not limited to, tube, rod, shell, cage, sphere, fiber, wire, stars, plate, sea archon, or a combination thereof.

[0102] The terms “cancer”, “tumour” and “carcinoma” refer to cells that exhibit relatively abnormal, uncontrolled, and / or autonomous growth, so, for example, they exhibit an aberrant growth phenotype characterized by a significant loss of control of cell proliferation. The term “cancer” is understood to be broad and encompass, for example, “tumour” and “carcinoma”. An “immunomodulator” refers to any modulator that can provide (e.g. change, start, stop, increase, decrease, etc.) an immune response (e.g. molecule, heat, radiation, etc.).

[0103] An “immunotherapeutic” as used herein refers to any type of compound which can be used in immunotherapy. Immunotherapy as used herein is the treatment of disease by inducing, enhancing, or suppressing an immune response. An “anti-cancer immunotherapy”, as used herein, stimulates the immune system to reject and destroy tumors. An “anti-cancer immunotherapeutic” as used herein thus includes such compounds as e.g. tumor-specific antigens (TSA), tumor-associated antigens (TAA), immune adjuvants, immune modulators, antibodies, modified immune cells, cytokines, immune checkpoint blockade molecules, viruses. These compounds are further described herein below.

[0104] The term “abscopal effect” refers to the treatment of a distant or metastatic cancer whereby shrinkage of untreated tumors occurs concurrently with shrinkage of tumors within the scope of the localized treatment. This is typically due to an enhancement of anti-tumour immunity.

[0105] As used herein, the terms “bind” and “bound” can refer to an association between atoms or molecules. The association can be direct or indirect. For example, bound atoms or molecules may be directly bound to one another, e.g., by a covalent bond or non-covalent bond (e.g. electrostatic interactions (e.g. ionic bond, hydrogen bond, halogen bond), van der Waals interactions (e.g., dipoledipole, dipole-induced dipole, London dispersion), ring stacking (pi effects), hydrophobic interactions and the like). As a further example, two molecules may be bound indirectly to one another by way of direct binding to one or more intermediate molecules, thereby forming a complex.

[0106] The term “conjugate” as used herein refers to a molecule (e.g. biomolecule) comprising at least two moieties. For example, and without limitation, the moieties can be connected via a linker (e.g. covalent and / or non-covalent linker).

[0107] As used herein, "treatment", “treating”, or “therapy” is an approach for obtaining beneficial or desired clinical results. For the purposes disclosed herein, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i. e. , not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" and “therapy” can also mean prolonging survival as compared to expected survival if not receiving treatment or therapy. Thus, "treatment" or “therapy” is an intervention performed with the intention of altering the pathology of a disorder. Specifically, the treatment or therapy may directly prevent, slow down or otherwise decrease the pathology of a disease or disorder such as cancer, or may render the cells more susceptible to treatment or therapy by other therapeutic agents.

[0108] The terms "therapeutically effective amount", "effective amount" or "sufficient amount" mean a quantity sufficient, when administered to a subject, including a mammal, for example a human, to achieve a desired result, for example an amount effective to treat cancer. Effective amounts of the metal nanorods disclosed herein may vary according to factors such as the disease state, age, sex, and weight of the subject. Dosage or treatment regimes may be adjusted to provide the optimum therapeutic response, as is understood by a skilled person.

[0109] Moreover, a treatment regime of a subject with a therapeutically effective amount may consist of a single administration, or alternatively comprise a series of applications. The length of the treatment period depends on a variety of factors, such as the severity of the disease, the age of the subject, the concentration of the agent / compound / medicament, the responsiveness of the patient to the agent / compound / medicament compound / medicament, or a combination thereof. It will also be appreciated that the effective dosage of the agent / compound / medicament used for the treatment may increase or decrease over the course of a particular treatment regime. Changes in dosage may result and become apparent by standard diagnostic assays known in the art. The conjugate compounds disclosed herein may, in embodiments, be administered before, during or after treatment with conventional therapies for the disease or disorder in question, such as cancer.

[0110] The term "subject" as used herein refers to any member of the animal kingdom, including birds, fish, invertebrates, amphibians, mammals, and reptiles. Typically, the subject is a human or non-human vertebrate. Non-human vertebrates include livestock animals, companion animals, and laboratory animals. Non-human subjects also specifically include non-human primates as well as rodents. Non- human subjects also specifically include, without limitation, poultry, chickens, horses, cows, pigs, goats, dogs, cats, guinea pigs, hamsters, mink, rabbits, crustaceans, and molluscs. Typically, the subject is a mammal. The term "mammal" refers to any animal classified as a mammal, including humans, other higher primates, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. Typically, the mammal is human.

[0111] The term “pharmaceutically acceptable” means that the compound or combination of compounds is compatible with the remaining ingredients of a formulation for pharmaceutical use, and that it is generally safe for administering to humans according to established governmental standards, including those promulgated by the United States Food and Drug Administration.

[0112] The term "pharmaceutically acceptable carrier" includes, but is not limited to solvents, dispersion media, coatings, antibacterial agents, antifungal agents, isotonic and / or absorption delaying agents and the like. The use of pharmaceutically acceptable carriers is well known.

[0113] The term “non-toxic” refers to the non-occurrence of pathological phenomena as a result of using pharmacological levels of the metal nanorods disclosed herein. The term substantially non-toxic is defined as including acceptably low toxicity as well as non-toxicity.

[0114] "Substantially free" of an element / feature herein means less than about 5%, typically less than about 2%, more typically less than about 1%, even more typically less than about 0.5%, most typically less than about 0.1% of the element / feature.

[0115] A “mixture” is not limited to two or more components that have been mixed. A mixture may be two or more components combined without having been mixed.

[0116] A “combination” may refer to a mixture or it may refer to discrete components that are for use simultaneously or sequentially.

[0117] The terms inhibit, reduced, prevented, minimized, or delayed, can be used interchangeably. These terms can refer to partially, substantially, or completely slowing, hindering, reducing, delaying, or preventing.

[0118] In understanding the scope of the present application, the articles “a”, “an”, “the”, and “said” are intended to mean that there are one or more of the elements.

[0119] The term "comprising" and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The foregoing also applies to words having similar meanings such as the terms, "including", "having" and their derivatives. It will be understood that any embodiments described as “comprising” certain components may also “consist of or “consist essentially of,” wherein “consisting of” has a closed- ended or restrictive meaning and “consisting essentially of” means including the components specified but excluding other components except for materials present as impurities, unavoidable materials present as a result of processes used to provide the components, and components added for a purpose other than achieving the technical effect of the invention. For example, a composition defined using the phrase “consisting essentially of encompasses any known pharmaceutically acceptable additive, excipient, diluent, carrier, and the like. Typically, a composition consisting essentially of a set of components will comprise less than 5% by weight, typically less than 3% by weight, more typically less than 1% by weight of non-specified components.

[0120] It will be understood that any component defined herein as being included may be explicitly excluded from the claimed invention by way of proviso or negative limitation, such as any specific compounds or method steps, whether implicitly or explicitly defined herein.

[0121] In addition, all ranges given herein include the end of the ranges and also any intermediate range points, whether explicitly stated or not.

[0122] Terms of degree such as "substantially", "about" and "approximately" as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree may be construed as including a deviation of at least ±10% of the modified term if this deviation would not negate the meaning of the word it modifies.

[0123] The abbreviation, “e.g.” is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g.” is synonymous with the term “for example.” The word “or” is intended to include “and” unless the context clearly indicates otherwise.

[0124] The phrase “at least one of” is understood to be one or more. The phrase “at least one of... and...” is understood to mean at least one of the elements listed or a combination thereof, if not explicitly listed. For example, “at least one of A, B, and C” is understood to mean A alone or B alone or C alone or a combination of A and B or a combination of A and C or a combination of B and C or a combination of A, B, and C.

[0125] The phrase “a combination thereof” in conjunction with a list of two or more features, for example, “A, B, C, or a combination thereof” is understood to mean at least one of the elements listed or a combination thereof, if not explicitly listed. For example, A alone or B alone or C alone or a combination of A and B or a combination of A and C or a combination of B and C or a combination of A, B, and C.

[0126] It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting. Patent applications, patents, and publications are cited herein to assist in understanding the aspects described. All such references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material. I. Combinations

[0127] Described herein are combinations comprising targeted hyperthermia therapy (THT) and tumour immunotherapy. Typically, the combined therapy synergistically treats a tumour. In additional or alternative aspects, the combined therapy results in an abscopal effect. Typically, the THT comprises administration of a metal nanoparticle (MNP) and application of infrared light to heat the MNP. In some aspects, the tumour immunotherapy involves the use of one agent or at least two separate agents, such as a cytokine and a checkpoint inhibitor, such as IL-2 and a PD-1 inhibitor.

[0128] MNP

[0129] The metal nanoparticle (MNP) can have an average particle size that is less than about 1 m in size, and typically from about 1 nm to about 900 nm in size and various ranges therebetween. In most embodiments, the average particle size is about 1 nm to about 100 nm. The particle can have one or more dimensions of the order of 100 nm or less. A nanoparticle can be made of a variety of materials, including but not limited to, transition metals or precious metals and are typically selected from gold, nickel, palladium, platinum, copper, silver, zinc, cadmium, or a combination thereof. With respect to combinations of metals, the metal nanoparticle can be a metal alloy (combines more than one metal or mixes a metal with other non-metallic elements). A metal nanoparticle can take a variety of shapes, including but not limited to, tube, rod, shell, cage, sphere, fiber, wire, stars, plate, sea archon, or a combination thereof. As examples, nanofibers are fibers with diameters less than 100 nanometers; nanowires are about 75 nm in diameter, and range from 1 pm to 10 microns in length; nanotubes are cylindrical nanoscale structures with length-to-diameter aspect ratios of up to 132,000:1.

[0130] In typical embodiments, the MNP is a metal nanorod (MNR). Dimensions of an MNR usually ranges from about 1 to about 100 nm. Typically the MNR has a diameter or cross-section of between about 5 nm and about 50 nm, such as from about 5 nm, about 10 nm, about 15 nm, about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 40 nm, or about 45 nm, to about 10 nm, about 15 nm, about 20 nm, about 25 nm, about 30 nm, about 35 nm, about 40 nm, about 45 nm, or about 50 nm. For example, the diameter may be from about 5 nm to about 30 nm or from about 15 nm to about 30 nm. The MNR may typically have an axial length of between about 20 nm and about 500 nm, such as from about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 125 nm, about 150 nm, about 175 nm, about 200 nm, about 250 nm, about 300 nm, about 350 nm, about 400 nm, or about 450 nm, to about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 125 nm, about 150 nm, about 175 nm, about 200 nm, about 250 nm, about 300 nm, about 350 nm, about 400 nm, about 450 nm, or about 500 nm. For example, the axial length may be from about 30 nm to about 500 nm, from about 50 nm to about 300 nm, or from about 80 nm to about 100 nm.

[0131] Furthermore, the MNR may typically have an aspect ratio (i.e., the ratio of the length of the major axis of the nanorod to the minor axis of the nanorod) of from about 1.1 to about 100, such as from about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, or about 90, to about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100. For example, the aspect ratio may be from about 1.1 to about 10. Substantially uniform length, diameter, and / or aspect ratio is used to refer to a population of metal nanorods wherein a majority of the metal nanorods have the same length, diameter, and / or aspect ratio within an acceptable variance for a subsequent analysis of the population. The population can be a single population in a sample or a subpopulation within a sample. In particular embodiments, the acceptable variance for the length, diameter, and / or aspect ratio of any given metal nanorod in the population or subpopulation can be at most 10%, 8%, 5%, 2%, 1% or 0.1% different from the average length, diameter, and / or aspect ratio for metal nanorods in the population. In embodiments, the population can be composed of at least 90%, 95%, 99% or 99.9% metal nanorods having a particular length, diameter, and / or aspect ratio.

[0132] In embodiments, at least a portion of the surface of the nanorod is substantially smooth. In embodiments, at least a portion of the surface of the nanorod is substantially smooth and / or at least a portion of the surface may be etched (e.g. symmetrically etched and / or asymmetrically etched). In certain embodiments, the nanorod may be symmetrically etched to provide a multi-harmonic shape (e.g. appears as wave in 2-D).

[0133] MNRs may be synthesized from metals or semiconducting materials or their combinations. A MNR can have two ends and a linear body between the two ends. The two ends are also called the transverse or shorter ends. Accordingly, the longitudinal surface of the linear body is also called the longitudinal or longer end. The methods, compositions, and MNRs disclosed herein have been exemplified with respect to gold as the metal, however, it will be understood that the methods are equally applicable to nanorods of other metals, particularly those listed above. Known methods may be used to make the metal nanorods such as, and without being limited thereto, those described in WO2019 / 084661 , which is incorporated by reference in its entirety.

[0134] MNPs that are compact in size, biocompatible, have tunable optical properties, efficient at conversion of light to heat, and / or capable of functionalization are typically used, and in particular, gold nanorods (GNRs).

[0135] In general, the MNP (e.g. MNR) can be bare, or can be capped with any suitable capping agents such as, and without being limited thereto, carboxylic acid, conventional citrate, and / or a positively charged ligand. These capping agents can readily be replaced with covalent and charge chemistries. The term “capping agent” refers to a chemical entity that is adsorbed on the surface of MNP and can provide stability against substantial aggregation of nanoparticles.

[0136] The MNP can be protected from exposure to various elements, such as whole blood. Polymers can be used to protect the MNP, such as synthetic polymers and / or natural polymers. Synthetic polymers are those suitable for use in mammals. Examples of synthetic polymers include PEG (polyethylene glycol), PVP (polyvinylpyrrolidone), PSS (polystyrene sulfonate), PLGA Poly lactic-co-glycolic acid, PNIPAM (poly(N-isopropylacrylamide)), ploxamer, diblock or triblock polymers, pH or thermo-responsive polymers, or a combination thereof. Natural polymers are those suitable for use in mammals. Examples of natural polymers include glycoproteins, albumin, gelatine, collagen, or a combination thereof. In addition to polymer(s) or separately, phospholipids (both low and high density), micelles, vesicles, liposomes, solid lipid nanoparticle (SLN), nanostructured lipid carriers (NLC), or a combination thereof. For the protection of the MNP with polymer(s), a selected polymer may have a thiol or disulphide functional group (e.g. at a terminal end) or has been modified to include this functional group. Nano-aggregates such as micelles, vesicles, liposomes, solid lipid nanoparticle (SLN), nanostructured lipid carriers (NLC) and / or supramolecular vesicles are described for encapsulation and / or surface adsorption of the MNP for intra-tumoral and intravenous injection or as a topical application.

[0137] Representative coatings that may be used with the MNPs disclosed herein can include molecules having, for example, hydrophobic segments such as PPO segments with molecular weights of at least about 1.8 kDa, or at least about 2 kDa, or at least about 2.4 kDa, or at least about 2.8 kDa, or at least about 3.2 kDa, or at least about 3.6 kDa, or at least about 4.0 kDa, or at least about 4.4 kDa, or at least about 4.8 kDa or at least about 5.2 kDa, or at least 5.6 kDa, or at least 6.0 kDa, or at least 6.4 kDa or more. In some embodiments, the coatings can have PPO segments with molecular weights of from about 1.8 kDa to about 10 kDa, or from about 2 kDa to about 5 kDa, or from about 2.5 kDa to about 4.5 kDa, or from about 2.5 kDa to about 3.5 kDa, or from about 3.0 kDa to about 5.0 kDa, or from about 3.0 kDa to about 6.0 kDa, or from about 4 kDa to about 6 kDa, or from 4.0 kDa to about 7.0 kDa. In some embodiments, at least about 10%, or at least about 25%, or at least about 50%, or at least about 75%, or at least about 90%, or at least about 95%, or at least about 99% or more of the hydrophobic regions in these coatings have molecular weights within these ranges. In some embodiments, the coatings are biologically inert. Compounds that generate both a hydrophilic surface and an uncharged or substantially neutrally-charged surface can be biologically inert.

[0138] Representative coatings that may be used with the MNP disclosed herein can include molecules having, for example, hydrophobic segments such as PEG segments with molecular weights of at least about 1.8 kDa, or at least about 2 kDa, or at least about 2.4 kDa, or at least about 2.8 kDa, or at least about 3.2 kDa, or at least about 3.6 kDa, or at least about 4.0 kDa, or at least about 4.4 kDa, or at least about 4.8 kDa, or at least about 5.2 kDa, or at least 5.6 kDa, or at least 6.0 kDa, or at least 6.4 kDa or more. In some embodiments, the coatings can have PEG segments with molecular weights of from about 1.8 kDa to about 10 kDa, or from about 2 kDa to about 5 kDa, or from about 2.5 kDa to about 4.5 kDa, or from about 2.5 kDa to about 3.5 kDa. In some embodiments, at least about 10%, or at least about 25%, or at least about 50%, or at least about 75%, or at least about 90%, or at least about 95%, or at least about 99% or more of the hydrophobic regions in these coatings have molecular weights within these ranges. In some embodiments, the coatings are biologically inert. Compounds that generate both a hydrophilic surface and an uncharged or substantially neutrally-charged surface can be biologically inert.

[0139] Representative coatings that may be used with the MNPs disclosed herein can include molecules having, for example, segments such as PLGA segments with molecular weights of at least about 4 kDa, or at least about 8 kDa, or at least about 12 kDa, or at least about 16 kDa, or at least about 20 kDa, or at least about 24 kDa, or at least about 28 kDa, or at least about 32 kDa, or at least about 36 kDa, or at least about 40 kDa, or at least about 44 kDa, of at least about 48 kDa, or at least about 52 kDa, or at least about 56 kDa, or at least about 60 kDa, or at least about 64 kDa, or at least about 68 kDa, or at least about 72 kDa, or at least about 76 kDa, or at least about 80 kDa, or at least about 84 kDa, or at least about 88 kDa or more. In some embodiments, at least about 10%, or at least about 25%, or at least about 50%, or at least about 75%, or at least about 90%, or at least about 95%, or at least about 99% or more of the regions in these coatings have molecular weights within these ranges. In some embodiments, the coatings are biologically inert. Compounds that generate both a hydrophilic surface and an uncharged or substantially neutrally-charged surface can be biologically inert. Immunotherapy

[0140] In aspects, the immunotherapy described herein comprises administration of a tumor-specific antigen (TSA), a tumor-associated antigen (TAA), an antibody, a modified immune cell, a cytokine, a cytokine agonist, a chemokine, a chemokine agonist, a toll-like receptor (TLR) agonist, an immune checkpoint blockade molecule, a virus, a nucleic acid, or any combination thereof.

[0141] As used herein, the term “antigen” is any structural substance that serves as a target for the receptors of an adaptive immune response. A “tumor-specific antigen” (TSA) as used herein refers to an antigen which is only present on tumor cells, but not on normal cells. By contrast, a “tumor-associated antigen” (TAA), as used herein, is an antigen which is present not only on tumor cells but also on some normal cells.

[0142] Preferred Tumor-Specific Antigen (TSA) and Tumor-Associated Antigen (TAA) include MAG-Tn3 (See US 20140171618, incorporated herein by reference), Melanoma Associated Antigen-A3 (MAGE- A3) as described in Gaugler et at, J. Exp. Med. 179:921-930 (1994) incorporated herein by reference, New York esophageal squamous cell carcinoma antigen (NY-ESO-1), HER-2 / neu, p53, melanoma- associated antigen recognized by T cells 1 (MART-1), glycoprotein (gp) 100, Alphafetoprotein (AFP), EGFRvlll-specific 14-amino acid peptide PEP-3 chemically conjugated to keyhole limpet hemocyanin (KLH), CA-125, MUC-1 , carcinoembryonic antigen (CEA), Epithelial tumor antigen (ETA), Tyrosinase, prostatic acid phosphatase (PAP), prostate-specific antigen (PSA), Sialyl-Tn, prostate specific membrane antigen (PSMA), and non-catalytic hTERT.

[0143] Other preferred TSA and TAA include epidermal growth factor receptor, survivin, ras, LAGE-1 , MAGE-A4, SSX-2, RCAS1 , and WT1. Other TSA and TAA are described in Melero, I. et al. (2014), Therapeutic vaccines for cancer: an overview of clinical trials Nat Rev Clin Oncol. 2014 September; 11(9):509-24, and Hong et al., World J Hepatol. 2015 Jun. 18; 7(11): 1581-1585, which are hereby incorporated herein by reference.

[0144] The TSAs and TAAs can be delivered as proteins / peptides, nucleic acids encoding these antigens, or using viral vectors.

[0145] Many tumors express mutations. These mutations potentially create new targetable antigens (neoantigens) for use in immunotherapy. A “neoantigen”, as used herein, is a newly formed antigen that has not been previously recognized by the immune system.

[0146] As used herein, an “immune adjuvant” is a component that potentiates the immune responses to an antigen towards the desired immune responses. An “immune modulator”, as used herein, is a component that modulates the immune responses to an antigen towards the desired immune responses.

[0147] Preferred immune adjuvants / immune modulators include TLR agonists, such as TLR-4 agonist, a TLR-7 agonist, a TLR-8 agonist, a TLR-9 agonist, a TLR-12 agonist or a combination thereof, typically a TLR-9 agonist, for example CpG and PF-3512676. See, e.g., Pashenkov, M., et al., J Clin Oncol 24, 5716-5724 (2006); Krieg, A. M., Nucleic Acid Ther22, 77-89 (2012), which are hereby incorporated by reference.

[0148] Typical adjuvants or immune modulators include GM-CSF, KLH, liposomal AS15, BCG, freeze dried BCG, MONTANIDE, IL2, KLH, or combinations thereof.

[0149] Also contemplated are isolated antibodies that bind specifically to TAAs, TSAs, and immune checkpoint proteins, and peptides derived therefrom. In some embodiments, purified proteins are used to produce antibodies by conventional techniques. In some embodiments, recombinant or synthetic proteins or peptides are used to produce antibodies by conventional techniques.

[0150] Antibodies can be synthetic, monoclonal, or polyclonal and can be made by techniques well known in the art. A typical antibody is comprised of two identical heavy chains and two identical light chains that are joined by disulfide bonds. Each heavy and light chain contains a constant region and a variable region. Each variable region contains three segments called “complementarity -determining regions” (“CDRs”) or “hypervariable regions”, which are primarily responsible for binding an epitope of an antigen. Such antibodies specifically bind to proteins and polypeptides via the CDRs, i.e. the antigenbinding sites of the antibody (as opposed to non-specific binding). Purified or synthetic proteins and peptides can be employed as immunogens in producing antibodies immunoreactive therewith. The proteins and peptides contain antigenic determinants or epitopes that elicit the formation of antibodies.

[0151] These antigenic determinants or epitopes can be either linear or conformational (discontinuous). Linear epitopes are composed of a single section of amino acids of the polypeptide, while conformational or discontinuous epitopes are composed of amino acids sections from different regions of the polypeptide chain that are brought into close proximity upon protein folding (C. A. Janeway, Jr. and P. Travers, Immuno Biology 3:9 (Garland Publishing Inc., 2nd ed. 1996)). Because folded proteins have complex surfaces, the number of epitopes available is quite numerous; however, due to the conformation of the protein and steric hindrances, the number of antibodies that actually bind to the epitopes is less than the number of available epitopes (C. A. Janeway, Jr. and P. Travers, Immuno Biology 2:14 (Garland Publishing Inc., 2nd ed. 1996)). Epitopes can be identified by any of the methods known in the art. Such epitopes or variants thereof can be produced using techniques well known in the art such as solid-phase synthesis, chemical or enzymatic cleavage of a polypeptide, or using recombinant DNA technology.

[0152] Antibodies are defined to be specifically binding if they bind proteins or polypeptides with a Ka of greater than or equal to about 107M-1. Affinities of binding partners or antibodies can be readily determined using conventional techniques, for example those described by Scatchard et al., Ann. N.Y. Acad. Set, 51:660 (1949).

[0153] Polyclonal antibodies can be readily generated from a variety of sources, for example, horses, cows, goats, sheep, dogs, chickens, rabbits, mice, or rats, using procedures that are well known in the art. In general, a purified protein or polypeptide that is appropriately conjugated is administered to the host animal typically through parenteral injection. The immunogenicity can be enhanced through the use of an adjuvant, for example, Freund's complete or incomplete adjuvant.

[0154] Following booster immunizations, small samples of serum are collected and tested for reactivity to proteins or polypeptides. Examples of various assays useful for such determination include those described in Antibodies: A Laboratory Manual, Harlow and Lane (eds. ), Cold Spring Harbor Laboratory Press, 1988; as well as procedures, such as countercurrent immuno-electrophoresis (CIEP), radioimmunoassay, radio-immunoprecipitation, enzyme-linked immunosorbent assays (ELISA), dot blot assays, and sandwich assays. See U.S. Pat. Nos. 4,376,110 and 4,486,530.

[0155] Monoclonal antibodies can be readily prepared using well known procedures. An antibody reactive with a specific antigen can be generated by recombinant methods such as selection of libraries of recombinant antibodies in phage or similar vectors, or by immunizing an animal with the antigen or an antigen-encoding nucleic acid. See, for example, the procedures described in U.S. Pat. Nos. RE 32,011, 4,902,614, 4,543,439, and 4,411,993; Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennet, McKeam, and Bechtol (eds.), 1980.

[0156] For example, the host animals, such as mice, can be injected intraperitoneally at least once and preferably at least twice at about 3 week intervals with isolated and purified proteins or conjugated polypeptides, for example a peptide comprising or consisting of the specific amino acids set forth above. Mouse sera are then assayed by conventional dot blot technique or antibody capture (ABC) to determine which animal is best to fuse. Approximately two to three weeks later, the mice are given an intravenous boost of the protein or polypeptide. Mice are later sacrificed and spleen cells fused with commercially available myeloma cells, such as Ag8.653 (ATCC), following established protocols. Briefly, the myeloma cells are washed several times in media and fused to mouse spleen cells at a ratio of about three spleen cells to one myeloma cell. The fusing agent can be any suitable agent used in the art, for example, polyethylene glycol (PEG). Fusion is plated out into plates containing media that allows for the selective growth of the fused cells. The fused cells can then be allowed to grow for approximately eight days. Supernatants from resultant hybridomas are collected and added to a plate that is first coated with goat anti-mouse Ig. Following washes, a label, such as a labeled protein or polypeptide, is added to each well followed by incubation. Positive wells can be subsequently detected. Positive clones can be grown in bulk culture and supernatants are subsequently purified over a Protein A column (Pharmacia).

[0157] The monoclonal antibodies of the invention can be produced using alternative techniques, such as those described by Atting-Mees et al., “Monoclonal Antibody Expression Libraries: A Rapid Alternative to Hybridomas”, Strategies in Molecular Biology 3:1-9 (1990), which is incorporated herein by reference. Similarly, binding partners can be constructed using recombinant DNA techniques to incorporate the variable regions of a gene that encodes a specific binding antibody. Such a technique is described in Larrick et al., Biotechnology, 7:394 (1989).

[0158] Antigen-binding fragments of such antibodies, which can be produced by conventional techniques, are also encompassed by the present invention. Examples of such fragments include, but are not limited to, Fab and F(ab')2 fragments. Antibody fragments and derivatives produced by genetic engineering techniques are also provided.

[0159] The monoclonal antibodies described herein include in particular chimeric antibodies and humanized antibodies, i.e. versions of murine monoclonal antibodies with reduced immunogenicity.

[0160] Such chimeric and humanized antibodies can be prepared by known techniques, and offer the advantage of reduced immunogenicity when the antibodies are administered to humans. A “chimeric antibody”, as used herein, is an antibody in which the constant region, or a portion thereof, is altered, replaced, or exchanged, so that the variable region is linked to a constant region of a different species, or belonging to another antibody class or subclass. “Chimeric antibody” also refers to an antibody in which the variable region, or a portion thereof, is altered, replaced, or exchanged, so that the constant region is linked to a variable region of a different species, or belonging to another antibody class or subclass. In one embodiment, a chimeric monoclonal antibody comprises the variable region of a murine antibody (or just the antigen binding site thereof) and a constant region derived from a human antibody. Alternatively, a chimeric antibody fragment can comprise the antigen binding site of a murine monoclonal antibody and a variable region fragment (lacking the antigen-binding site) derived from a human antibody. Procedures for the production of chimeric and further engineered monoclonal antibodies include those described in Riechmann et al. (Nature 332:323, 1988), Liu et al. (PNAS 84:3439, 1987), Larrick et al. (Bio / Technology 7:934, 1989), and Winter and Harris (TIPS 14:139, May, 1993).

[0161] A “humanized antibody” as used herein refers to an antibody that contains CDR regions derived from an antibody of nonhuman origin, the other parts of the antibody molecule being derived from one (or several) human antibodies. In addition, some of the skeleton segment residues (called FR) can be modified to preserve binding affinity (Jones et al., Nature, 321 :522-525, 1986; Verhoeyen et al., Science, 239:1534-1536, 1988; Riechmann et al., Nature, 332:323-327, 1988).

[0162] The goal of humanization is a reduction in the immunogenicity of a xenogenic antibody, such as a murine antibody, for introduction into a human, while maintaining the full antigen binding affinity and specificity of the antibody. The humanized antibodies of the invention or fragments of same can be prepared by techniques known to a person skilled in the art (such as, for example, those described in Singer et al., J. Immun., 150:2844-2857, 1992; Mountain et al., Biotechnol. Genet. Eng. Rev., 10:1-142, 1992; and Bebbington et al., Bio / Technology, 10:169-175, 1992). Such humanized antibodies are preferred for their use in methods involving in vitro diagnoses or preventive and / or therapeutic treatment in vivo. Antibodies can be humanized using a variety of other techniques including CDR-grafting (EP 0 451 261, EP 0 682 040, EP 0 939 127, EP 0 566 647 or U.S. Pat. Nos. 5,530,101 , 6,180,370, 5,585,089 and 5,693,761), veneering or resurfacing (EP 0 592 106; EP 0 519 596; Padlan E. A., 1991, Mol Immunol, 28(4 / 5): 489-498; Studnicka G. M. et al., 1994, Protein Engineering 7(6): 805-814; Roguska M. A. et al., 1994, Proc. Natl. Acad. Sci. U.S.A., 91 : 969-973), and chain shuffling (U.S. Pat. No. 5,565,332). Another preferred method of humanization of antibodies, based on the identification of flexible residues, has been described in PCT application WO 2009 / 032661.

[0163] In certain embodiments both the variable and constant regions of the antibodies, or antigenbinding fragments, variants, or derivatives thereof are fully human. Fully human antibodies can be made using techniques that are known in the art. For example, fully human antibodies against a specific antigen can be prepared by administering the antigen to a transgenic animal which has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled. Exemplary techniques that can be used to make such antibodies are described in U.S. Pat. Nos. 6,150,584; 6,458,592; 6,420,140. Other techniques are known in the art. Fully human antibodies can likewise be produced by various display technologies, e.g., phage display or other viral display systems. See also U.S. Pat. Nos. 4,444,887, 4,716,111 , 5,545,806, and 5,814,318; and international patent application publication numbers WO 98 / 46645, WO 98 / 50433, WO 98 / 24893, WO 98 / 16654, WO 96 / 34096, WO 96 / 33735, and WO 91 / 10741 (said references incorporated by reference in their entireties). Procedures to generate antibodies transgenically can be found in GB 2,272,440, U.S. Pat. Nos. 5,569,825 and 5,545,806.

[0164] Antibodies produced by genetic engineering methods, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, can be used. Such chimeric and humanized monoclonal antibodies can be produced by genetic engineering using standard DNA techniques known in the art, for example using methods described in Robinson et al. International Publication No. WO 87 / 02671 ; Akira, et al. European Patent Application 0184187; Taniguchi, M., European Patent Application 0171496; Morrison et al. European Patent Application 0173494; Neuberger et al. PCT International Publication No. WO 86 / 01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al. European Patent Application 0125023; Better et al., Science 240: 1041 1043, 1988; Liu et al., PNAS 84:3439 3443, 1987; Liu et al., J. Immunol. 139:3521 3526, 1987; Sun et al. PNAS 84:214 218, 1987; Nishimura et al., Cane. Res. 47:999 1005, 1987; Wood et al., Nature 314:446 449, 1985; and Shaw et al., J. Natl. Cancer Inst. 80:1553 1559, 1988); Morrison, S. L, Science 229:1202 1207, 1985; Oi et al., BioTechniques 4:214, 1986; Winter U.S. Pat. No. 5,225,539; Jones et al., Nature 321 :552 525, 1986; Verhoeyan et al., Science 239:1534, 1988; and Beidler et al., J. Immunol. 141 :4053 4060, 1988.

[0165] In connection with synthetic and semi-synthetic antibodies, such terms are intended to cover but are not limited to antibody fragments, isotype switched antibodies, humanized antibodies (e.g., mousehuman, human-mouse), hybrids, antibodies having plural specificities, and fully synthetic antibody-like molecules.

[0166] In one embodiment, the anti-cancer immunotherapeutic comprises a monoclonal antibody that targets CD28, CD52, EGFR, VEGF, HER-2, CD20, CD16, 0X40, CD137, CD27, GITR, CD40, CD19, CD272, CD279, CD274, PAP, CD38, CD47, or GD2.

[0167] In one embodiment, the anti-cancer immunotherapeutic comprises a monoclonal antibody that targets a transmembrane programmed cell death 1 protein (PDCD1 , PD-1; also known as CD279) or its ligand, PD-1 ligand 1 (PD-L1 , CD274), such as for example Nivolumab described in Pardoll, D M Nature reviews of Mar. 22, 2012, incorporated herein by reference. In one embodiment, the anti-cancer immunotherapeutic comprises a monoclonal antibody that targets B7-H3, CTLA-4 (e.g., Ipilimumab), GITR, 0X40, LAG-3, CTLA-4 (CD152, or TIM-3 / Tim-3L.

[0168] In one embodiment, the anti-cancer immunotherapeutic comprises multiple antibodies, including combinations of 2 or 3 of any of the antibodies detailed herein. In a preferred embodiment, the anticancer immunotherapeutic comprises antibodies targeting PD-1 and LAG-3.

[0169] In still another embodiment, the anti-cancer immunotherapeutic comprises an anti-CD47 antibody, for example as described in Keith Syson Chan et at, Proc Natl Acad Sci USA. 2009 Aug. 18; 106(33): 14016-14021 , incorporated herein by reference.

[0170] In still another embodiment, the anti-cancer immunotherapeutic comprises an anti-GD3 or anti- GD2 antibody, for example as described in Ahmed, M; Cheung, N K (Jan. 21 , 2014). “Engineering anti- GD2 monoclonal antibodies for cancer immunotherapy.”. FEBS Letters 588 (2): 288-97, incorporated herein by reference. In another embodiment, the anti-cancer immunotherapeutic comprises Bec2, an anti-idiotypic antibody that mimics GD3, a ganglioside antigen, preferably with Bacillus Calmette-Guerin (BCG), as described in Giaccone et al., J Clin Oncol. 2005 Oct. 1 ; 23(28):6854-64, incorporated herein by reference.

[0171] Also contemplated herein are modified immune cells. The immune cells of the invention encompass any type of cell of the immune system. In a preferred embodiment, the immune cells of the invention are dendritic cells. A modified immune cell as used herein is an immune cell which has been engineered in order to modify its properties and, as a consequence, the behavior of the immune system (see e.g., Porter, D. L. et al. N. Engl. J. Med. doi:10.1056 / nejmoa1103849, 2011 ; Kalos, M. et al. Sci. Transl. Med. 3, 95ra73; 2011 ; Brentjens, R. J. et al. Sci. Transl. Med. 5, 177ra38, 2013). Preferably, said modified immune cell has been genetically modified. More preferably, said genetically modified immune cell expresses a protein or an RNA. The immune cells can be loaded with a protein. See U.S. Pat. No. 7,414,108. The immune cells can be loaded with an RNA. See U.S. Pat. No. 7,105,157, which is hereby incorporated by reference.

[0172] The immune cell therapy can be, for example, SIPLEUCEL T, BELAGENPUMATUCEL-L, or TERGENPUMATUCEL-L (Villaruz et al. Transt Lung Cancer Res. 2014 February; 3(1 ): 2-14.), incorporated herein by reference.

[0173] In a preferred embodiment, the modified immune cell is a dendritic cell that expresses a PAP antigen.

[0174] The invention encompasses modified immune cells including T cells expressing chimeric antigen receptors (CARs) and T cells modified through altering the specificity of the T cell receptor (TCRs) targeting and TAAs, particularly those detailed herein. TCRs, and CARs and immune cells expressing them, can be produced using routine techniques in the art, for example, those set forth in U.S. Pat. Nos. 8,088,379, 8,785,601, 5,359,046 and 8,389,282, which are hereby incorporated by reference.

[0175] In a preferred embodiment, the modified immune cell expresses a chimeric antigen receptor (CAR) or is a TCR directed against CD19, melanoma-associated antigen recognized by T cells 1 (MART- 1 ), glycoprotein (gp) 100, carcinoembryonic antigen (CEA), p53, MAGE-A3, or New York esophageal squamous cell carcinoma antigen (NY-ESO-1 ).

[0176] In preferred embodiments, the modified immune cell expresses a chimeric antigen receptor (CAR) or is a TCR directed against folate receptor (FR) (preferably in ovarian cancer), carbonic anhydrase IX (CAIX) (preferably in renal cell carcinoma), L1 -cell adhesion molecule (L1-CAM; CD171), CD20 (preferably in indolent non-Hodgkin lymphoma), and diasialoganglioside GD2 (preferably in neuroblastoma).

[0177] In preferred embodiments, the modified immune cell expresses a chimeric antigen receptor (CAR) or is a TCR directed against CD19, HER-2, or CEA.

[0178] Preferably, the CAR comprises a single chain antibody, preferably a humanized scFv or an scFv derived from a human monoclonal antibody, directed against a tumor TSA or TAA.

[0179] In a preferred embodiment, the immune cell has been modified with a vector, particularly a plasmid, a poxvirus, an adenovirus, an adeno-associated virus, an integrative or non-integrative lentivirus, or a measles virus vector. In a particularly preferred embodiment, the lentivirus technology set forth in U.S. Pat. No. 8,460,678 is used to construct the modified immune cell.

[0180] By “cytokine”, it is herein referred to a group of cell signaling proteins that aid cell to cell communication in immune responses and stimulate the movement of cells towards sites of inflammation, infection and trauma. Cytokines are classified as being proinflammatory (T helper 1 , Th1 ) or antiinflammatory (T helper 2, Th2) depending on their effects on the immune system.

[0181] The invention encompasses the use of cytokines as anti-cancer immunotherapeutics. Particularly preferred cytokines include interleukin or interferon. Particularly preferred cytokines are GM-CSF, IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12 IL-15, IL-17, IL-23, IL-1 , TNF-a, IFN-a, IFN-|3, IFN-y, an agonist of any thereof, or any combination thereof.

[0182] An “immune checkpoint” as used herein refers to an inhibitory pathway hardwired into the immune system that is crucial for maintaining self-tolerance and modulating the duration and amplitude of physiological immune responses in peripheral tissues in order to minimize collateral tissue damage. The expression of immune-checkpoint proteins is known to be dysregulated in at least some tumors as an important immune resistance mechanism, in particular against T cells. An “immune checkpoint blockade molecule”, as used herein, is a molecule which blocks said immune checkpoint, thus overriding the immune resistance of the tumor. Immune checkpoints and immune checkpoint blockade molecule are well-known in the literature. See e.g., Nature Reviews Cancer 12: 252-264, April 2012).

[0183] In various embodiments, the invention encompasses an immune checkpoint blockade molecule, preferably a monoclonal antibody, which targets CTLA-4, PD-L1 or PD-1.

[0184] In various embodiments, the anti-cancer immunotherapeutic comprises a monoclonal antibody that targets CD27, CD28, CD40, CD122, CD137, B7-H3, B7-H4, A2R2, ICOS, VISTA, B7-H3, KIR, IDO, BTLA, GITR, 0X40, LAG-3 or TIM-3 / Tim-3L.

[0185] In some embodiments, the anti-cancer immunotherapeutic comprises multiple antibodies, including combinations of 2 or 3 antibodies targeted against CD27, CD28, CD40, CD122, CD137, B7-H3, B7-H4, A2R2, ICOS, VISTA, B7-H3, KIR, IDO, BTLA, CTLA-4, PD-L1 , PD-1 , GITR, 0X40, LAG-3 or TIM- 3 / Tim-3L. In a preferred embodiment, the anti-cancer immunotherapeutic comprises antibodies targeting PD-1 and LAG-3.

[0186] In preferred embodiments, the monoclonal antibody is NIVOLUMAB, an lgG4 anti-PD-1 monoclonal antibody that acts as an immunomodulator by blocking ligand activation of the programmed cell death 1 (PD-1 ) receptor on activated T cells. Preferably, it is used for patients with metastatic melanoma or for the treatment of squamous non-small cell lung cancer.

[0187] PEMBROLIZUMAB is a humanized monoclonal antibody that targets the programmed cell death 1 (PD-1 ) receptor. Preferably, it is for use following treatment with IPILIMUMAB, or after treatment with IPILIMUMAB and a BRAF inhibitor in advanced melanoma patients who carry a BRAF mutation.

[0188] Further contemplated are nucleic acid vectors expressing an antigen such as one of the antigens described above.

[0189] In a preferred embodiment, the vector is a plasmid vector.

[0190] Vectors are well-known in the art and include measles virus, lentivirus, retrovirus, adenovirus, poxvirus, herpes virus, measles virus, foamy virus or adeno-associated virus (AAV). Viral vectors can be replication-competent, or can be genetically disabled so as to be replication-defective or replication- impaired. Suitable vectors can be integrative or non-integrative.

[0191] In one embodiment, the vector is an Alphavirus vector. Alphaviruses are single-stranded positivesense RNA viruses that replicate in the cytoplasm of infected cells. In various embodiments, the vector is a Venezuelan equine encephalitis virus (VEE), Sindbis virus (SIN), Semliki forest virus (SFV), and VEE- SIN chimera vector.

[0192] In various embodiments, the vector is a poxvirus, preferably a vaccinia virus, vector. In one embodiment, the poxvirus vector expresses a tumor antigen, such as prostate-specific antigen (PSA) or CEA, and multiple human T-cell co-stimulatory molecules (B7.1 , LFA-3, and intracellular adhesion molecule-1 ). In various embodiments, the poxvirus vector is a replicating poxviral vector selected from attenuated modified vaccinia virus Ankara (MVA), NYVAC (derived from the Copenhagen strain of vaccinia), and ALVAC (canarypoxviral vector) strains.

[0193] In various embodiments, the vector is a lentiviral vector. Preferred vectors are the DNA Flap vectors as described in WO 99 / 055892, U.S. Pat. No. 6,682,507 and WO 01 / 27300, and U.S. Pat. No. 8,460,678, which are hereby incorporated by reference.

[0194] In a preferred embodiment, the virus expresses a 5T4 tumor-associated antigen. The nucleic acid vector can be an mRNA. Preferable, the mRNA is a modified mRNA, preferably in a nanoparticle. See, e.g., U.S. Pat. Nos. 8,664,194, 8,754,062, and 8,999,380, which are hereby incorporated by reference.

[0195] In some specific aspects, a triple system is described in which the combination therapy comprises THT, a cytokine, and a checkpoint inhibitor. Typically, the THT is MNP-mediated, the cytokine comprises IL-2, and / or the checkpoint inhibitor is a PD-1 inhibitor, such as an anti-PD-1 antibody, such as pembrolizumab.

[0196] The combinations disclosed herein are in embodiments non-toxic and / or pharmaceutically acceptable and are therefore suitable for in vivo use in treating diseases or disorders such as cancer, or for diagnostic or imaging purposes. Compositions comprising the combinations disclosed herein are also contemplated, including pharmaceutically acceptable compositions (e.g. formulations). Various types of pharmaceutical compositions can be used, depending on the desired form of administration. For example, aqueous compositions comprise an effective amount of the metal nanoparticles disclosed herein dissolved and / or dispersed in a pharmaceutically acceptable carrier and / or aqueous medium. The pharmaceutical compositions disclosed herein can further comprise supplementary active ingredients, such as an anti-cancer agent, such as an anti-cancer immunotherapy.

[0197] Other delivery methods of the present invention comprise compositions comprising one or more lipids associated with the combination. A lipid is a substance that is characteristically insoluble in water and extractable with an organic solvent. Lipids include, for example, the substances comprising the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which are well known to those of skill in the art which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes. These examples are not meant to be limiting, and compounds other than those specifically disclosed herein that are understood by one of skill in the art as lipids are also encompassed by the compositions and methods disclosed herein.

[0198] For example, a lipid may be naturally occurring or synthetic (i.e., designed or produced by man). However, a lipid is usually a biological substance. Biological lipids are well known, and include for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulphatides, lipids with ether and ester-linked fatty acids and polymerizable lipids, and combinations thereof. In particular embodiments, a lipid comprises a liposome. A liposome is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes may be characterized as having vesicular structures with a bilayer membrane, generally comprising a phospholipid, and an inner medium that generally comprises an aqueous composition. A multilamellar liposome has multiple lipid layers separated by aqueous medium. They form spontaneously when lipids comprising phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures, entrapping water and dissolved solutes between the lipid bilayers. Lipophilic molecules or molecules with lipophilic regions may also dissolve in or associate with the lipid bilayer.

[0199] In particular embodiments, one or more components of the combinations may be, for example, encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the component, entrapped in a liposome, complexed with a liposome, etc. A liposome used as disclosed herein may be made by different methods, as would be known to one of ordinary skill in the art. Phospholipids can form a variety of structures other than liposomes when dispersed in water, depending on the molar ratio of lipid to water. At low ratios the liposome is the typical structure. The size of a liposome varies depending on the method of synthesis. Liposomes disclosed herein can have a variety of sizes. In certain embodiments, the liposomes are small, e.g., less than about 100 nm, about 90 nm, about 80 nm, about 70 nm, about 60 nm, or less than about 50 nm in external diameter. In preparing such liposomes, any protocol disclosed herein, or as would be known to one of ordinary skill in the art may be used. Additional non-limiting examples of preparing liposomes are described in U.S. Patent Nos. 4,728,575, 4,737,323, 4,533,254, 4,162,282, 4,310,505, and 4,921 ,706; A comprehensive review of lipid vesicles and methods for their preparation are described in "Liposome Technology" (1984. Gregoriadis G. ed. ORC Press Inc Boca Raton Florida Vol I II & III).

[0200] Liposomes interact with cells to deliver agents via four different mechanisms: Endocytosis by phagocytic cells of the reticuloendothelial system such as macrophages and / or neutrophils; adsorption to the cell surface, either by nonspecific weak hydrophobic and / or electrostatic forces, and / or by specific interactions with cell-surface components; fusion with the plasma cell membrane by insertion of the lipid bilayer of the liposome into the plasma membrane, with simultaneous release of liposomal contents into the cytoplasm; and / or by transfer of liposomal lipids to cellular and / or subcellular membranes, and / or vice versa, without any association of the liposome contents. Varying the liposome formulation can alter which mechanism is operative, although more than one may operate at the same time.

[0201] A skilled person realizes that the combinations and methods disclosed herein can be employed in a variety of types of experimental, therapeutic and diagnostic procedures, including in vitro or in vivo procedures. In another embodiment, systems, devices, materials, and techniques are described for minimally invasive active targeting, fluorescent imaging, and NIR photothermal treatment of tumours, for example, which can be applied to a variety of cancer types.

[0202] Protecting the MNP upon exposure to various elements, such as whole blood, are described herein. Polymers can be used to protect the MNP, such as synthetic polymers and / or natural polymers. Synthetic polymers are those suitable for use in mammals. Examples of synthetic polymers include PEG (polyethylene glycol), PVP (polyvinylpyrrolidone), poly lactic-co-glycolic acid (PLGA), PSS (polystyrene sulfonate), PNIPAM (poly(N-isopropylacrylamide)), ploxamer, diblock or triblock polymers, pH or thermo- responsive polymers, or a combination thereof. Natural polymers are those suitable for use in mammals. Examples of natural polymers include glycoproteins, albumin, gelatine, collagen, or a combination thereof. In addition to polymer(s) or separately, phospholipids (both low and high density), micelles, vesicles, liposomes, solid lipid nanoparticle (SLN), nanostructured lipid carriers (NLC), or a combination thereof. For the protection of the MNP with polymer(s), a selected polymer may have a thiol or disulphide functional group (e.g. at a terminal end) or has been modified to include this functional group. Nanoaggregates such as micelles, vesicles, liposomes, solid lipid nanoparticle (SLN), nanostructured lipid carriers (NLC) and / or supramolecular vesicles are described for encapsulation and / or surface adsorption of the MNP for intratumoral injection.

[0203] According to certain embodiments, the pharmaceutical composition or combinations described herein are formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, sub-cutaneous, intralesional, and intraperitoneal routes. Typically, such compositions are prepared either as liquid solutions or suspensions; solid forms suitable for using to prepare solutions and / or suspensions upon the addition of a liquid prior to injection can also be prepared; and the preparations can also be emulsified. The combinations disclosed herein can be formulated into a composition in a neutral and / or salt form for example. Any pharmaceutically acceptable salt known to a person skilled in the art can be used, providing it would not interfere with the function of the combination or individual components thereof.

[0204] Sterile injectable solutions are generally prepared by incorporating the active compounds in the required amount in the appropriate solvent with other ingredients, as required, followed by filtered sterilization.

[0205] Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and / or the required other ingredients as disclosed herein above. In the case of sterile powders for the preparation of sterile injectable solutions, the typical methods of preparation are vacuum-drying and / or freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The preparation of more, and / or highly, concentrated solutions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small target area.

[0206] In another method, the MNP may be wrapped in a polymer. In embodiments, the wrapping polymers may include proteins, gelatin, bovine serum albumin, polystyrene sulfonate, polyethylene oxides, thiolated polyethylene oxides, thiolated polyethyene oxides with terminating carboxylic acid functionalities, thiolated polyethyene oxides with terminating amine acid functionalities, and combinations thereof.

[0207] In an embodiment, the wrapping polymer may form covalent and / or non-covalent bonds with other polymers, proteins, etc. In certain embodiments, the carboxylic ending of thiolated polyethylene oxides is bound to protein(s), polypeptide(s), antibodie(s), antibody fragment(s), IgG class of antibody, a polyclonal antibody, a monoclonal antibody, and combinations thereof. In another embodiment, the amine ending thiolated polyethylene oxide is bound to protein(s), polypeptide(s), antibodie(s), antibody fragment(s), IgG class of antibody, a polyclonal antibody, a monoclonal antibody, and combinations thereof. Such covalently bounded bioconjugates may be formed, for example, from any metal nanorods disclosed herein, a polymer, and, for example, an antibody, protein(s), polypeptide(s), antibodie(s), antibody fragment(s), IgG class of antibody, a polyclonal antibody, a monoclonal antibody. In an embodiment, single or double stranded nucleic acid may be tethered to MNPs with metal-thiol bonds. In an embodiment, an oligonucleotide may be tethered to metal nanorods with metal-thiol bonds.

[0208] In embodiments, the wrapping polymer is covalently bonded to the MNP. In embodiments, the wrapping polymer is non-covalently bonded to the MNP.

[0209] The MNP can comprise a variety of capping agents are described. In an embodiment, methods of capping the MNP with a non-surfactant involve first removal of a solvent or excess surfactant from the metal nanorod solution followed by addition of an aqueous solution of capping agent(s). In typical embodiments, about 95% to about 98% of the solvent is removed and a similar quantity of the aqueous solution of the new capping agent(s) is added. In a more specific embodiment, the method comprises removal of about 95% to about 98% of solvent from the metal nanorod solution, followed by the addition of a similar amount of an aqueous solution of a first capping agent (e.g. an ionic polymer, typically an anionic polymer), and allowing the solution to equilibrate for a period of time. For example, the mixture may be equilibrated to a temperature of about 4°C to about 25°C for at least about 1 hour. Then removing about 95% to about 98% of resultant solvent from the resultant nanoparticle pellets, for example, by using a centrifugal method, and additional dispersion of the resultant MNP into an aqueous solution of a second capping agent (e.g. same or different from first capping agent).

[0210] In embodiments, the capping agent of MNP in a colloidal solution form may be a mixture of surfactant and a thiolated polymer (polyethylene glycol of mwt. of about 200Da to about 50kDa) or it can be poly lactic-co-glycolic acid (PLGA) of mwt. of about 200Da to about 50kDa. Other ranges include for example, about 600Da to about 50kDa, about 700Da to about 50kDa, about 800Da to about 50kDa, about 900Da to about 50kDa, about 1 kDa to about 50kDa, about 1 kDa to about 40kDa, about 1 kDa to about 30kDa, about 1 kDa to about 20kDa, or about 1 kDa to about 10kDa. I n an embodiment, the capping agent of MNP in a colloidal solution form may be a mixture of surfactant from a surfactant solution and a thiolated polymer (polyethylene glycol of mwt. of about 500Da to about 50kDa, Other ranges include for example, about 600Da to about 50kDa, about 700Da to about 50kDa, about 800Da to about 50kDa, about 900Da to about 50kDa, about 1 kDa to about 50kDa, about 1 kDa to about 40kDa, about 1 kDa to about 30kDa, about 1 kDa to about 20kDa, or about 1 kDa to about 10kDa). In an embodiment, the capping agent of MNP may be a mixture of surfactant, a co-surfactant, and small biomolecules. The small biomolecules may be selected from a general class of flavonoids, antioxidants, aromatic acids, amino acids, monohydroxybenzoic acid, monosaccharides, disaccharides, bile salt, nucleotides, or combinations thereof. In an embodiment, co-capping agent(s) may be added such as quercetin, epigallocatechin gallate, curcumin, glutathione, ascorbic acid, citric acid, anthranilic acid, cinnamic acid, bile acid, and p-hydroxybenzoic acid, metal anionic salts of biological acid(s), or combinations thereof.

[0211] / / . Methods of Treatment and Uses Thereof

[0212] In embodiments, the combinations described herein are used for sensitizing a cancer to cancer immunotherapy. In other embodiments, the combinations described herein are used for exposing tumour antigens for cancer immunotherapy. In other embodiments, the combinations described herein are used for increasing CD8+ T cell infiltration into a tumour, increasing CD8+ memory T cell subsets, and / or increasing PD-1 expression on CD+ T cells. In other embodiments, the combinations described herein are used for treating cancer.

[0213] In other embodiments, MNP-mediated THT is used to stimulate an immune response. Thus, a method for stimulating an immune response comprises administering MNP-mediated THT to a subject in need thereof.

[0214] In other embodiments, MNP-mediated THT is used to treat a tumour, wherein the THT is for administration at least twice, wherein each THT administration is separated by a period of time such as from about 1 day to about 3 months, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, or 14 days, with or without further administration of MNPs.

[0215] The methods can comprise administering a therapeutically effective amount of the combinations disclosed herein, or a pharmaceutical composition thereof, to a subject in need thereof. In other embodiments, the use of the combinations or the pharmaceutical composition thereof for treatment of cancer in a subject is provided. The methods and uses of the combinations disclosed herein or the pharmaceutical composition thereof may improve therapeutic outcomes while minimizing side-effects in a subject, as further described below. In specific embodiments, the combinations disclosed herein can be used to treat a tumour. The method can comprise administering a therapeutically effective amount of the combinations disclosed herein, or a pharmaceutical composition thereof, to a subject in need thereof for treating a tumour. In other embodiments, the use of the combinations or the pharmaceutical composition thereof for treatment of a tumour in a subject is provided. The methods and uses disclosed herein or the pharmaceutical composition thereof may improve therapeutic outcomes while minimizing side-effects in a subject, as further described below.

[0216] Therefore, cancer may be treatable and / or preventable (e.g. prevention of further cancer development and / or metastasis) by administration or delivery of the combinations as disclosed herein, or a pharmaceutical composition thereof.

[0217] In some aspects, the combinations disclosed herein may be administered to a cell (e.g. cancerous cells) or tissue (e.g. tumour) using targeting schemes involving specific chemical interactions (e.g., antigen-antibody binding, etc.), such as for example, through the use of a tumour-targeting agent so as to directly target one or more component of the combination to the tumour, or may consist of the direct delivery of one or more component of the combination to the desired area (e.g. tumours). The combinations disclosed herein may be delivered in the form of a pharmaceutical composition. Typically, the MNPs are administered intratumourally or by i.v. and will accumulate in a tumour. In aspects, the immunotherapy may also be administered intratumourally or by other known routes of administration as described herein. In some aspects, intratumoural administration of the MNPs and / or the immunotherapy advantageously results in treatment of the local tumour as well as treatment of a distant (e.g. metastatic) tumour to which the MNPs and / or immunotherapy were not directly administered.

[0218] The MNP disclosed herein has optical properties, in that it can absorb light in the NIR of about 700 nm to about 1000 nm. Laser light at this wavelength is typically used, as a high percentage of this wavelength of light can pass through biological tissue. As a result, the MNP can be injected directly into the bloodstream or via other methods as described herein, such as intratumorally, and their location / distribution can be determined using a safe laser light, also referred to as infrared irradiation. In this way, the methods and uses disclosed herein using EMR-NIR can also increase the subject’s level of comfort during the treatment thereof.

[0219] In embodiments, the EMR is used to irradiate the MNP or composition(s) disclosed herein. In embodiments, the MNP absorbs the EMR and generates heat to temperatures of about 40°C or greater, such as, for example, in a range of about 42 °C to about 45 °C, and more particularly about 44 °C, which heats the target tissue (e.g. the tumour of the subject) to similar temperatures to inhibit growth / reduce the tumour / generate neoantigens.

[0220] The EMR may be generated by, for example, a light-emitting diode (LED) or a laser generator. LEDs and laser generators that are capable of generating EMR, and more particularly NIR-EMR, are available. As non-limiting examples, such laser generators may include semiconductor laser generators (also referred to as laser diodes) with a non-limiting example being a vertical-cavity surface-emitting laser (VCSEL), and solid-state laser generators (i.e. , a laser that uses a solid gain medium) with a non-limiting example being a titanium sapphire laser generator. Devices capable of delivery EMR are those described in US 63 / 562461 , the entirety of which is incorporated by reference.

[0221] In certain embodiments, the EMR functions to excite the MNP to generate heat. In embodiments, the MNP generate heat to inhibit / reduce growth of tumour tissue and generate neo antigens. To this regard, the MNP disclosed herein can heat-up when irradiated. This property results in their ability to increase the temperature locally, for example in the immediate vicinity of a specific target. Thus, when the target is a tumour or individual cancer cells, the tumour or cell will be damaged or destroyed. This allows for non-invasive anti-cancer therapy using the MNP disclosed herein.

[0222] The cancer disclosed herein may be any cancer. A subject that has a cancer or a tumour is a subject that can have objectively measurable cancer cells present. In some embodiments, a tumour may be or comprise cells that are precancerous (e.g. , benign), malignant, pre-metastatic, metastatic, and / or non-metastatic. In some embodiments, the cancer may be characterized by a solid tumour, and in other embodiments, the relevant cancer may be characterized by a hematologic tumour. Examples of different types of cancers include, for example, hematopoietic cancers including leukemias, lymphomas (Hodgkin's and non-Hodgkin's), myelomas and myeloproliferative disorders; sarcomas, melanomas, adenomas, carcinomas of solid tissue, squamous cell carcinomas mouth, throat, larynx, and lung, liver cancer, genitourinary cancers such as prostate, cervical, bladder, uterine, and endometrial cancer and renal cell carcinomas, bone cancer, pancreatic cancer, skin cancer, cutaneous or intraocular melanoma, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, head and neck cancers, breast cancer, brain cancer, kidney cancer, gastro-intestinal cancers and nervous system cancers, benign lesions such as papillomas, and the like.

[0223] In embodiments, the cancer is selected from brain tumour, nerve sheath tumour, breast cancer, colon cancer, colorectal cancer, skin cancer, lung cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, renal cell carcinoma, carcinoma, melanoma, lymphoma, head and neck cancer, brain cancer, and prostate cancer, including but not limited to androgen-dependent prostate cancer and androgen-independent prostate cancer. In embodiments, the cancer is prostate cancer, and in other embodiments, the cancer is breast cancer.

[0224] The tumour tissue and / or tumour to be treated may be of any source. In embodiments, the tumour tissue or the tumour, is derived from a peripheral tumour, breast cancer, colon cancer, colorectal cancer, skin cancer, lung cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, renal cell carcinoma, carcinoma, melanoma, lymphoma, head and neck cancer, brain cancer, and prostate cancer, including but not limited to androgen-dependent prostate cancer and androgenindependent prostate cancer. In embodiments, the tumour is derived from prostate cancer, and in other embodiments, the tumour is derived from breast cancer.

[0225] The subject referred to herein is, typically, a mammal, and most typically a human that that is suffering from cancer and / or has a tumour that can be treated by the methods and uses disclosed herein. Thus, the subject has detectable cancer cells which can be treated by the methods or uses disclosed herein. The methods and uses disclosed herein may also have a preventative function, in that, if cancer cells are detected in the subject, and the subject is subsequently treated by the methods and uses disclosed herein, further development of cancer, or metastasis may be prevented through application of the methods and uses disclosed herein. Thus, the uses and methods disclosed herein can be used to treat, such as, to destroy a tumour, if for example, the combinations are provided (e.g. administered) to the subject who suffers from cancer and / or has a tumour disclosed herein. In alternative embodiments, further cancer and / or tumour development may be halted by treating the cancer and / or tumour with the methods disclosed herein. For purposes of the present disclosure, the subject may have a single cancer cell, a single cancer, a single tumour, multiple cancer cells, multiple cancers (e.g. breast and skin cancer) and / or multiple tumours, that are to be treated by the uses and methods disclosed herein.

[0226] In some embodiments, treatment refers to an increased survival (e.g. an increased survival time). For example, treatment can result in an increased life expectancy of a subject. In some embodiments, treatment results in an increased life expectancy of a subject by more than about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 105%, about 110%, about 115%, about 120%, about 125%, about 130%, about 135%, about 140%, about 145%, about 150%, about 155%, about 160%, about 165%, about 170%, about 175%, about 180%, about 185%, about 190%, about 195%, about 200% or more, as compared to the average life expectancy of one or more control individuals with similar disease without treatment. In some embodiments, treatment according to the present invention results in an increased life expectancy of a patient (subject) by more than about 6 month, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years or more, as compared to the average life expectancy of one or more control individuals with a similar disease without treatment. In some embodiments, treatment results in long term survival of a patient. As used herein, the term “long term survival” refers to a survival time or life expectancy longer than about 20 years, 30 years, 40 years, 50 years, 60 years, or longer.

[0227] Administration "in combination with" one or more further therapeutic agents can include simultaneous (concurrent) and consecutive administration in any order.

[0228] In embodiments, a therapeutically effective amount of at least one MNP in combination with an immunotherapy may be used. Administration may comprise a single administration, or alternatively, comprise a series of administrations or systematic administration of combinations with different MNPs and immunotherapies. The length of the treatment period can depend on factors, such as the severity of the disease, the age of the subject, the components of the combination, the dosage of the combination, the cancer stage, frequency of administration, intensity of light, duration of light, a combination thereof. It will also be appreciated that the effective dosage of the agent used for the treatment may increase or decrease over the course of a particular treatment regime. Changes in dosage may result and become apparent by standard diagnostic assays known in the art. The combination disclosed herein may, in embodiments, be administered before, during or after treatment with conventional therapies for the disease or disorder in question, such as cancer.

[0229] In some aspects, the THT is administered a single time or a plurality of times, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 times, with each THT administration being separated by a period of time. This period of time may be from about 1 day to about 3 months, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, or 14 days, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks, or 1, 2, or 3 months, or any combination or range thereinbetween. The THT may be used with or without further administration of MNPs.

[0230] Similarly, in some aspects, the immunotherapy is administered a single time or a plurality of times and the immunotherapy may be administrated as a single agent or a combination of agents. For example, the immunotherapy may comprise the use of a cytokine such as IL-2 and a checkpoint inhibitor, such as a PD-1 inhibitor. When the immunotherapy is administered a plurality of times, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 times, with each immunotherapy administration being separated by a period of time. This period of time may be from about 1 day to about 3 months, such as about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, or 14 days, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12 weeks, or 1, 2, or 3 months, or any combination or range thereinbetween.

[0231] In exemplary aspects, the THT is administered a single time and the immunotherapy is administered a plurality of times, before, during, and / or after the THT, such as at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times, typically two or three times following the THT, wherein each immunotherapy administration is separated by a period of time such as from about 1 hour to about 1 month, such as about 1 , 2, 3, 4, 5, 6, or 7 days.

[0232] In other embodiments, the combination disclosed herein can be used in the manufacture of a medicament, and typically the medicament is for the prevention (e.g. further cancer development and / or metastasis) and / or treatment of the cancers disclosed herein. In typical embodiments, the combinations, as a medicament, are for administration to a subject (e.g. mammals, typically humans) in need thereof.

[0233] The combination can be administered to mammals, typically humans. When administered as a pharmaceutical composition, the combination may be provided in combination with pharmaceutically acceptable carriers or diluents, optionally with pharmaceutically acceptable adjuvants, such as alum. The combination may, therefore, be suitably formulated into a pharmaceutical composition for administration to human subjects in a biologically compatible form suitable for administration in vivo.

[0234] Accordingly, in embodiments, the pharmaceutical composition comprises one or more components of the combination, in admixture with a suitable diluent or carrier. The compositions can be prepared by known methods for the preparation of pharmaceutically acceptable compositions which can be administered to subjects, such that an effective quantity of the components is combined in a mixture with a pharmaceutically acceptable carrier. Suitable carriers are described, for example, in Remington's Pharmaceutical Sciences (2003 - 20th edition), in The United States Pharmacopeia: The National Formulary (USP 24 NF19) published in 1999 and in the Handbook of Pharmaceutical Additives (compiled by Michael and Irene Ash, Gower Publishing Limited, Aidershot, England (1995)), the references of which are incorporated by reference in their entirety. On this basis, the composition may include solution(s) of the components in association with one or more pharmaceutically acceptable carrier(s) or diluent(s), and contained in buffered solution(s) with a suitable pH and iso-osmotic with the physiological fluids. Solution(s) can be prepared in water suitably mixed with suitable excipients. Under ordinary conditions of storage and use, these preparations can contain a preservative to prevent the growth of microorganisms. A person skilled in the art would know how to prepare suitable formulations / compositions. In this regard, reference can be made to U.S. Patent No. 5,843,456, which is incorporated herein by reference.

[0235] The combination may be administered alone or in combination with other components / ingredients / actives. For example, the combination may be administered as a pharmaceutical composition. The described combination and / or compositions thereof, may be administered to a subject in a variety of forms depending on the selected route of administration, as will be understood by those skilled in the art. The combination, and the pharmaceutical composition(s) thereof, may be administered, for example, by oral, parenteral (e.g. intravenous, intraperitoneal, subcutaneous, intramuscular, transepithelial, nasal, intrapulmonary, intrathecal, rectal and topical (e.g. ointment) modes of administration), intratumoural, buccal, sublingual, patch, pump or transdermal administration. In typical embodiments, the combinations disclosed herein are administered, or are for administration, using parenteral routes of administration. Typically, the combinations are administered intratumourally.

[0236] If the combinations are administered orally, the combinations may be administered, for example, in the form of tablets or capsules, or as an aqueous solution or suspension. Examples may include: ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. For tablet dosage forms, depending on dose, the components may make up from 1 wt % to 80 wt % of the dosage form, more typically from 5 wt % to 60 wt % of the dosage form. Moreover, carriers which are commonly used include lactose and corn starch, and lubricating agents, such as magnesium stearate, calcium stearate, zinc stearate, sodium stearyl fumarate, and mixtures of magnesium stearate with sodium lauryl sulphate, are commonly added. In addition, tablets generally contain a disintegrant. Examples of disintegrants include sodium starch glycolate, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, croscarmellose sodium, crospovidone, polyvinylpyrrolidone, methyl cellulose, microcrystalline cellulose, lower alkyl substituted hydroxypropyl cellulose, starch, pregelatinized starch and sodium alginate. Useful diluents include lactose (monohydrate, spray dried monohydrate, anhydrous and the like), mannitol, xylitol, dextrose, sucrose, sorbitol, microcrystalline cellulose, starch and dibasic calcium phosphate dihydrate, and suitable binders include microcrystalline cellulose, gelatin, sugars, polyethylene glycol, natural and synthetic gums, polyvinylpyrrolidone, pregelatinized starch, hydroxypropyl cellulose and hydroxypropyl methylcellulose. Other conventional ingredients include antioxidants, colorants, flavoring agents, preservatives and taste masking agents. Tablet blends may be compressed directly or by roller to form tablets. Tablet blends or portions of blends may alternatively be wet, dry, or melt granulated, melt congealed, or extruded before tableting. The final formulation may include one or more layers and may be coated or uncoated; or encapsulated. The formulation of tablets is discussed in detail in "Pharmaceutical Dosage Forms: Tablets, Vol. 1", by H. Lieberman and L. Lachman, Marcel Dekker, N.Y., N.Y., 1980 (ISBN 0 8247 6918 X), the disclosure of which is incorporated herein by reference in its entirety.

[0237] If the combinations are administered orally, the combinations may be administered, for example, in the form of an aqueous solution or suspension. When aqueous suspensions are prepared for oral use, the active ingredient can be combined with emulsifying and suspending agents. If desired, certain sweetening and / or flavoring agents may be added. The combination may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be incorporated directly into food. For oral therapeutic administration, the combination may be incorporated with excipient(s) and used in the form of ingestible elixirs, suspensions, syrups, and the like.

[0238] If the combinations are administered parenterally, the parenteral administration may be by continuous infusion, bolus, or intermittent bolus and may be over a selected period of time. Suitable examples of devices for parenteral administration include needle (including micro needle) injectors, needle free injectors and infusion techniques. For intramuscular, intraperitoneal, subcutaneous and intravenous use, sterile solutions of the active ingredient are usually prepared, and the pH of the solutions should be suitably adjusted and buffered. For intravenous use, the total concentration of solutes may be controlled in order to render the preparation isotonic. Thus, in embodiments, one or more of the combinations disclosed herein may be prepared in isotonic medium and administered intravenously.

[0239] The pharmaceutical forms suitable for injectable use may include sterile aqueous solutions or dispersion and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In embodiments, the form is sterile and the fluid is easily syringeable. The preparation of parenteral kits for reconstitution at point-of-care under sterile conditions, for example, by lyophilization, may readily be accomplished using standard pharmaceutical techniques known to those skilled in the art.

[0240] Combinations, including a pharmaceutical composition thereof, for nasal administration may conveniently be formulated as aerosols, drops, gels and powders. Aerosol formulations typically comprise a solution or fine suspension of the active substance in a physiologically acceptable aqueous or non-aqueous solvent and are usually presented in single or multidose quantities in sterile form in a sealed container, which can take the form of a cartridge or refill for use with an atomizing device (e.g. nebuliser, for example, to create a mist-like dispersion of combination such as an aqueous vehicle (e.g. saline)). Alternatively, the sealed container may be a unitary dispensing device such as a single dose nasal inhaler or an aerosol dispenser fitted with a metering valve which is intended for disposal after use. Where the dosage form comprises an aerosol dispenser, it will contain a propellant which can be a compressed gas such as compressed air or an organic propellant such as fluorochlorohydrocarbon. The aerosol dosage forms can also take the form of a pump-atomizer.

[0241] Combinations, including a pharmaceutical composition thereof, suitable for buccal or sublingual administration include tablets, lozenges, and pastilles, wherein the active ingredient is formulated with a carrier such as sugar, acacia, tragacanth, or gelatin and glycerine. Compositions for rectal administration are conveniently in the form of suppositories containing a conventional suppository base such as cocoa butter. In embodiments, a delivery system can be used to deliver the combination (e.g. formulations or pharmaceutical compositions). It is understood that the delivery system itself may include a device such as an implantable device.

[0242] The combination may be combined with soluble macromolecular entities, such as cyclodextrin and suitable derivatives thereof or polyethylene glycol containing polymers, in order to improve their solubility, dissolution rate, taste masking, bioavailability and / or stability for use in any of the aforementioned modes of administration. Regardless of the route of administration selected, the combination which may be used in a suitable hydrated form, and / or the pharmaceutical compositions thereof, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.

[0243] In embodiments, the above noted pharmaceutical compositions may be in the form of a controlled release composition, sustained release composition, extended release composition, modified release composition, pulsed release composition, delayed release composition, targeted release composition, site-specific release composition, time release composition, or a combination thereof. To this regard, the term “controlled release” may be variously characterized by "sustained release", “sustained action”, “extended release”, “modified release”, "pulsed release", "delayed release", “targeted release”, “site-specific release”, and “timed release”, which may be used interchangeably herein refer to the time of release, the extent of release, the rate of release, the site of release and / or release of an active ingredient from a composition at such a rate that when a dose of the active ingredient is administered in the sustained release, extended release, pulsed release, timed release, delayed release or controlled-release composition, concentrations (levels) of the active ingredient are maintained within a desired range but below toxic levels over a selected period of time. In the case of in vivo administration, concentrations (levels) of the active ingredient could be measured in blood or plasma, for example. When administered in v / vo the sustained release, extended release, pulsed release, timed release, delayed release or controlled-release composition allows for a timely onset of action and useful plasma concentration of an active ingredient to be maintained for longer than in the case of immediate-release forms. The skilled person would understand how the above described formulations may be made into the controlled release composition, sustained release composition, extended release composition, modified release composition, pulsed release composition, delayed release composition, targeted release composition, site-specific release composition, or time release composition.

[0244] Actual dosage levels of the combination may be varied so as to obtain an amount which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration. To this regard, the dosage of the combination can depend upon the pharmacokinetic and pharmacodynamic properties of the combination and its mode and route of administration; the rate of release of the combination, the age, sex, health, medical condition, the nature and extent of the symptoms and weight of the recipient, the renal and hepatic function of the patient; the frequency of the treatment and the type of concurrent treatment, if any, and the clearance rate of the combination in the subject to be treated and the effect desired. The selected dosage level may also depend on the additional factors including the activity of the particular combination and pharmaceutical compositions disclosed herein, the time of administration, the rate of excretion or metabolism of the particular combination being employed, the rate and extent of absorption, the duration of the treatment, other drugs that may be administered to the patient, compounds and / or materials used in combination with the particular combination employed and like factors well known in the medical arts. One of skill in the art can determine the appropriate dosage based on the above factors.

[0245] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the combination or pharmaceutical composition thereof. For example, the physician or veterinarian could start doses of the combination employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, a suitable daily dose of the combination will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.

[0246] The components of the combination may be administered initially in a suitable dosage that may be adjusted as required, depending on the clinical response. In some embodiments, the MNP and / or immunotherapy may be administered in an amount from about 0.001 mg / kg of body weight to about 1000 mg / kg of body weight per day; such as from about 0.01 mg / kg of body weight to about 500 mg / kg of body weight per day; from about 0.01 mg / kg of body weight to about 250 mg / kg of body weight per day; or 0.01 mg / kg of body weight to about 100 mg / kg of body weight per day, and any intermediate ranges or specific amounts, such as from about 0.001 mg / kg, about 0.01 mg / kg, about 0.1 mg / kg, about 1 mg / kg, about 10 mg / kg, about 25 mg / kg, about 50 mg / kg, about 75 mg / kg, about 100 mg / kg, about 150 mg / kg, about 200 mg / kg, about 250 mg / kg, about 300 mg / kg, about 350 mg / kg, about 400 mg / kg, about 450 mg / kg, about 500 mg / kg, about 600 mg / kg, about 700 mg / kg, about 800 mg / kg, about 900 mg / kg, or about 1000 mg / kg, to about 0.001 mg / kg, about 0.01 mg / kg, about 0.1 mg / kg, about 1 mg / kg, about 10 mg / kg, about 25 mg / kg, about 50 mg / kg, about 75 mg / kg, about 100 mg / kg, about 150 mg / kg, about 200 mg / kg, about 250 mg / kg, about 300 mg / kg, about 350 mg / kg, about 400 mg / kg, about 450 mg / kg, about 500 mg / kg, about 600 mg / kg, about 700 mg / kg, about 800 mg / kg, about 900 mg / kg, or about 1000 mg / kg of body weight per day, per hour, per week, or per dose. If intravenous administration is desired, in typical embodiments, the doses will range from about 0.01 to about 10 mg / kg / minute during a constant rate infusion.

[0247] If formulated as a fixed dose, such combination products employ the components within the dosage range described above and the other pharmaceutically active agent(s) within its approved dosage range. The components may alternatively be used sequentially with known pharmaceutically acceptable agent(s) when a combination formulation is inappropriate.

[0248] Therapeutically effective amounts of the combination will generally range up to the maximally tolerated dosage, but may vary widely. The precise amounts employed by the attending physician will vary, of course, depending on the combination, route of administration, physical condition of the patient (e.g. age, weight, and response of the individual patient, as well as the severity of the patient's symptoms) and other factors. The daily dosage may be administered as a single dosage or may be divided into multiple doses, such as two, three, or four times daily, for administration. Alternatively, the doses may be provided on a weekly, biweekly, or monthly basis. These doses can represent a dosing schedule for a combination therapy of phototherapy and immunotherapy to treat the cancer or the tumour as disclosed herein. In some embodiments, reduced dosages may be used as compared to conventional therapeutic dosages of known agents.

[0249] The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific Examples. The Examples are described solely for purposes of illustration and are not intended to limit the scope of the invention. Changes in form and substitution of equivalents are contemplated as circumstances may suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.

[0250] EXAMPLES

[0251] Example 1 - A Method and Kit for Immune System Activation to Kill Cancer Cells Using Combined Targeted Hyperthermia Therapy and Immunotherapy

[0252] Abstract

[0253] Described herein is a cancer treatment method that combines targeted hyperthermia therapy (THT) and immunotherapy. For THT, intratumoural injection of gold nanorods (a transducer for conversion of infrared light into heat) and exposure to infrared light generated by a light-generating device was used. For immunotherapy, intratumoural injection of cytokines (interleukin-2 (IL-2)) was used.

[0254] Experimental detail

[0255] Subcutaneous Tumor Injection: Female mice received subcutaneous (s.c.) bilateral tumor injections of 4T1 breast cancer or B16 melanoma cells. For 4T1 , 1 * 10A5 cells in 100 pL of PBS were be injected into the right and left mammary pads. For B16, 5 * 10A5 cells in 100 pL of PBS were injected into the right and left flanks. The mice were anesthetized with isoflurane during the procedure to minimize pain and discomfort.

[0256] Selection of Treated and Untreated Tumors: Upon tumors reaching palpability (5x5x1 mm), approximately 10 days post cell implantation, mice were stratified into distinct treatment groups, designating one of their two tumors for treatment and the other as an untreated control, using a randomized and balanced approach. Prior to tumor injection, mice underwent established ear clipping for individual identification. Treatment Administration: the tumors selected for treatment received different interventions, including intratumoral (i.t.) administration of gold nanorods (GNRs, 100 l 20 mg / kg, and / or i.t. injections of interleukin-2 (IL-2, 60,000 U / 50 pl), PBS was used as a control. GNR, IL-2, and PBS we injected using two injections into the tumor a site at the superior and inferior of the tumor were used.

[0257] The GNRs-treated tumors went through infrared laser light treatment (1-1.5 W / cm2for 5 minutes, and a thermal camera [HIKMICRO] was used to ensure that the temperature was maintained under 48°C) at 24 hours after GNR administration. The laser used: LDX Laser (Model: LDX-3520-860-HHLFC, Minnetronix) was used to deliver laser irradiance to tumors in mice. The mice were anesthetized with isoflurane during the procedure to minimize pain and discomfort. Three i.t. IL2 injections were performed 24, 72, and 96 hours after the laser treatment.

[0258] The growth of the treated and untreated tumors was monitored and measured regularly (every 2- 3 days) using Vernier calipers. Additionally, body weight was collected every 2-3 days to support the general health of each test animal.

[0259] As an endpoint experiment was terminated at Day 25. At the end of the experiment, the mice were euthanized, and samples was collected for further analysis of draining lymph nodes, spleen, lungs, and tumors.

[0260] Flow cytometry was used to estimate the presence and abundance of the following flow panel for flow cytometric studies:

[0261] Lymphocyte Myeloid

[0262] CD3 F4 / 80

[0263] CD4 CD45

[0264] APC CD273

[0265] CD8 CD206

[0266] CD25 PDL1

[0267] FOXP3 MHCII la / IE

[0268] CD27 CD11c

[0269] CD19 CD86

[0270] CD44 CD11b

[0271] CD62L CD80

[0272] CD56 Ly6G

[0273] PD1 Ly6C

[0274] Results Details

[0275] The results herein show cancer treatment efficacy enhancement from the combined effect of THT and immune therapy by intratumoral injection of a transducer (GNRs capable of converting light energy into heat) and an immune modulator (cytokine, IL-2). The effect of combined therapy is reported for the treated tumor and in distant tumors (untreated). Immune activation and anti-tumor response is observed within the mouse models of colorectal cancer, breast cancer, and melanoma using THT alone, immunological agents alone, and combinations of both.

[0276] In the cohort of animals that were treated with THT and immunotherapy, there was an average reduction in tumor volume of 80% in comparison with untreated ‘control’. Within a same animal tumor shrinkage in distant, untreated tumors was also observed in the treatment group supporting the hypothesis of that the combination treatment resulted in systemic immunogenic responses. THT alone was able to shrink the tumor within 24 hours however the tumor grew back. Similarly, immunotherapy using IL-2 treatment alone slowed tumor growth. However, the combination of the two treatments greatly reduced tumour size by synergistically killing the tumor and maintained the tumor at a very small size (Fig. 1 ). Notably, the treatment was not associated with toxicities such as skin ulcers seen in some other studies.

[0277] Immune system activation was evaluated from flow cytometry analysis. Fluorescence-Activated Cell Sorting (FACS) analysis of the tumor infiltrating cells demonstrated a statistically significant T-cell infiltrate in both the treated tumors and the untreated (contralateral) tumors confirming a systemic, abscopal immune response in the mine treated with combined therapy. This was not seen in the other groups (THT or immunotherapy alone). Also notable is the fact that T-cells in treated tumors expressed significantly more PD-1 , indicating the potential for additional benefit from the addition of a systemic PD-1 inhibitor such as pembrolizumab in this model. Finally, treated tumors also demonstrated significantly more memory T-cells consistent with a systemic immune response (Fig. 2).

[0278] It is assumed that at the immune system level, the combination of the two therapies increased the infiltration of activate cytotoxic CD8+ T cells. This hypothesis is supported by the high levels of memory CD8+ T cells, suggesting that this treatment induced long term immune memory. Additionally, NK and B cell infiltration into the tumor was increased by the combination treatment.

[0279] In other words, these (FACS) data confirm the interpretation of the tumor volume data. The response generated by the combination therapy was due to activation of a tumor-specific systemic immune response.

[0280] Example 2

[0281] Fig. 3 shows the upregulation of inflammatory gene expression in the STING / cGAS / TLR pathway following GNR-induced hyperthermia in both 4T1 tumours and B16 tumours.

[0282] Fig. 4 shows that GNR-induced hyperthermia upregulated the number of several innate immune cells including dendritic cells (CD45+CD11 b+F4 / 80-MHCII+), Macrophages (CD45+CD11 b+F4 / 80+), M1 macrophages (CD45+CD11b+F4 / 80+CD80+CD86+), NK cells (CD3-CD56+) in 4T1 and B16 tumours.

[0283] Fig. 5 shows tumour volume of 4T 1 and B16 tumors over time. Treatments include i.t. GNR injection, followed 24 hours by laser / NIR application, and a series of 3 i.t. IL-2 injections. These are compared to controls (PBS / NIR only, GNR / NIR only, IL2-only / N I R). Combination of GNR / NIR and II-2 enhanced tumour reduction as compared to controls.

[0284] Fig. 6 shows enhanced tumour reduction with double NIR treatment in a B16 model. With a small cohort of B16 mice (n=3), were reapplied laser / NIR 6 days after the first laser application. The second laser further reduced tumor volume of the B16 tumor as compared to tumours with only one laser / NIR application.

[0285] Fig. 7 shows enhanced infiltration of CD8+ T cells into 4T 1 and B16 tumours treated with a combination of GNR and IL-2. Furthermore these CD8+ T cells had higher proportion of central memory (CD44+CD62L+) and PD-1+ expression. Suggesting a memory phenotype was induced by combination therapy. Also, that higher expression of PD-1 suggests that this combination would be further enhanced with the use of a checkpoint inhibitor, such as anti-PD1. Fig. 8 shows abscopal effects of the therapy combining GNR and IL-2, where increased CD8+ T cells were observed in a contralateral tumour following the combination therapy. Contralateral tumor volumes were reduced in mice that underwent combination therapy as compared to controls, suggesting that we achieved a systemic response with the combination therapy.

[0286] From these data, it is clear the GNR-induced hyperthermia triggers a STING / innate immune response, reducing tumour volume drastically. Combination of GNR with intratumoural IL-2 therapy led to lasting tumour reduction, more CD8+ T cell infiltration, increased CD8+ T cell memory subsets, and higher PD-1 expression on CD8+ T cells. Further, an abscopal effect was observed on a non-treated contralateral tumour with combination therapy.

[0287] Example 3 - Gold Nanorod-Induced Hyperthermia Enhances Immunogenic Response and Synergizes with IL-2 Immunotherapy to Reduce Tumor Growth in Preclinical Models of Melanoma and Breast Cancer

[0288] Abstract

[0289] Integrating targeted hyperthermia therapy (THT) with immunotherapy holds significant promise in cancer treatment. Herein, the potential of gold nanorod (GNR)-mediated targeted hyperthermia combined with intratumoral lnterleukin-2 (IL-2) and / or systemic PD-1 inhibiting immunotherapy was investigated in preclinical 4T 1 and B16-F10 tumor models. GNRs were activated using near-infrared (NIR) laser achieving temperatures in the mild hyperthermia range of 42-48°C to induce localized tumor cell death through THT. THT alone effectively induced local tumor immunity and reduced tumor volume within 24- 48 hours, however tumor regrowth was observed after 72 hours following a single treatment with THT. T o enhance the immunity initiated through THT, intratumoral IL-2 was administered in three doses, 48 hours apart. The combination therapy not only sustained tumor reduction but also significantly amplified the immune response, as evidenced by increased CD8+ T cell infiltration and the development of memory subtypes. Furthermore, we observed upregulation of PD-1 expression on infiltrating CD8+ T cells, indicating potential for further immune enhancement with immune checkpoint inhibitors. Importantly, this combination therapy demonstrated systemic effects, reducing tumor volume at distant sites, thereby indicating a robust systemic anti-tumor immune response. These findings confirm in two pre-clinical models that GNR-induced targeted hyperthermia induces a local, tumor-associated immune response that, when combined with additional local or systemic immunotherapy, could serve as a powerful new strategy in cancer treatment, in a broad range of cancers in the clinical setting.

[0290] Introduction

[0291] Cancer remains the second leading cause of mortality globally, surpassed only by cardiovascular disease. Despite significant advancements in chemotherapies, radiation techniques, and the introduction of targeted molecular therapies and check-point targeting immunotherapies, these approaches alone or in combination continue to fall short. Limitations such as inadequate tumor targeting and severe systemic toxicity top the list of a multitude of reasons for failures, underscoring the need for less toxic, more effective therapies. The approach described herein to overcome these limitations is the application of targeted hyperthermia therapy (THT), which leverages near-infrared (NIR) lasers to induce localized heating of tumors (Behrouzkia et al., 2016). Unlike traditional hyperthermia, which can cause widespread tissue damage, THT provides a controlled rise in temperature (targeting between 42°C and 48°C) that selectively induces apoptotic cell death in tumor cells while minimizing harm to surrounding healthy tissues (Huang et al., 2021 ). The molecular mechanisms underlying this process involve the activation of heat shock proteins, disruption of cellular homeostasis, and induction of oxidative stress, leading to irreversible damage to cancer cells (Skitzki et al., 2009). Additionally, THT modulates the tumor microenvironment (TME), enhancing tumor perfusion, oxygenation, and immune cell infiltration, which collectively improves the delivery and efficacy of concurrent therapies (Griffin et al., 2010).

[0292] Gold nanoparticles (GNPs), especially gold nanorods (GNRs), have emerged as highly effective agents for THT due to their unique optical properties, which enable efficient conversion of NIR light into heat (Kesharwani et al., 2023). GNRs can be engineered to absorb light at specific wavelengths, making them ideal for deep tissue penetration and targeted tumor destruction. Among GNRs, non-toxic, CTAB- free GNRs such as those described in International Patent Application Publication No. WO 2019 / 084661 , incorporated herein by reference in its entirety, offer superior biocompatibility, making them particularly suitable for in vivo applications.

[0293] While THT alone can effectively reduce tumor burden, its benefits can be further amplified when combined with immunotherapy (Chen et al., 2022). Immunotherapies, such as intratumoral injections of interleukin-2 (IL-2), have shown promise in enhancing the body's immune response against cancer by activating T-cells and promoting the destruction of cancer cells (Melero et al., 2021 ). The synergy between THT and immunotherapy lies in their complementary mechanisms: while THT induces tumor cell death and exposes tumor antigens, IL-2 enhances the immune system's ability to recognize newly exposed antigens and attack remaining cancer cells (Payne et al., 2020). Furthermore, THT-induced hyperthermia has been shown to alter the immunosuppressive TME, facilitating a more effective immune response and overcoming resistance to immunotherapy (Li et al., 2020). However, although both THT and immunotherapies like IL-2 have individually shown promise in cancer treatment, the synergistic potential of combining these therapies has not been explored. This gap in research highlights the need to better understand how these therapies can work together to enhance anti-tumor immune efficacy and achieve more comprehensive therapeutic outcomes.

[0294] The integration of THT with immunotherapy represents a novel and potentially transformative approach to cancer treatment. By leveraging the unique properties of GNRs for targeted hyperthermia and combining them with immune-activating therapies, this strategy aims to not only eradicate primary tumors but also treat metastasis and prevent recurrence. Herein, it is demonstrated in two distinctly different and widely accepted preclinical models that combining GNR-mediated THT combined with standard immunotherapies significantly enhances the efficacy of current cancer immunotherapies.

[0295] Materials and Methods

[0296] Animals

[0297] Female Balb / c mice and female C57BL / 6 mice were purchased from Charles River Laboratories (Montreal, Canada) and acclimated for one week at the Carleton Animal Care Facility at Dalhousie University, Halifax, NS, Canada. Mice were housed in ventilated rack cages under a standard 12-hour I ig ht / dark cycle, with a controlled room temperature of 22°C and humidity of 55-60%. Animals were fed a standard diet of rodent chow and water ad libitum. This study was conducted in accordance with the guidelines and standards set forth by the Canadian Council on Animal Care and approved by the University Committee on Laboratory Animals at Dalhousie University (#23-081 ). Before tumor implantation, fur was shaved in the areas where tumors were to be implanted.

[0298] Cell Culture 4T 1 and B16-F10 cells (ATCC) were cultured under standard conditions in a humidified incubator at 37°C with 5% CO2. 4T1 cells were maintained in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin (Gibco). B16-F10 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Gibco) supplemented with 10% FBS and 1% penicillinstreptomycin.

[0299] Tumor Establishment

[0300] 6-8 week old Balb / c mice, were subcutaneously (s.c.) injected into the mammary pad on the left side with 1 x 105 4T 1 cells in 100 L of PBS, while 5 x 105 B16-F10 melanoma cells in 100 L of PBS were injected s.c. into the left flank of 6-8 week old C57BL / 6 mice, all under isoflurane anesthesia. After 72 hours, 5 x 104 4T1 cells were injected into the fourth mammary pad on the right side of the Balb / c mice to generate a contralateral tumor. T umor volume was measured using a digital caliper and calculated as an ellipsoid (length x width x height x 1 / 2).

[0301] T reatment Administration

[0302] Upon tumor palpability, typically around 50 mm3and approximately 10 days post-implantation, treatments were administered via intratumoral (i.t.) injections of either GNRs (100 pl, 230 pg [SONA]) or PBS (100 pl). T umors treated with GNRs were subsequently exposed to NIR laser irradiation (1-1.5 W / cm2for 5 minutes), with a thermal camera (HIKmicro) monitoring and internal temperature probes (OMEGA) inserted intratumorally ensuring temperature remained below 48°C (42-48°C). Mouse IL-2 (60,000 U / 50 pL, [Biolegend]) was administered intratumorally. PBS (Gibco) served as a control. In the 4T1 model, three i.t. IL-2 injections were performed at 24, 72, and 120 hours after the laser treatment. Similarly, in the B16-F10 model, three i.t. IL-2 injections were performed immediately after the laser, 48, and 96 hours after the laser treatment.

[0303] Laser Protocol

[0304] The NIR laser treatment was administered using a LDX Laser (Model: LDX-3520-860-HHLFC, Minnetronix) 24 hours after the initial GNR / PBS injections. The laser was positioned approximately 2 cm above the tumor, and the internal temperature of the tumor was maintained between 42°C and 48°C for five minutes. To mitigate damage to mouse skin, aloe vera was applied to the treated area to protect the skin before treatment. Temperature was closely monitored throughout, and the laser was adjusted as necessary to maintain the desired temperature range.

[0305] Flow Cytometry

[0306] Tumors were harvested and processed into single cells using a mouse tumor dissociation kit and gentleMACS™ Dissociator (Miltenyi Biotec) for flow cytometry analysis. Cells were prepared for flow cytometric analysis using lymphocyte and myeloid panels with the appropriate fluorochrome-conjugated antibodies, as detailed in Table 1. Stained cells were washed in FACS buffer (PBS containing 2% FBS) and resuspended in HorizonTM Brilliant Stain Suffer (BD) for analysis. Flow cytometry was performed on a FACSCelesta (BD Biosciences), and data was collected. The collected data were analyzed using FlowJo software (FlowJo LLC), with gating strategies applied to identify and quantify the relevant cell populations.

[0307] Table 1 :

[0308] Quantitative PCR

[0309] Tumors were harvested and immediately snap-frozen in liquid nitrogen for subsequent RNA extraction. RNA was isolated from the samples using the PureLink™ RNA Mini Kit (Invitrogen) following the manufacturer's protocol. The quantity and purity of the isolated RNA were assessed using a NanoDrop™ spectrophotometer (Thermo Fisher Scientific). Subsequently, RNA was reverse-transcribed into complementary DNA (cDNA) using the iScript™ cDNA Synthesis Kit (Bio-Rad) according to the manufacturer's instructions.

[0310] For qPCR analysis, specific primers were designed and validated for genes of interest, as listed in Table 2. Reactions were set up using SYBR™ Green PCR Master Mix (Invitrogen) protocol. The relative expression levels were normalized to GAPDH and calculated using the 2A(-AACt) method (Livak & Schmittgen, 2001 ).

[0311] Table 2:

[0312] Statistical Analysis

[0313] Statistical analyses were performed on the collected data to assess the significance of differences between treatment effects. For comparisons between two groups, an unpaired two-tailed Student’s t-test was used. When comparing three or more groups, one-way analysis of variance (ANOVA) was conducted, followed by Tukey's multiple comparison test to identify specific group differences. All statistical tests were performed using GraphPad Prism software (version 10, GraphPad Software). A p-value of less than 0.05 was considered statistically significant. Data are presented as mean ± standard deviation (SD) unless otherwise specified.

[0314] Results

[0315] GNR Activation by NIR is Required to Generate THT

[0316] To confirm the efficacy of the SON A GNRs to create THT, we compared temperature profiles between GNR-injected tumors and controls exposed to NIR. The setup, involving a BALB / c mouse with a 4T1 tumor and two internal temperature probes, is illustrated in Figure 9A.

[0317] GNR-injected 4T1 tumors exhibited a rapid and significant temperature increase upon NIR exposure, reaching a peak internal temperature of 48°C within 60-100 seconds. This hyperthermic state was maintained on average for 20 seconds post-exposure, demonstrating the effectiveness of GNRs in facilitating thermal conversion. Subsequent NIR stimulations required only 20 seconds to achieve a surface temperature of 48°C, and this temperature (range 42-48°C) was sustained for 5 minutes (Figure 9B). In contrast, tumors in the NIR-only control group exposed to the same intensity and duration of NIR exhibited minimal temperature elevation, always remaining below 43°C, insufficient to trigger apoptotic cell death (Figure 9B).

[0318] Quantitative analysis of surface temperatures revealed that in 4T1 tumors, GNR-treated mice achieved a maximum surface temperature of 48.92°C (±2.8°C), compared to 36.32°C (±2.89°C) in controls. Similarly, in B16-F10 tumors, GNR-treated mice reached a maximum temperature of 50.65°C (±5.13°C), while controls reached only 41.17°C (±1.69°C). Notably, control tumors in the B16F10 model exhibited higher temperature elevations than 4T1 controls (Figure 9C-D). The difference in the control temperature observed between the Balb / c and the C57BL / 6 may be related to pigmentation and water absorption (Sabino et al., 2016).

[0319] These results confirm that GNRs are required and responsible for the THT related to NIR exposure in tumors and highlights their potential for the precise application of THT in cancer treatment.

[0320] GNR-Mediated THT Significantly Reduces Tumor Volume in 4T1 and B16-F10 Models and Activates Innate Immunity Within 48 Hours Post-NIR Activation

[0321] To evaluate the therapeutic effects of single modality GNR-mediated THT on tumor progression, we measured tumor volumes in 4T 1 and B16-F10 tumor models within 72 hours post-NIR activation. In the 4T 1 tumor model, significant tumor reduction was observed in the GNR-treated group within 48 hours of treatment (Figure 10A). Tumor volume decreased to 19.9 mm3(±15.6 mm3) at 48 hours post- NIR activation, down from 43.1 mm3(±42.9 mm3) at 24 hours post-NIR activation. In contrast, the control group showed an increase in tumor volume, with measurements rising to 76.2 mm3(±51.2 mm3) at 48 hours post-NIR activation, up from 59.2 mm3(±42.0 mm3) at 24 hours post-NIR activation. This reduction was statistically significant compared to the untreated control group at both 48- and 72-hours post-NIR activation (Figure 10B). A representative photo of a control mouse and THT treated mouse 48 hours post-NIR activation is shown in Figure 10B.

[0322] Flow cytometry analysis (Figure 10C) revealed significantly higher levels of non-viable cells in 4T 1 tumors treated with THT at 24- and 48-hours post-treatment compared to controls. This increase in cell death aligns with the observed tumor volume reduction and underscores the cytotoxic effects of GNR-mediated THT. Additionally, extracellular levels of calreticulin were significantly elevated at 48 hours post-THT in 4T1 tumors, as shown in Figure 10D. This suggests the induction of immunogenic cell death (ICD), further highlighting the potential for GNR-mediated THT to not only directly kill tumor cells but also to stimulate an immune response (Kielbik et al., 2021 ). Moreover, THT-treated 4T1 tumors exhibited a higher percentage of CD45+ infiltrating immune cells at 48 hours post-treatment compared to controls (Figure 10E). Interestingly, there was also a significant increase in M2 macrophage levels in THT-treated tumors at 48 hours post-treatment compared to controls (Figure 10F).

[0323] Within 48 hours post-NIR activation, tumor volumes of B16-F10 tumors treated were significantly smaller (62.0 mm3±68.1 mm3, 66.6 mm3±60.6 mm3) compared to controls (133.3 mm3±110.2 mm3, 161.13±116.1 mm3) at 48- and 72-hours post-NIR activation, respectively (Figure 10G). These findings indicate that THT as a single modality effectively shrinks tumors and activates innate immunity in multiple preclinical models.

[0324] Single Treatment with GNR-lnduced THT is Insufficient to Sustain Tumor Regression

[0325] Following the initial response to a single treatment with THT, tumor regrowth was observed in both the 4T 1 and B16-F10 models over time. In the 4T 1 model, THT led to a significant reduction in tumor size within the first 48 hours post-NIR activation. However, tumor volumes began to increase over the subsequent days, with regrowth evident by day 6. By day 9, tumors had returned to control sizes (Figure 11 A). B16-F10 tumor volumes were significantly different between control and THT-treated tumors at 3 days post-NIR activation. However, after 3 days, tumor volume was restored to control levels (Figure 11 B).

[0326] Interestingly, analysis of gene expression 8 days post-NIR activation revealed a significant upregulation of genes involved in the STING (Stimulator of Interferon Genes) cGAS (cyclic GMP-AMP synthase) pathway in tumors subjected to THT, as illustrated in Figures 11C (4T1) and 11 D (B16-F10). This upregulation suggests an enhanced recognition of tumor-associated antigens and a subsequent activation of immune signaling (Gan et al., 2021). The increased expression of STING cGAS pathway genes indicates that THT not only induces localized hyperthermia and tumor cell death but also triggers an innate immune response. However, the observed tumor regrowth suggests that while the innate immune response is activated, it may not be sufficient to prevent tumor recurrence or completely eradicate the remaining tumor cells.

[0327] Complementing these findings, additional flow cytometry analysis revealed increased innate immune cell populations in THT-treated tumors. Specifically, in 4T1 tumors, there was a significant increase in dendritic cells (Figure 11 E) and a notable rise in M1 macrophages (Figure 11G), as well as a trend towards increased macrophages and NK cells (Figures 11 F and 11 H). B16-F10 tumors exhibited no significant difference in dendritic cell counts between THT and control groups (Figure 111), with a trend towards increased macrophages and M1 macrophages (Figures 11 J and 11 K), and a significant increase in NK cells (Figure 11 L). These results highlight that while THT stimulates immune cell recruitment and activation, the observed variations between tumor models underscore the complexity of the immune response and its role in tumor regrowth.

[0328] Intratumoral IL-2 Treatments Prevent Tumor Regrowth Following GNR-lnduced THT

[0329] The findings from Section 4.3 identified a critical challenge in our approach in using a single application THT: while GNR-induced THT effectively reduced tumor size and stimulated innated immunity initially, tumor regrowth occurred over time. We hypothesized that the intensity of the initial immune response was insufficient to overcome the growth rate of the surviving tumor cells. What’s more on day 2, we observed significantly more M2 macrophages in the TME that could potentially enable further tumor growth. In consideration of these observations, we attempted to further stimulate the THT-initiated immune response with the addition of intratumoral IL2.

[0330] IL-2 is known for its ability to activate and expand T-cells (Raeber et al., 2023). Mice bearing 4T 1 or B16-F10 tumors were subjected to GNR-mediated THT, followed by a series of intratumoral IL-2 injections administered immediately post-THT.

[0331] In the 4T1 model, the combination of THT and IL-2 injections resulted in sustained tumor regression, with no significant tumor regrowth observed throughout the 14-day monitoring period post- NIR activation. Tumor volumes in the THT+IL-2 treated group were significantly reduced compared to controls starting at 2 days post-NIR treatment, significantly reduced compared to the IL-2 only group starting at 6 days post-NIR, and significantly reduced compared to THT alone starting at 8 days post- NIR. The THT+IL-2 group maintained a low average tumor volume, peaking at 60.1 mm3(±55.7 mm3) on day 9 post-laser, compared to 230 mm3(±96.1 mm3) in the control group, 157.1 mm3(±112.7 mm3) in the IL-2 only group, and 166.9 mm3(±148.8 mm3) in the THT only group (Figure 12A). Similarly, final tumor weights of THT+IL2 treated tumors were significantly reduced as compared to control and IL2 treated tumors (Figure 12B).

[0332] In the B16-F10 model, similar trends were observed. At day 8 post-NIR treatment, the average tumor volume in the THT+IL-2 group (120.7 mm3(±29.6 mm3)) was significantly lower compared to 235.7 mm3(±111.1 mm3) in the control group, 281.6 mm3(±150.5 mm3) in the IL-2 only group, and 271.5 mm3(±169.8 mm3) in the THT only group (Figure 12C). Notably, up to day 8, there was minimal separation between the groups. Final tumor weights of THT+ 1 L2 treated tumors were reduced as compared to controls but this reduction did not reach statistical significance due to small sample size (Figure 12D). These results demonstrate that intratumoral IL-2 injections effectively prevent tumor regrowth following GNR-induced THT, likely by promoting a strong adaptive immune response. This approach offers a promising strategy for enhancing the long-term efficacy of THT-based cancer treatments.

[0333] IL-2 Treatment Enhances CD8+ T Cell Infiltration, Central Memory Differentiation, and PD- 1 Expression in Tumors Following GNR-induced THT

[0334] Intratumoral IL-2 injections administered after GNR-induced THT markedly enhanced CD8+ T cell infiltration into the TME. Flow cytometry analysis at the study endpoint demonstrated a substantial increase in CD8+ T cell numbers in tumors treated with both GNR-induced THT and IL-2 compared to those treated with THT alone. In both the 4T 1 (Figure 13A-D) and B16-F10 models (Figure 13E-H), the THT+IL2 group showed a significant increase in CD8+ T cell count relative to the control group (Figure 13A and 13E).

[0335] 4T1 tumors treated with THT alone exhibited significantly elevated levels of PD-1+ CD8+ T cells. However, the addition of IL-2 did not significantly increase PD-1+ CD8+ T cell levels compared to the control (Figure 13B). In the B16-F10 model, the combination of THT and IL-2 but not THT alone resulted in significantly higher PD-1 expression compared to control (Figure 13F). This indicates that while THT alone enhances PD-1 expression in 4T 1 tumors, the combination with IL-2 is necessary to further elevate PD-1 levels in B16-F10 tumors.

[0336] The THT group demonstrated a significant increase in CD8+ central memory (CM) cells in the 4T 1 model. The addition of IL-2 trended towards higher CM cell levels compared to the control (Figure 13C). In the B16-F10 model, the percentage of CD8+ T cells exhibiting CM phenotype was significantly elevated in the THT+IL2 group compared to the control (Figure 13G).

[0337] In the 4T 1 model, there was a non-significant trend towards increased M2 macrophage levels in the THT-alone group compared to controls, suggesting that hyperthermia may promote an immunosuppressive environment. However, the addition of IL-2 to the THT treatment resulted in lower M2 macrophage levels compared to THT alone, potentially counteracting this immunosuppressive effect (Figure 13D). A similar trend was observed in the B16-F10 model, where M2 macrophage counts were elevated in the THT-only group, with the THT+IL2 combination reducing M2 macrophage levels relative to THT alone (Figure 13H). These observations suggest that while THT may induce an increase in M2 macrophages, the addition of IL-2 could mitigate this effect, helping to reduce the immunosuppressive influence of M2 macrophages in the TME.

[0338] To follow up on the observation of increased PD1 expression on CD8+ T cells, we explored the impact of anti-PD1 monotherapy and the combination of anti-PD1 and THT on 4T 1 tumor growth. Anti- PD1 was administered intraperitoneally (i.p.) at 200 pg per injection, with three injections given 48 hours apart. Anti-PD1 treatment alone did not significantly alter tumor growth compared to the control group. The combination of anti-PD1 and THT resulted in a tumor volume reduction similar to that achieved with THT and IL2 (Figure 13H).

[0339] In summary, IL-2 administration following GNR-induced THT significantly enhances CD8+ T cell infiltration in both tumor models. While IL-2 promotes differentiation into central memory cells and increases PD-1 expression in the B16-F10 model, THT alone is sufficient to induce central memory cell differentiation and increase PD-1 expression in the 4T1 model. Additionally, the combination of IL-2 with THT mitigates the increase in immunosuppressive M2 macrophages observed with THT alone, highlighting its role in counteracting tumor-promoting immune cells. These results underscore the efficacy of combining IL-2 with hyperthermia-based treatments to bolster immune modulation and improve cancer therapeutic outcomes.

[0340] GNR-lnduced THT Combined with IL-2 Reduces Contralateral 4T1 Tumor Size and Enhances CD8+ T Cell Infiltration

[0341] To assess the systemic effects of combining GNR-induced THT with intratumoral IL-2 injections, we evaluated the impact on contralateral (untreated) 4T 1 tumors in mice with bilateral tumors. The combination treatment led to a significant reduction in contralateral tumor size compared to controls at day 6 and 7 post-NIR activation and significantly reduced as compared to THT alone at day 6 post NIR activation (Figure 14A). Flow cytometry revealed a significant increase in CD8+ T cells within the contralateral tumors of both THT and THT+IL2 -treated mice (Figure 14B). This indicates that GNR- induced THT not only enhances local tumor immunity but also triggers a systemic immune response that extends beyond the primary tumor site.

[0342] Discussion

[0343] The efficacy of GNRs in inducing tumor hyperthermia upon NIR activation underscores their potential as a therapeutic device in cancer treatment. The data herein demonstrate that GNRs produced hyperthermia that can achieve therapeutic temperatures sufficient to induce tumor cell apoptosis (Figure 9). This observation is consistent with existing literature highlighting the ability for nanoparticle-mediated hyperthermia in tumors (Ghafarkhani et al., 2021). Furthermore, GNRs have been shown to induce hyperthermia-induced apoptosis and autophagy in cancer cell lines, including neuroblastoma (Ghafarkhani et al., 2021 ). Our findings with hyperthermia-induced tumor responses are consistent with other published findings.

[0344] We hypothesized that the apoptotic cell death associated with THT would result in activation of innate immune responses leading to tumor neoantigen presentation and novel tumor-specific immunity. Our study confirmed the essential role for hyperthermia to induce the ICD as demonstrated by elevated calreticulin levels seen in our THT-treated 4T1 tumors (Figure 10D). Hyperthermia-induced ICD relies on the generation of reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress, producing "enabler" and "eat me" signals that attract immune cells into the TME, thus creating a favorable immunogenic TME (Dudek et al., 2013; Feng et al., 2019; Garg et al., 2016; Huaqi et al., 2023; Knippertz et al., 2011 ; Li et al., 2020; Ostberg & Repasky, 2006). Further confirmation of enhanced innate immunity is shown in the upregulation of the STING cGAS immune pathways observed in 4T1 tumors that have undergone THT (Figure 11 ). This combined with the observation of increased immune cell infiltration, particularly CD45+ cells, confirms the relationship between THT and enhanced intratumoral immunity (Figure 10E).

[0345] Interestingly, we also observed an increase in classically immunosuppressive M2 macrophages (Figure 10F), which aligns with findings that fever-range hyperthermia can induce M2-like polarization in macrophage cell lines (Kozlowski et al., 2023). This increase in M2 macrophages may reflect a complex balance between pro-inflammatory and immunosuppressive responses. This observation led to our hypothesis that the addition of intratumoral IL2 would drive the balance toward a more pro-inflammatory or anti-tumor TME.

[0346] Here, we demonstrate that intratumoral GNRs activated with a single exposure of NIR to generate mild hyperthermia alone initiates a robust innate immune response that when combined with either intratumoral or systemic immune modulators leads to a highly effective tumor-specific immunity. To our knowledge, this is the first such application of GNR-induced THT combined with immunotherapy to generate effective immunity in otherwise traditionally immunogenically cold tumors. One group, applying a similar principle combined direct NIR induced-hyperthermia with intratumoral immunomodulation to achieve a similar effect (L. Huang et al., 2019). However, their method did not use metal nanorods and required multiple applications of NIR radiation over longer treatment periods to achieve the desired outcomes. By using intratumoral GNRs, we were able to induce hyperthermia more precisely, with significantly shorter exposure time, and in a single treatment, thereby reducing local toxicity while increasing treatment efficacy. Moreover, we believe that we have demonstrated for the first time that the initial response to mild hyperthermia induced tumor cell death is associated with a complex pro- and antitumor immune response that highlights the need for further immune modulation to drive the process to adaptive immunity.

[0347] Our findings highlight the potential for targeted-tumor immunomodulation combining THT with immunotherapies to generate effective local and systemic anti-tumor immunity while limiting the toxicity associated with current immunotherapy strategies.

[0348] Example 4

[0349] Laser Intensity Adjustment Across Different Mouse Models Due to inherent differences in skin pigmentation and tumor characteristics, different laser intensities were used to achieve the desired hyperthermic temperature range across mouse models. In the Balb / c mice bearing 4T1 tumors, a laser intensity of 3000 mA was applied, corresponding to an output power of 0.98 W / cm2Conversely, C57BL / 6 mice with B16F10 tumors were subjected to a lower laser intensity of 1200 mA, yielding a power of 0.2 W / cm2. The necessity for these adjustments stems from the differential absorption properties between the models: the lighter skin pigmentation of Balb / c mice permits tolerance to higher laser intensities, while the darker pigmentation and tumor color in C57BL / 6 mice result in greater laser energy absorption, leading to elevated skin surface temperatures. Consequently, reduced laser intensity was employed for C57BL / 6 mice to prevent excessive heating and potential thermal damage. This phenomenon may be attributed to the variation in water absorption between the two strains, with Balb / c mice exhibiting higher water absorption in their skin (Sabino et al., 2016). Aloe vera was applied to the tumor region to further protect the skin during treatment.

[0350] Laser Cycling Protocol The laser was positioned approximately 2 cm above the tumor site, with the internal tumor temperature maintained between 42°C and 48°C for a duration of five minutes. To prevent thermal damage to the skin of C57BL / 6 mice, aloe vera was applied to the treatment area as a protective measure. Continuous temperature monitoring was conducted, and the laser was adjusted as needed to sustain the target temperature range. Initially, the laser was activated for 1 minute to elevate the tumor temperature to the hyperthermic range. Two internal probes were inserted, with one placed near the tumor's apex and the other near its base, to provide precise internal temperature measurements. Additionally, surface temperature was monitored using a thermal camera, with a surface temperature threshold of 52°C, at which point the laser was deactivated. An internal tumor temperature exceeding 48°C also triggered laser deactivation. Conversely, when the internal temperature dropped to 43°C, the laser was reactivated. Practically, this protocol entailed an initial 1 -minute laser activation, followed by alternating 20-second on / off cycles, maintained for a total of 5 minutes.

[0351] Double laser in B16 model In this study, we assessed the efficacy of gold nanorod-induced hyperthermia (THT) in the B16-F10 tumor model, comparing the effects of a single laser treatment to those of a double laser treatment. Mice were divided into three groups: a control group, a group treated with a single laser of THT, and a group treated with a double laser regimen, where the second laser was administered 6 days after the first, without additional gold nanorods.

[0352] Tumor growth in the control group progressed steadily over time, with no significant alterations in tumor volume. The single laser THT treatment led to a significant reduction in tumor volume compared to the control group; however, tumor regrowth was observed over time, consistent with previous findings. In contrast, the double laser regimen resulted in a marked reduction in tumor volume following both treatments. Notably, in three mice from this group, there was a substantial drop in tumor volume, indicating a potential benefit from the additional laser application. This reduction in tumor volume persisted after the second laser, suggesting that the double laser treatment might enhance the efficacy of THT (Figure 15).

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[0375] Skitzki, J. J., Repasky, E. A., & Evans, S. S. (2009). Hyperthermia as an immunotherapy strategy for cancer. Current Opinion in Investigational Drugs (London, England: 2000), 10(6), 550-558.

[0376] It will be understood that certain of the above-described structures, functions, and operations of the above-described embodiments are not necessary to practice the present invention and are included in the description simply for completeness of an exemplary embodiment or embodiments. In addition, it will be understood that specific structures, functions, and operations set forth in the above-described referenced patents and publications can be practiced in conjunction with the present invention, but they are not essential to its practice. It is therefore to be understood that the invention may be practiced otherwise than as specifically described without actually departing from the spirit and scope of the present invention as defined by the appended claims.

Claims

\Ne Claim:1 . A combination comprising targeted hyperthermia therapy (THT) and immunotherapy, wherein the combination is for synergistically treating a cancer.

2. A combination comprising targeted hyperthermia therapy (THT) and immunotherapy, wherein the combination causes an abscopal effect.

3. The combination of claim 2, wherein the combination is synergistic for treating a cancer.

4. The combination of claim 2, wherein the combination is additive for treating a cancer.

5. The combination of any one of claims 1 to 4, wherein the THT comprises administration of a metal nanoparticle (MNP) and application of infrared light to heat the MNP.

6. The combination of any one of claims 1 to 5, wherein the MNP is a tube, rod, shell, cage, sphere, fiber, wire, stars, plate, sea archon, or a combination thereof.

7. The combination of any one of claims 1 to 6, wherein the MNP has an average particle size that is less than about 1 m in size, and typically from about 1 nm to about 900 nm in size and various ranges therebetween.

8. The combination of any one of claims 1 to 7, wherein the average particle size of the MNP is about 1 nm to about 100 nm.

9. The combination of any one of claims 1 to 8, wherein the MNP has one or more dimensions of the order of 100 nm or less.

10. The combination of any one of claims 1 to 9, wherein the MNP is a metal nanorod (MNR).

11. The combination of any one of claims 1 to 10, wherein the MNP comprises a transition metal, a precious metal, or a combination thereof.

12. The combination of any one of claims 1 to 11 , wherein the MNP comprises gold, nickel, palladium, platinum, copper, silver, zinc, cadmium, or any combination thereof.

13. The combination of any one of claims 1 to 12, wherein the MNP is a gold nanoparticle.

14. The combination of any one of claims 1 to 13, wherein the MNP is a gold nanorod (GNR).

15. The combination of any one of claims 1 to 14, wherein the MNP is capped with a capping agent, such as carboxylic acid, citrate, a positively charged ligand, or any combination thereof.

16. The combination of any one of claims 1 to 15, wherein the metal nanoparticles have a surfactant monolayer, or a surfactant bilayer wrapped in a polymer.

17. The combination of claim 16, wherein the polymer comprises proteins, gelatin, bovine serum albumin, polystyrene sulfonate, polyethylene oxides, thiolated polyethylene oxides, thiolated polyethyene oxides with terminating carboxylic acid functionalities, thiolated polyethyene oxides with terminating amine acid functionalities, or any combination thereof.

18. The combination of claim 16 or 17, wherein the polymer forms covalent or non-covalent bonds with at least one of a protein, a polypeptide, an antibody, an antibody fragment, an IgG class of antibody, a polyclonal antibody, a monoclonal antibody, or any combination thereof.

19. The combination of any one of claims 1 to 18, wherein the metal nanoparticles comprise pegylated GNRs.

20. The combination of any one of claims 1 to 19, wherein the metal nanoparticles are conjugated to the immunotherapy.21 . The combination of any one of claims 1 to 20, wherein the metal nanoparticles are not conjugated to the immunotherapy.

22. The combination of any one of claims 1 to 21 , wherein the immunotherapy comprises administration of a tumor-specific antigen (TSA), a tumor-associated antigen (TAA), an antibody, a modified immune cell, a cytokine, a cytokine agonist, a chemokine, a chemokine agonist, a toll-like receptor (TLR) agonist, an immune checkpoint blockade molecule, a virus, a nucleic acid, or any combination thereof.

23. The combination of claim 22, wherein the tumor-specific antigen (TSA) or tumor-associated antigen (TAA) comprises MAG-Tn3, MAGE-A3, New York esophageal squamous cell carcinoma antigen (NY-ESO-1 ), HER-2 / neu, p53, melanoma-associated antigen recognized by T cells 1 (MART-1), glycoprotein (gp) 100, alphafetoprotein (AFP), EGFRvI Il-specific 14-amino acid peptide, PEP-3 chemically conjugated to keyhole limpet hemocyanin (KLH), CA-125, MUC-1, carcinoembryonic antigen (CEA), epithelial tumor antigen (ETA), tyrosinase, prostatic acid phosphatase (PAP), prostate-specific antigen (PSA), Sialyl-Tn, prostate specific membrane antigen (PSMA), non-catalytic hTERT, or any combination thereof.

24. The combination of claim 22 or 23, wherein the antibody is a monoclonal antibody.

25. The combination of any one of claims 22 to 24, wherein the antibody targets CD52, EGFR, VEGF, HER-2, CD20, CD16, 0X40, CD137, CD27, GITR, CD40, CD19, CD272, CD279, CD274, PAP, CD38, CD47, GD2, or any combination thereof.

26. The combination of any one of claims 22 to 25, wherein the modified immune cell comprises a dendritic cell that expresses a PAP antigen.

27. The combination of any one of claims 22 to 26, wherein the modified immune cell expresses a chimeric antigen receptor (CAR).

28. The combination of claim 27, wherein the CAR is directed against CD19, melanoma-associated antigen recognized by T cells 1 (MART-1), glycoprotein (gp) 100, carcinoembryonic antigen (CEA), p53, MAGE-A3, New York esophageal squamous cell carcinoma antigen (NY-ESO-1 ), or any combination thereof.

29. The combination of any one of claims 22 to 28, wherein the cytokine comprises an interferon, an interleukin, or a combination thereof.

30. The combination of any one of claims 22 to 28, wherein the cytokine comprises IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-12 IL-15, IL-17, IL-23, IL-i p, TNF-a, IFN-a, IFN-p, IFN-y, an agonist of any thereof, or any combination thereof.31 . The combination any one of claims 22 to 30, wherein the chemokine comprises CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11 , CXCL12, CXCL14, CCL2, CCL5, an agonist of any thereof, or any combination thereof.

32. The combination of any one of claims 22 to 31 , wherein the TLR agonist comprises a TLR-4 agonist, a TLR-7 agonist, a TLR-8 agonist, a TLR-9 agonist, a TLR-12 agonist, or a combination thereof.

33. The combination of any one of claims 22 to 32, wherein the immune checkpoint blockade molecule comprises a monoclonal antibody.

34. The combination of claim 33, wherein the antibody targets CTLA-4, PD-L1 , PD-1 , or any combination thereof.

35. The combination of any one of claims 22 to 34, wherein the virus expresses a 5T4 tumor- associated antigen.35a. The combination of any one of claims 1 to 35, comprising THT, IL-2, and a checkpoint inhibitor.35b. The combination of claim 35a, wherein the checkpoint inhibitor comprises an anti-PD-1 antibody.

36. The combination of any one of claims 1 to 35, further comprising an adjuvant, an immune modulator, or a combination thereof.

37. The combination of claim 36, wherein the adjuvant or immune modulator comprises GM-CSF, KLH, liposomal AS15, BCG, freeze dried BCG, MONTANIDE, IL-2, or any combination thereof.

38. The combination of any one of claims 1 to 37, wherein the cancer is brain cancer, nerve sheath cancer, breast cancer, colon cancer, colorectal cancer, skin cancer, lung cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, renal cell carcinoma, carcinoma, melanoma, lymphoma, head and neck cancer, brain cancer, and prostate cancer, or any combination thereof.

39. The combination of claim 38, wherein the cancer is breast cancer, colon cancer, or melanoma.

40. The combination of claim 39, wherein the breast cancer is triple-negative breast cancer.41 . The combination of any one of claims 1 to 40, wherein the cancer is metastatic.

42. The combination of any one of claims 1 to 41 , wherein the THT and the immunotherapy are independently administered intratumourally or systemically.

43. The combination of claim 42, wherein the THT is administered intratumourally and treats a distant tumour.43a. The combination of any one of claims 1 to 43, wherein the THT is administered a single time or a plurality of times, wherein each THT administration is separated by a period of time such as from about 1 day to about 3 months, such as about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, or 14 days, with or without further administration of MNPs.43b. The combination of any one of claims 1 to 43a, wherein the immunotherapy is administered a single time or a plurality of times.43c. The combination of any one of claims 1 to 43b, wherein the THT is administered a single time and the immunotherapy is administered a plurality of times, before, during, and / or after the THT, such as at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times, wherein each immunotherapy administration is separated by a period of time such as from about 1 hour to about 1 month, such as about 1, 2, 3, 4, 5, 6, or 7 days.

44. The combination of any one of claims 1 to 43, wherein the immunotherapy is for administration before, after, or concurrently with the THT.

45. A pharmaceutical composition or kit comprising the combination of any one of claims 1 to 44.

46. A method for sensitizing a cancer to cancer immunotherapy, the method comprising administering the combination, composition, or kit of any one of claims 1 to 45.

47. A method for exposing cancer antigens for immunotherapy, the method comprising administering the combination, composition, or kit of any one of claims 1 to 45.

48. A method for treating cancer, the method comprising administering the combination, composition, or kit of any one of claims 1 to 45.

49. A method for increasing CD8+ T cell infiltration into a tumour, increasing CD8+ memory T cell subsets, and / or increasing PD-1 expression on CD+ T cells, the method comprising administering the combination, composition, or kit of any one of claims 1 to 45.

50. The method of any one of claims 46 to 49, wherein the cancer is selected from brain tumour, nerve sheath tumour, breast cancer, colon cancer, colorectal cancer, skin cancer, lung cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer,bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, renal cell carcinoma, carcinoma, melanoma, lymphoma, head and neck cancer, brain cancer, and / or prostate cancer, including but not limited to androgen-dependent prostate cancer and androgen-independent prostate cancer.51 . The method of any one of claims 46 to 50, wherein the cancer is selected from breast cancer, colon cancer, and melanoma.

52. The method of any one of claims 46 to 51 , wherein the cancer is metastatic.

53. The method of claim 52, wherein treatment of a primary tumour further treats one or more metastatic lesions.

54. The method of any one of claims 46 to 53, wherein the mammal is a human.

55. The method of any one of claims 46 to 54, wherein administering comprises parenteral administration (e.g. formulated for injection via the intravenous, intramuscular, sub-cutaneous, intralesional, and / or intraperitoneal routes).

56. Use of the combination, composition, or kit of any one of claims 1 to 45 for sensitizing a cancer to cancer immunotherapy.

57. Use of the combination, composition, or kit of any one of claims 1 to 45 for exposing cancer antigens for immunotherapy.

58. Use of the combination, composition, or kit of any one of claims 1 to 45 for treating cancer.

59. Use of the combination, composition, or kit of any one of claims 1 to 45 for increasing CD8+ T cell infiltration into a tumour, increasing CD8+ memory T cell subsets, and / or increasing PD-1 expression on CD+ T cells.

60. The use of any one of claims 56 to 59, wherein the cancer is selected from brain tumour, nerve sheath tumour, breast cancer, colon cancer, colorectal cancer, skin cancer, lung cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, renal cell carcinoma, carcinoma, melanoma, lymphoma, head and neck cancer, brain cancer, and / or prostate cancer, including but not limited to androgen-dependent prostate cancer and androgen-independent prostate cancer.61 . The use of any one of claims 56 to 60, wherein the cancer is selected from breast cancer, colon cancer, and melanoma.

62. The use of any one of claims 56 to 61 , wherein the cancer is metastatic.

63. The use of claim 62, wherein treatment of a primary tumour further treats one or more metastatic lesions.

64. The use of any one of claims 56 to 63, wherein the mammal is a human.

65. The use of any one of claims 56 to 64, wherein administering comprises parenteral administration (e.g. formulated for injection via the intravenous, intramuscular, sub-cutaneous, intralesional, and / or intraperitoneal routes).

66. Use of MNP-mediated THT to stimulate an immune response.

67. A method for stimulating an immune response, the method comprising administering MNP- mediated THT to a subject in need thereof.

68. Use of MNP-mediated THT to treat a tumour, whererin the THT is for administration at least twice, wherein each THT administration is separated by a period of time such as from about 1 day to about 3 months, such as about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, with or without further administration of MNPs.

68. A method of treating a tumour using MNP-mediated THT, the method comprising administering the THT at least twice, wherein each THT administration is separated by a period of time such as from about 1 day to about 3 months, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, or 14 days, with or without further administration of MNPs.

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