Treatment of immune-evasive tumors

Abstract

To provide a method for treating any cancer.SOLUTION: A method of treating cancer in a subject comprising administering a surface functionalized particle alone or in combination with a cancer therapeutic, wherein the subject has one or more immune evasive tumors.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 018,026, filed April 30, 2020, and U.S. Provisional Patent Application No. 62 / 881,326, filed July 31, 2019, the contents of each of which are incorporated herein by reference in their entirety for all purposes.

[0002] Field of Disclosure FIELD OF THE DISCLOSURE

[0002] The present disclosure relates generally to methods for treating tumors using surface-functionalized particles, alone or in combination with anti-cancer therapeutic agents. [Background technology]

[0003] background Research over the past decade has clearly confirmed the long-held hypothesis that the immune system can mount an efficient antitumor response (1-3). Successful immune-mediated elimination of tumor cells depends on the collective activity of several immune cell types, including antigen-presenting cells (APCs) (e.g., macrophages and dendritic cells), other myeloid cells (e.g., monocytes and neutrophils), and effector cells (e.g., B cells, T cells, NKT cells, and NK cells) (3-9). However, tumor cells can evolve mechanisms that result in immune system evasion. One major mechanism promoting tumor immune evasion is the expression of proteins and soluble factors (e.g., PD-L1, CD47, and TGF-β) that suppress antitumor immune function (10-12). These findings have led to the development of immunotherapies aimed at modulating the immune system to enable sustained antitumor immune function. Although numerous immunotherapeutic agents have been successfully developed for clinical application and have revolutionized cancer treatment, only a fraction of patients respond to immunotherapy, and many of those who respond develop resistance to the treatment. For example, only 20–30% of patients respond to checkpoint inhibitor (anti-PD1 / L1) treatment. Understanding the mechanisms underlying the low response rates to immunotherapy has led to the identification of cellular and molecular determinants that influence treatment success. Key among these determinants are the immunological state of the tumor, immune cell infiltrate, and tumor gene mutation burden (13–15).

[0004] The immunological status of a tumor is determined by the expression of immune cells (e.g., CD4 + T cells, CD8 + T cells, NK1.1 + The degree of tumor infiltration (NK cells, APCs, monocytes, and neutrophils), immune cell phenotype (e.g., PD-1 + , PD-L1 + , and PD-L2 + Tumors that exhibit a higher degree of immune cell infiltration are referred to as immunologically "hot" tumors. Immunotherapy relies on the presence of immune cells in the TME. Therefore, immunologically "hot" tumors usually respond well to immunotherapy and are associated with favorable outcomes. In contrast, tumors that exhibit low levels of immune infiltrate are called immunologically "cold" (also known as immune evasive, immunologically protected, microsatellite stable, microsatellite instability-low, low immune infiltrate, low tumor mutational burden, and / or heterogeneity) and respond poorly to immunotherapy (16, 17). The balance between pro- and anti-inflammatory mediators in the tumor microenvironment (TME) also plays a role in determining the immunological status of a tumor. For example, the abundance of myeloid-derived suppressor cells (MDSCs) (CD11b) in the TME has been shown to be related to the immunological status of tumors. + Ly6C HI or CD11b + Ly6G + ), M2 tumor-associated macrophages (TAMs) (CD11b + F4 / 80 + CD206 + MHCII LO ), and MARCO + The increased presence of anti-inflammatory cells, such as TAMs, leads to immunosuppression in the TME and suppression of antitumor immune function, leading to resistance to treatment and unfavorable disease outcomes. Related to (18-22).

[0005] Tumor mutation burden (TMB) is an important tumor-specific genetic factor that influences therapeutic response to immunotherapy. TMB is defined as the total number of mutations per coding base pair in the tumor genome. Tumors with high mutation burden are considered highly immunogenic. The presence of frequent genetic mutations generates tumor neoantigens that can be recognized by the immune system, leading to the activation of a cascade of events that culminates in the induction of an antigen-specific antitumor response. Consistent with this hypothesis, studies have shown that high TMB correlates with a higher response rate to immunotherapy compared with low TMB (14, 15, 23, 24). Therefore, determining a tumor's TMB status can be beneficial in cancer diagnosis and treatment. Current clinical practice relies on microsatellite instability (MSI) testing to gain insight into a tumor's TMB status (25). MSI is a state of genetic hypermutability resulting from a defect in the DNA mismatch repair system. High MSI causes tumors to accumulate a high mutation burden, making them highly immunogenic. Such tumors are called MSI-high (MSI-h) tumors. Therefore, MSI-h tumors are more likely to respond to immunotherapy, and several have been specifically approved for the treatment of MSI-h tumors. In contrast, mismatch repair-competent microsatellite stable (MSS) tumors have been found to be poor responders to immunotherapy (12, 26, 27). Summary of the Invention [Problem to be solved by the invention]

[0006] Some of the most commonly diagnosed cancers (e.g., bladder, colorectal, ovarian, and pancreatic cancers) are also some of the most difficult to treat. The majority of patients diagnosed with cancer have tumors that respond poorly or not at all to first-line (e.g., radiation and / or combination chemotherapy) and second-line (e.g., anti-PD-1 / L1) treatments. Common characteristics of such tumors are that they are immunologically "cold," immunologically protected, contain anti-inflammatory and immunosuppressive mediators within the TME, have low TMB, or are microsatellite stable (MSS) / MSI-low. Tumor heterogeneity further complicates treatment, as therapeutically unresponsive tumors are often composed of heterogeneous populations of tumor cells that exhibit varying degrees of factors (e.g., immune infiltrate, TMB, and MSI) that play a role in determining response to therapy (28, 29). [Means for solving the problem]

[0007] overview Surface-functionalized nanoparticles (SFPs) are demonstrated herein to be effective against tumors that are immunologically "cold," immunoevasive, immunoprotected, immunologically "cold," microsatellite stable ("MSS"), low microsatellite instability ("MSI"), have low immune infiltrate, have low tumor mutational burden, and / or exhibit heterogeneity. For example, without limitation, such tumors may have low TMB, be MSS / MSI-low, and / or exhibit cellular and molecular factors within the TME that are anti-inflammatory or immunosuppressive. SFPs can inhibit tumor growth and induce tumor cell death. Administered alone or in combination with other cancer therapeutics, SFPs can be used as an effective treatment option for various types of cancer. Indeed, as described herein, SFPs are not limited to cancer type and can be used to treat any cancer that can be characterized as being immunologically "cold," immunoevasive, immunologically protected, immunologically "cold," microsatellite stable, microsatellite low instability, having a low immune infiltrate, having a low tumor mutational burden, and / or exhibiting heterogeneity, or a combination thereof.

[0008] In various embodiments, the present disclosure provides surface-functionalized particles, alone or in combination with cancer therapeutic agents. and administering to the subject a combination of the compounds of formula (I) and formula (II), wherein the subject has one or more tumors characterized as being immunoevasive, immunologically protected, immunologically "cold," microsatellite stable, microsatellite low instability, containing a low immune infiltrate, containing a low tumor mutational burden, and / or exhibiting heterogeneity.

[0009] In various embodiments, the surface-functionalized particles are negatively charged particles that do not contain attached peptide or antigenic moieties or other bioactive agents.

[0010] In various embodiments, the present disclosure provides methods of treating cancer in a subject comprising administering surface-functionalized particles, alone or in combination with a cancer therapeutic, to the subject, the subject having one or more tumors with low immune infiltrate. In various embodiments, administration to a subject having one or more tumors with low immune infiltrate alters the tumor immune infiltrate. In various embodiments, the tumor immune infiltrate comprises antigen-presenting cells, myeloid cells, and lymphoid cells. In various embodiments, the antigen-presenting cells in the tumor immune infiltrate comprise macrophages and / or dendritic cells. In various embodiments, the myeloid cells in the tumor immune infiltrate comprise monocytes, neutrophils, myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages (TAMs). In various embodiments, the TAMs in the tumor immune infiltrate comprise M1 macrophages, M2 macrophages, and MARCOs. + In various embodiments, lymphoid cells in the tumor immune infiltrate include T cells, B cells, NKT cells, and NK cells.

[0011] In various embodiments, the present disclosure provides a method of treating cancer in a subject comprising administering surface-functionalized particles, alone or in combination with a cancer therapeutic, to a subject, wherein the subject has one or more immune-evasive tumors. In various embodiments, the subject has one or more immunologically protected tumors. In various embodiments, the subject has one or more microsatellite-stable tumors. In various embodiments, the subject has one or more microsatellite-low instability tumors. In various embodiments, the subject has one or more tumors with intermediate microsatellite instability. In various embodiments, the subject has one or more tumors with low tumor mutation burden. In various embodiments, the subject has one or more tumors with intermediate tumor mutation burden. In various embodiments, the subject has one or more tumors that are resistant to treatment. In various embodiments, the subject has one or more immunologically heterogeneous tumors. In various embodiments, the subject has genetically heterogeneous tumors. In various embodiments, the subject has one or more refractory tumors. In one or more embodiments, the subject has a tumor that develops resistance to therapy during the course of treatment.

[0012]

[0012] In various embodiments, the present disclosure provides methods of treating cancer in a subject comprising administering surface-functionalized particles, alone or in combination with a cancer therapeutic, to the subject, the subject having one or more immune-evasive tumors. In various embodiments, the administration alters the tumor immune infiltrate. In various embodiments, the administration alters the anti-tumor immune response. In various embodiments, the administration alters the tumor microenvironment, including tumor cells, immune cells, cancer stem cells, and stroma. In various embodiments, the administration converts an immunologically cold tumor to an immunologically hot tumor. In various embodiments, the administration reduces tumor size and / or inhibits tumor growth. In various embodiments, the administration induces tumor cell death, apoptosis, and / or necrosis through direct particle uptake by tumor cells.

[0013] In various embodiments, the present disclosure provides methods of treating cancer in a subject comprising administering to the subject surface-functionalized particles, alone or in combination with a cancer therapeutic agent, the subject having one or more cancers characterized as immunologically protected and / or immunoevasive. In various embodiments, the administration alters tumor-associated stroma, including fibroblasts, cancer-associated fibroblasts, adipocytes, pericytes, endothelium, vasculature, lymphatic vessels, tumor-associated vasculature, mesenchymal stromal cells, mesenchymal stem cells, and extracellular matrix.

[0014]

[0014] In various embodiments, the surface-functionalized particles are polyglycolic acid (PGA) particles, polylactic acid (PLA) particles, poly(lactic-co-glycolic acid) (PLGA) particles, polystyrene particles, diamond particles, or iron, zinc, cadmium, gold, or silver particles, or combinations thereof.

[0015] In some embodiments, the surface-functionalized particles are poly(lactic-co-glycolic acid) (PLGA) particles. In various embodiments, the particles comprise about 50:50, or about 80:20 to about 100:0 polylactic acid:polyglycolic acid, or about 50:50, or about 80:20 to about 100:0 polyglycolic acid:polylactic acid. In various embodiments, the particles comprise 50:50 polylactic acid:polyglycolic acid. In various embodiments, the particles comprise polylactic acid:polyglycolic acid in a ratio of about 99:1 to about 1:99, e.g., about 99:1, about 95:5, about 90:10, about 85:15, about 80:20, about 75:25, about 70:30, about 65:35, about 60:40, about 55:45, about 50:50, about 45:55, about 40:60, about 35:65, about 30:70, about 25:75, about 20:80, about 15:85, about 10:90, about 5:95, and about 1:99, including all values ​​and ranges therebetween.

[0016] In various embodiments, surface functionalization is achieved by carboxylation. Carboxylation can create a negative charge on an otherwise neutral particle or increase the negative charge of a negatively charged particle. Without being bound by theory, carboxylation creates a negatively charged surface, and this negative charge induces a therapeutic response in immune-evasive tumors. In some embodiments, the surface-functionalized particles do not contain an embedded or attached therapeutic agent, such as a cancer therapeutic. In further embodiments, surface functionalization is achieved by the addition of a targeting agent. In some embodiments, the targeting agent includes a polypeptide, an antibody, a carbohydrate, a nucleic acid, a lipid, a small molecule, and a surfactant. In various embodiments, the surface-functionalized nanoparticles are preferentially targeted to monocytes, neutrophils, macrophages, T cells, B cells, NK cells, NKT cells, fibroblasts, cancer-associated fibroblasts, endothelial cells, adipocytes, pericytes, endothelium, vasculature, lymphatic vessels, tumor-associated vasculature, mesenchymal stromal cells, mesenchymal stem cells, and / or extracellular matrix.

[0017] In various embodiments, the particles have a zeta potential between -100 mV and -1 mV. In various embodiments, the particles have a zeta potential between -80 mV and -30 mV. In some embodiments, the zeta potential of the particles is about -100 mV to about -40 mV, about -75 mV to about -40 mV, about -70 mV to about -30 mV, about -60 mV to about -35 mV, or about -50 mV to about -40 mV. In various embodiments, the zeta potential is about -30mV, -35mV, -40mV, -45mV, -50mV, -55mV, -60mV, -65mV, -70mV, -75mV, -80mV, -85mV, -90mV, -95mV, or -100mV (including all values ​​and ranges therebetween).

[0018] In various embodiments, the negatively charged particles have a diameter between 0.1 μm and 10 μm. In various embodiments, the particles have an average diameter between about 0.2 μm and about 2 μm, between about 0.3 μm and about 5 μm, between about 0.5 μm and about 3 μm, or between about 0.5 μm and about 1 μm. In some embodiments, the particles have a diameter of about 100-1500 nm, about 200-2000 nm, about 100-1000 nm, about 300-1000 nm, about 400-800 nm, or about 200-700 nm. In various embodiments, Thus, the particles have an average diameter of about 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1100 nm, 1200 nm, 1300 nm, 1400 nm, 1500 nm, or 2000 nm (including all values ​​and ranges therebetween). In some embodiments, the diameter of the negatively charged particles is between 400 nm and 800 nm.

[0019]

[0019] In various embodiments, the particles are PLGA particles having a zeta potential between -80 and -30 mV and a diameter between 200 and 2000 nm, and are optionally surface functionalized by carboxylation.

[0020]

[0020] In various embodiments, the present disclosure provides a method for treating cancer in a subject, comprising administering to the subject a composition comprising negatively charged PLGA particles, alone or in combination with a cancer therapeutic agent, wherein the particles do not comprise a peptide, antigenic moiety, or other bioactive agent and have a diameter between 400 nm and 800 nm and a zeta potential between -1 mV and -100 mV, and the subject has one or more immune-evasive tumors.

[0021] In various embodiments, the subject has a cancer selected from the group consisting of brain cancer, skin cancer, eye cancer, breast cancer, prostate cancer, lung cancer, esophageal cancer, head and neck cancer, cervical cancer, liver cancer, colon cancer, bone cancer, uterine cancer, ovarian cancer, bladder cancer, stomach cancer, oral cancer, thyroid cancer, kidney cancer, testicular cancer, leukemia, lymphoma, and mesothelioma. Additional cancers contemplated by the present methods are disclosed in the detailed description.

[0022]

[0022] In various embodiments, the cancer therapeutic agent is a chemotherapeutic agent selected from the group consisting of growth inhibitors, DNA replication inhibitors, kinase inhibitors, signal transduction cascade inhibitors, angiogenesis inhibitors, metabolism inhibitors, amino acid synthesis inhibitors, selective inhibitors of oncogenic proteins, metastasis inhibitors, inhibitors of anti-apoptotic factors, apoptosis inducers, nucleoside signal transduction inhibitors, enzyme inhibitors and DNA damaging agents.

[0023] In various embodiments, the cancer therapeutic agent comprises one or more biologic agents selected from the group consisting of cytokines, angiogenesis inhibitors, immune checkpoint modulators, enzymes, and monoclonal antibodies.

[0024] In various embodiments, the cytokine is selected from the group consisting of a transforming growth factor, a tumor necrosis factor, an interferon, and an interleukin. Exemplary cytokines include, but are not limited to, IFN-alpha, IFN-beta, IFN-gamma, IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, members of the transforming growth factor beta superfamily (including TGF-β1, TGF-β2, and TGF-β3), tumor necrosis factor alpha, granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0025] In various embodiments, the cancer therapeutic agent comprises an enzyme. In various embodiments, the cancer therapeutic agent comprises an enzyme that targets T cells, B cells, APCs, monocytes, MDSCs, TAMs, neutrophils, other monocyte-derived cells, tumor-associated stroma, cancer stem cells, mesenchymal stem cells, extracellular matrix, and amino acids. In various embodiments, the cancer therapeutic agent comprises an enzyme selected from the group including asparaginase, kynurininase, L-arginine deiminase, L-methionine-γ-lyase, one or more amino acid-degrading enzymes, and one or more nucleoside-degrading enzymes.

[0026] In various embodiments, the monoclonal antibody is a monospecific, bispecific, trispecific or bispecific T cell engager (BiTE) antibody.

[0027] In various embodiments, the monoclonal antibody is an immune cell costimulatory molecule agonist that induces an anti-tumor immune response. Exemplary costimulatory molecules include, but are not limited to, ICOS (inducible T cell costimulator) (CD278), OX40 (CD134), GITR (glucocorticoid-induced tumor necrosis factor receptor), CD40, and CD27.

[0028] In various embodiments, the monoclonal antibody is selected from the group including alemtuzumab, bevacizumab, brentuximab, cetuximab, denosumab, ibritumomab, trastuzumab, panitumumab, pertuzumab, and rituximab. In various embodiments, the monoclonal antibody targets receptor tyrosine kinases, EGFR, VEGF, VEGFR, PDGF, PDGFR, TGF-β, TGF-β-LAP, SIRP-α, CD47, CD39, CD73, and fibroblast activation protein (FAP).

[0029] In various embodiments, the immune checkpoint modulator targets programmed cell death protein 1 (PD1), programmed cell death protein ligand-1 (PD-L1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), T-cell immunoglobulin and mucin domain-containing 3 (TIM-3), lymphocyte-activation gene 3 (LAG-3), and / or TIGIT (T-cell immunoreceptor with Ig and ITIM domains). In various embodiments, the immune checkpoint modulator is an antibody selected from the group consisting of ipilimumab, tremelimumab, pembrolizumab, nivolumab, atezolizumab, avelumab, cemiplimab, and durvalumab.

[0030]

[0030] In various embodiments, the cancer therapeutic agent comprises one or more cell-based therapies selected from the group consisting of adoptive cell transfer, tumor-infiltrating leukocyte therapy, chimeric antigen receptor T-cell therapy (CAR-T), NK cell therapy, and stem cell therapy.

[0031] In various embodiments, the cell-based therapy is the adoptive transfer of autologous cells from the patient. In various embodiments, the cell-based therapy is the adoptive transfer of allogeneic cells from a donor.

[0032] In various embodiments, the cell-based therapy is the transfer of cells derived from universal donors or induced pluripotent stem cells that are not patient-specific and suitable for long-term storage. Such therapies are also referred to as "off-the-shelf" therapies.

[0033] In various embodiments, the cancer therapeutic is a hormone therapy. In various embodiments, the cancer therapeutic comprises one or more antibody-drug conjugates. In various embodiments, the cancer therapeutic comprises one or more cancer vaccines. In various embodiments, the cancer vaccine is a protein, polypeptide, and / or nucleic acid vaccine.

[0034] In various embodiments, the cancer therapeutic agent is an immunotherapy selected from the group including oncolytic viruses, bacteria, oncolytic bacteria or other bacterial consortia, tumor cell lysates, bacterial cell lysates, lipopolysaccharide (LPS), Bacillus Calmette-Guerin (BCG), microbiome modulating agents, and / or Toll-like receptor (TLR) agonists. In various embodiments, the TLR agonists are TLR3, TLR4, TLR5, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, and / or TLR13 agonists. In various embodiments, the TLR agonists are The immunotherapy may be viral, bacterial, and / or synthetically produced. In various embodiments, the immunotherapy is a STING pathway modulator.

[0035] In various embodiments, the cancer therapeutic agent comprises a viral or bacterial vector. In various embodiments, the viral vector is selected from the group including adenovirus, adeno-associated virus (AAV), herpes simplex virus, lentivirus, retrovirus, alphavirus, flavivirus, rhabdovirus, measles virus, Newcastle disease virus, poxvirus, vaccinia virus, modified Ankara virus, vesicular stomatitis virus, picornavirus, tobacco mosaic virus, potato virus x, comovirus, or cucumber mosaic virus. In various embodiments, the virus is an oncolytic virus. In various embodiments, the virus is a chimeric virus, synthetic virus, mosaic virus, or pseudotyped virus.

[0036]

[0036] Additional cancer therapeutic agents contemplated for use in the present methods are described in the detailed description.

[0037]

[0037] In various embodiments, the surface-functionalized particles and / or cancer therapeutic agent are administered once a day, twice a day, three times a day, seven times a week, six times a week, five times a week, four times a week, three times a week, twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once every two months, once every three months, once every six months or once a year. In various embodiments, the surface-functionalized particles and / or cancer therapeutic agents are administered for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 weeks or more.

[0038] In various embodiments, the particles and / or cancer therapeutic agent are administered intravenously, orally, intranasally, intramuscularly, intraocularly, transdermally, or subcutaneously.

[0039] In various embodiments, the subject is a mammal. In various embodiments, the subject is a human.

[0040] In various embodiments, administration ameliorates one or more symptoms of cancer or a proliferative disorder. In various embodiments, the one or more symptoms are selected from the group consisting of tumor size or tumor burden, tumor metastasis, and levels of inflammatory cells within a tumor in a subject. In various embodiments, administration reduces tumor size or tumor burden by about 10%, 20%, 30% or more. In various embodiments, administration reduces tumor size or tumor burden by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% (including all values ​​and ranges therebetween).

[0041] In various embodiments, the particles are formulated in a composition comprising a pharmaceutically acceptable carrier, diluent, or excipient. In various embodiments, the cancer therapeutic agent is formulated in a composition comprising a pharmaceutically acceptable carrier, diluent, or excipient. In various embodiments, the particles and the cancer therapeutic agent can be formulated in the same composition or in separate compositions.

[0042]

[0042] Also contemplated is a composition comprising any of the above surface-functionalized particles or cancer therapeutic drug compositions of the present disclosure, or its use in the preparation of a medicament for the treatment of any of the disorders described herein associated with inflammation and cancer and / or proliferative diseases.

[0043]

[0043] Each feature or embodiment or combination described herein is an aspect of the present invention. It is understood that any of the features or embodiments described herein are non-limiting, illustrative examples and are therefore intended to be combined with any other feature or embodiment or combination described herein. For example, when a feature is described with terms such as "various embodiments," "one embodiment," "some embodiments," "particular embodiments," "further embodiments," "specific exemplary embodiments," and / or "another embodiment," each of these types of embodiments is a non-limiting example of a feature that is intended to be combined with any other feature or combination of features described herein, and not all possible combinations need be listed. Such features or combinations of features apply to any aspect of the present disclosure. When example values ​​falling within a range are disclosed, these examples are all contemplated as endpoints of the possible range, and any and all numerical values ​​between such endpoints are contemplated, with any and all combinations of upper and lower endpoints being envisioned. [Brief explanation of the drawings]

[0044] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1A]

[0044] Figure 1 shows the efficacy of SFP against immunologically "cold" tumors with low TMB. The indicated effect of treatment on cell viability in tumors (n = 5). One-way ANOVA tests were performed to determine statistical significance (ns = P > 0.5; * = p < 0.05; ** = p < 0.01; **** = p < 0.0001). [Figure 1B]

[0044] Figure 1 shows the efficacy of SFP against immunologically "cold" tumors with low TMB. The effect of the indicated treatments on B16F10 tumor growth. Treatment was initiated after the formation of palpable tumors (day 0) (n=10). One-way ANOVA tests were performed to determine statistical significance (ns=P>0.5; *=p≦0.05; **=p≦0.01; ****=p≦0.0001). [Figure 1C]

[0044] Efficacy of SFP against immunologically "cold" tumors with low TMB. Effect of the indicated treatments administered after the formation of palpable tumors (day 0) on the survival of B16F10 tumor-bearing mice (n=10). One-way ANOVA tests were performed to determine statistical significance (ns=P>0.5; *=p≦0.05; **=p≦0.01; ****=p≦0.0001). [Figure 1D]

[0044] Figure 1 shows the efficacy of SFP against immunologically "cold" tumors with low TMB. The effect of the indicated treatments on the frequency of MDSCs in tumors (n = 5). One-way ANOVA tests were performed to determine statistical significance (ns = P > 0.5; * = p ≤ 0.05; ** = p ≤ 0.01; **** = p ≤ 0.0001). [Figure 1E]

[0044] Figure 1 shows the efficacy of SFP against immunologically "cold" tumors with low TMB. The effect of the indicated treatments on the frequency of TAM in tumors (n = 5). One-way ANOVA tests were performed to determine statistical significance (ns = P > 0.5; * = p ≤ 0.05; ** = p ≤ 0.01; **** = p ≤ 0.0001). [Figure 1F]

[0044] Figure 1 shows the efficacy of SFP against immunologically "cold" tumors with low TMB. The effect of the indicated treatments on the frequency of NK cells in the tumor (n = 5). One-way ANOVA tests were performed to determine statistical significance (ns = P > 0.5; * = p < 0.05; ** = p < 0.01; **** = p < 0.0001). [Figure 2A]

[0045]

[0023] Figure 1 shows the efficacy of surface-functionalized particle treatment to inhibit primary growth following orthotopic 4T1 tumor inoculation. Tumor volumes were measured throughout the experiment. Growth curves are shown for each treatment group. [Figure 2B]

[0045] This figure shows the efficacy of treatment with surface-functionalized particles after orthotopic 4T1 tumor inoculation in inhibiting primary growth. The mean tumor size at day 21 after tumor inoculation in groups treated with saline, anti-PD1, or CNP-301 is shown. CNP-301 treatment was initiated at different time points (days 1, 2, 4, or 5) after tumor inoculation. The mean tumor size at day 21 was significantly different between the saline group and the CNP-301-treated groups on both days 1 and 2 (p = 0.006 and p = 0.0295, respectively). The mean tumor size of the anti-PD1 group was also larger than the CNP-301-treated group on day 1 (p = 0.0194). Tumor sizes were compared using one-way ANOVA with Tukey's multiple comparison test. N = 7-8 per group. [Figure 3A]

[0046] This shows that treatment with surface-functionalized particles inhibits metastasis to the lung. Bioluminescence imaging of lung metastatic lesions using IVIS® demonstrates that CNP-301 inhibits tumor metastasis and the growth of metastatic lesions. Images acquired during evaluation of lung metastatic lesions in animals treated with saline, anti-PD1, or CNP-301 are shown. CNP-301 treatment was initiated at different time points (days 1, 2, 4, or 5) after tumor inoculation. [Figure 3B]

[0046] We demonstrate that treatment with surface-functionalized particles inhibits metastasis to the lung. Total flux from the images in Figure 3A was quantified and is shown in Figure 3B, where the dotted red line indicates the approximate flux cutoff for detectable metastasis. N=7-8 / group. [Figure 4A]

[0047] Figure 1 shows that treatment with surface-functionalized particles, CNP-301, inhibits existing metastases. 4T1 primary tumors were inoculated into the mammary fat pad and allowed to grow for 11 days. [Figure 4B]

[0047] Treatment with surface-functionalized particles, CNP-301, inhibits existing metastases. On day 11, primary tumors were surgically removed, and treatment with saline or CNP-301 (1 mg / mouse) was initiated. Lung metastases were assessed on day 42 by assaying bioluminescence signaling using IVIS®. N = 9-10 / group. [Figure 5A]

[0048] This figure shows the effect of treatment with surface-functionalized particles, CNP-301, on cytokine / chemokine levels in the blood of B16F10 tumor-bearing mice. C57BL / 6 mice were subcutaneously injected with B16F10 tumor cells. Treatment with saline or CNP-301 was initiated after the formation of palpable tumors. Treatment was administered by intravenous injection once every three days. Blood levels of the indicated cytokines and chemokines (MIP-1β) were measured on days 8 (before the first dose), 14 (24 hours after the third dose), and 20 (24 hours after the fifth dose). [Figure 5B]

[0048] This figure shows the effect of treatment with surface-functionalized particles, CNP-301, on cytokine / chemokine levels in the blood of B16F10 tumor-bearing mice. C57BL / 6 mice were subcutaneously injected with B16F10 tumor cells. Treatment with saline or CNP-301 was initiated after the formation of palpable tumors. Treatment was administered by intravenous injection once every three days. Blood levels of the indicated cytokines and chemokines (TNFα) were measured on days 8 (before the first dose), 14 (24 hours after the third dose), and 20 (24 hours after the fifth dose). [Figure 5C]

[0048] This figure shows the effect of treatment with surface-functionalized particles, CNP-301, on cytokine / chemokine levels in the blood of B16F10 tumor-bearing mice. C57BL / 6 mice were subcutaneously injected with B16F10 tumor cells. Treatment with saline or CNP-301 was initiated after the formation of palpable tumors. Treatment was administered by intravenous injection once every three days. Blood levels of the indicated cytokines and chemokines (RANTES (CCL5)) were measured on days 8 (before the first dose), 14 (24 hours after the third dose), and 20 (24 hours after the fifth dose). [Figure 5D]

[0048] This figure shows the effect of treatment with surface-functionalized particles, CNP-301, on cytokine / chemokine levels in the blood of B16F10 tumor-bearing mice. C57BL / 6 mice were subcutaneously injected with B16F10 tumor cells. Treatment with saline or CNP-301 was initiated after the formation of palpable tumors. Treatment was administered by intravenous injection once every three days. Blood levels of the indicated cytokines and chemokines (IFNγ) were measured on days 8 (before the first dose), 14 (24 hours after the third dose), and 20 (24 hours after the fifth dose). [Figure 5E]

[0048] This figure shows the effect of treatment with surface-functionalized particles, CNP-301, on cytokine / chemokine levels in the blood of B16F10 tumor-bearing mice. C57BL / 6 mice were subcutaneously injected with B16F10 tumor cells. Treatment with saline or CNP-301 was initiated after the formation of palpable tumors. Treatment was administered by intravenous injection once every three days. Blood levels of the indicated cytokines and chemokines (MCP-1) were measured on days 8 (before the first dose), 14 (24 hours after the third dose), and 20 (24 hours after the fifth dose). [Figure 6A]

[0049] This figure shows the effect of treatment with surface-functionalized particles, CNP-301, on immune cells in the blood. C57BL / 6 mice were subcutaneously injected with B16F10 tumor cells. Treatment with saline or CNP-301 was initiated after the formation of palpable tumors. Treatment was administered by intravenous injection once every three days. The frequency of PD-L1+ monocytes (CD11b+Ly6C+Ly6G-) in the blood was assayed on days 8 (before the first dose), 14 (24 hours after the third dose), and 20 (24 hours after the fifth dose). Immune cells from the blood were assayed by flow cytometry. Statistical significance was determined by two-way ANOVA with Bonferroni's multiple comparison test. N=5 / group. [Figure 6B]

[0049] This figure shows the effect of treatment with surface-functionalized particles, CNP-301, on immune cells in the blood. C57BL / 6 mice were subcutaneously injected with B16F10 tumor cells. Treatment with saline or CNP-301 was initiated after the formation of palpable tumors. Treatment was administered by intravenous injection once every three days. The frequency of PD-L1+ granulocytes (CD11b+Ly6C+Ly6G+) in the blood was assayed on days 8 (before the first dose), 14 (24 hours after the third dose), and 20 (24 hours after the fifth dose). Immune cells from the blood were assayed by flow cytometry. Statistical significance was determined by two-way ANOVA with Bonferroni's multiple comparison test. N=5 / group. [Figure 6C]

[0049] This figure shows the effect of treatment with surface-functionalized particles, CNP-301, on immune cells in the blood. C57BL / 6 mice were subcutaneously injected with B16F10 tumor cells. Treatment with saline or CNP-301 was initiated after the formation of palpable tumors. Treatment was administered by intravenous injection once every three days. The frequency of cell surface IL-15+ cells (CD45+) in the blood was assayed on days 8 (before the first dose), 14 (24 hours after the third dose), and 20 (24 hours after the fifth dose). Immune cells from the blood were assayed by flow cytometry. Statistical significance was determined by two-way ANOVA with Bonferroni's multiple comparison test. N=5 / group. [Figure 6D]

[0049] This shows the effect of treatment with surface-functionalized particles, CNP-301, on immune cells in the blood. C57BL / 6 mice were subcutaneously injected with B16F10 tumor cells. Treatment with saline or CNP-301 was initiated after the formation of palpable tumors. Treatment was administered by intravenous injection once every three days. The frequency of total NK cells (CD3-NK1.1+) in the blood was assayed on days 8 (before the first dose), 14 (24 hours after the third dose), and 20 (24 hours after the fifth dose). Immune cells from the blood were assayed by flow cytometry. Statistical significance was determined by two-way ANOVA with Bonferroni's multiple comparison test. N=5 / group. [Figure 6E]

[0049] This shows the effect of treatment with surface-functionalized particles, CNP-301, on immune cells in the blood. C57BL / 6 mice were subcutaneously injected with B16F10 tumor cells. Treatment with saline or CNP-301 was initiated after the formation of palpable tumors. Treatment was administered by intravenous injection once every three days. The frequency of granzyme+ NK cells (CD3-NK1.1+) in the blood was assayed on days 8 (before the first dose), 14 (24 hours after the third dose), and 20 (24 hours after the fifth dose). Immune cells from the blood were assayed by flow cytometry. Statistical significance was determined by two-way ANOVA with Bonferroni's multiple comparison test. N=5 / group. [Figure 6F]

[0049] This shows the effect of treatment with surface-functionalized particles, CNP-301, on immune cells in the blood. C57BL / 6 mice were subcutaneously injected with B16F10 tumor cells. Treatment with saline or CNP-301 was initiated after the formation of palpable tumors. Treatment was administered by intravenous injection once every three days. The frequency of perforin+ NK cells (CD3-NK1.1+) in the blood was assayed on days 8 (before the first dose), 14 (24 hours after the third dose), and 20 (24 hours after the fifth dose). Immune cells from the blood were assayed by flow cytometry. Statistical significance was determined by two-way ANOVA with Bonferroni's multiple comparison test. N=5 / group. [Figure 6G]

[0049] This shows the effect of treatment with surface-functionalized particles, CNP-301, on immune cells in the blood. C57BL / 6 mice were subcutaneously injected with B16F10 tumor cells. Treatment with saline or CNP-301 was initiated after the formation of palpable tumors. Treatment was administered by intravenous injection once every three days. The frequency of CD244+ NK cells (CD3-NK1.1+) in the blood was assayed on days 8 (before the first dose), 14 (24 hours after the third dose), and 20 (24 hours after the fifth dose). Immune cells from the blood were assayed by flow cytometry. Statistical significance was determined by two-way ANOVA with Bonferroni's multiple comparison test. N=5 / group. [Figure 7A]

[0050] This figure shows the effect of treatment with surface-functionalized particles, CNP-301, on immune cells in tumors. C57BL / 6 mice were subcutaneously injected with B16F10 tumor cells. Treatment with saline or CNP-301 was initiated after the formation of palpable tumors. Treatment was administered by intravenous injection once every three days. The frequency of cell surface IL-15+ cells (CD45+) in the blood was assayed on days 8 (before the first dose), 14 (24 hours after the third dose), and 20 (24 hours after the fifth dose). Immune cells from the blood were assayed by flow cytometry. Statistical significance was determined by two-way ANOVA with Bonferroni's multiple comparison test. N=5 / group. [Figure 7B]

[0050] This shows the effect of treatment with surface-functionalized particles, CNP-301, on immune cells in tumors. C57BL / 6 mice were subcutaneously injected with B16F10 tumor cells. Treatment with saline or CNP-301 was initiated after the formation of palpable tumors. Treatment was administered by intravenous injection once every three days. The frequency of granzyme+ NK cells (CD3-NK1.1+) in the blood was assayed on days 8 (before the first dose), 14 (24 hours after the third dose), and 20 (24 hours after the fifth dose). Immune cells from the blood were assayed by flow cytometry. Statistical significance was determined by two-way ANOVA with Bonferroni's multiple comparison test. N=5 / group. [Figure 7C]

[0050] This shows the effect of treatment with surface-functionalized particles, CNP-301, on immune cells in tumors. C57BL / 6 mice were subcutaneously injected with B16F10 tumor cells. Treatment with saline or CNP-301 was initiated after the formation of palpable tumors. Treatment was administered by intravenous injection once every three days. The frequency of perforin+ NK cells (CD3-NK1.1+) in the blood was assayed on days 8 (before the first dose), 14 (24 hours after the third dose), and 20 (24 hours after the fifth dose). Immune cells from the blood were assayed by flow cytometry. Statistical significance was determined by two-way ANOVA with Bonferroni's multiple comparison test. N=5 / group. [Figure 7D]

[0050] This shows the effect of treatment with surface-functionalized particles, CNP-301, on immune cells in tumors. C57BL / 6 mice were subcutaneously injected with B16F10 tumor cells. Treatment with saline or CNP-301 was initiated after the formation of palpable tumors. Treatment was administered by intravenous injection once every three days. The frequency of CD244+ NK cells (CD3-NK1.1+) in the blood was assayed on days 8 (before the first dose), 14 (24 hours after the third dose), and 20 (24 hours after the fifth dose). Immune cells from the blood were assayed by flow cytometry. Statistical significance was determined by two-way ANOVA with Bonferroni's multiple comparison test. N=5 / group. [Figure 8A]

[0051] This figure shows the effect of treatment with surface-functionalized particles, CNP-301, on cytokine / chemokine levels in the blood of MC38 tumor-bearing mice. C57BL / 6 mice were subcutaneously injected with MC38 tumor cells. Treatment with saline or CNP-301 was initiated after the formation of palpable tumors. Treatment was administered by intravenous injection once every three days. Blood levels of the indicated cytokines and chemokines (MIP-1β) were measured on days 8 (before the first dose), 14 (24 hours after the third dose), and 20 (24 hours after the fifth dose). Cytokines / chemokines from blood were assayed by ELISA. Statistical significance was determined by two-way ANOVA with Bonferroni's multiple comparison test. N=5 / group. [Figure 8B]

[0051] This figure shows the effect of treatment with surface-functionalized particles, CNP-301, on cytokine / chemokine levels in the blood of MC38 tumor-bearing mice. C57BL / 6 mice were subcutaneously injected with MC38 tumor cells. Treatment with saline or CNP-301 was initiated after the formation of palpable tumors. Treatment was administered by intravenous injection once every three days. Blood levels of the indicated cytokines and chemokines (TNFα) were measured on days 8 (before the first dose), 14 (24 hours after the third dose), and 20 (24 hours after the fifth dose). Cytokines / chemokines from blood were assayed by ELISA. Statistical significance was determined by two-way ANOVA with Bonferroni's multiple comparison test. N=5 / group. [Figure 8C]

[0051] This figure shows the effect of treatment with surface-functionalized particles, CNP-301, on cytokine / chemokine levels in the blood of MC38 tumor-bearing mice. C57BL / 6 mice were subcutaneously injected with MC38 tumor cells. Treatment with saline or CNP-301 was initiated after the formation of palpable tumors. Treatment was administered by intravenous injection once every three days. Blood levels of the indicated cytokines and chemokines (RANTES (CCL5)) were measured on days 8 (before the first dose), 14 (24 hours after the third dose), and 20 (24 hours after the fifth dose). Cytokines / chemokines from blood were assayed by ELISA. Statistical significance was determined by two-way ANOVA with Bonferroni's multiple comparison test. N=5 / group. [Figure 8D]

[0051] This figure shows the effect of treatment with surface-functionalized particles, CNP-301, on cytokine / chemokine levels in the blood of MC38 tumor-bearing mice. C57BL / 6 mice were subcutaneously injected with MC38 tumor cells. Treatment with saline or CNP-301 was initiated after the formation of palpable tumors. Treatment was administered by intravenous injection once every three days. Blood levels of the indicated cytokines and chemokines (IFNγ) were measured on days 8 (before the first dose), 14 (24 hours after the third dose), and 20 (24 hours after the fifth dose). Cytokines / chemokines from blood were assayed by ELISA. Statistical significance was determined by two-way ANOVA with Bonferroni's multiple comparison test. N=5 / group. [Figure 8E]

[0051] This figure shows the effect of treatment with surface-functionalized particles, CNP-301, on cytokine / chemokine levels in the blood of MC38 tumor-bearing mice. C57BL / 6 mice were subcutaneously injected with MC38 tumor cells. Treatment with saline or CNP-301 was initiated after the formation of palpable tumors. Treatment was administered by intravenous injection once every three days. Blood levels of the indicated cytokines and chemokines (MCP-1) were measured on days 8 (before the first dose), 14 (24 hours after the third dose), and 20 (24 hours after the fifth dose). Cytokines / chemokines from blood were assayed by ELISA. Statistical significance was determined by two-way ANOVA with Bonferroni's multiple comparison test. N=5 / group. [Figure 9A]

[0052] This figure shows the effect of treatment with surface-functionalized particles, CNP-301, on immune cells in the blood. C57BL / 6 mice were subcutaneously injected with MC38 tumor cells. Treatment with saline or CNP-301 was initiated after the formation of palpable tumors. Treatment was administered by intravenous injection once every three days. The frequency of PD-L1+ monocytes (CD11b+Ly6C+Ly6G-) in the blood was assayed on days 8 (before the first dose), 14 (24 hours after the third dose), and 20 (24 hours after the fifth dose). Immune cells from the blood were assayed by flow cytometry. Statistical significance was determined by two-way ANOVA with Bonferroni's multiple comparison test. N=5 / group. [Figure 9B]

[0052] This figure shows the effect of treatment with surface-functionalized particles, CNP-301, on immune cells in the blood. C57BL / 6 mice were subcutaneously injected with MC38 tumor cells. Treatment with saline or CNP-301 was initiated after the formation of palpable tumors. Treatment was administered by intravenous injection once every three days. The frequency of PD-L1+ granulocytes (CD11b+Ly6C+Ly6G+) in the blood was assayed on days 8 (before the first dose), 14 (24 hours after the third dose), and 20 (24 hours after the fifth dose). Immune cells from the blood were assayed by flow cytometry. Statistical significance was determined by two-way ANOVA with Bonferroni's multiple comparison test. N=5 / group. [Figure 9C]

[0052] This shows the effect of treatment with surface-functionalized particles, CNP-301, on immune cells in the blood. C57BL / 6 mice were subcutaneously injected with MC38 tumor cells. Treatment with saline or CNP-301 was initiated after the formation of palpable tumors. Treatment was administered by intravenous injection once every three days. The frequency of cell surface IL-15+ cells (CD45+) in the blood was assayed on days 8 (before the first dose), 14 (24 hours after the third dose), and 20 (24 hours after the fifth dose). Immune cells from the blood were assayed by flow cytometry. Statistical significance was determined by two-way ANOVA with Bonferroni's multiple comparison test. N=5 / group. [Figure 9D]

[0052] This shows the effect of treatment with surface-functionalized particles, CNP-301, on immune cells in the blood. C57BL / 6 mice were injected subcutaneously with MC38 tumor cells. Treatment with saline or CNP-301 was initiated after the formation of palpable tumors. Treatment was administered by intravenous injection once every three days. The frequency of total NK cells (CD3-NK1.1+) in the blood was assayed on days 8 (before the first dose), 14 (24 hours after the third dose), and 20 (24 hours after the fifth dose). Immune cells from the blood were assayed by flow cytometry. Statistical significance was determined by two-way ANOVA with Bonferroni's multiple comparison test. N=5 / group. [Figure 9E]

[0052] This shows the effect of treatment with surface-functionalized particles, CNP-301, on immune cells in the blood. C57BL / 6 mice were subcutaneously injected with MC38 tumor cells. Treatment with saline or CNP-301 was initiated after the formation of palpable tumors. Treatment was administered by intravenous injection once every three days. The frequency of granzyme+ NK cells (CD3-NK1.1+) in the blood was assayed on days 8 (before the first dose), 14 (24 hours after the third dose), and 20 (24 hours after the fifth dose). Immune cells from the blood were assayed by flow cytometry. Statistical significance was determined by two-way ANOVA with Bonferroni's multiple comparison test. N=5 / group. [Figure 9F]

[0052] This shows the effect of treatment with surface-functionalized particles, CNP-301, on immune cells in the blood. C57BL / 6 mice were subcutaneously injected with MC38 tumor cells. Treatment with saline or CNP-301 was initiated after the formation of palpable tumors. Treatment was administered by intravenous injection once every three days. The frequency of perforin+ NK cells (CD3-NK1.1+) in the blood was assayed on days 8 (before the first dose), 14 (24 hours after the third dose), and 20 (24 hours after the fifth dose). Immune cells from the blood were assayed by flow cytometry. Statistical significance was determined by two-way ANOVA with Bonferroni's multiple comparison test. N=5 / group. [Figure 9G]

[0052] This shows the effect of treatment with surface-functionalized particles, CNP-301, on immune cells in the blood. C57BL / 6 mice were subcutaneously injected with MC38 tumor cells. Treatment with saline or CNP-301 was initiated after the formation of palpable tumors. Treatment was administered by intravenous injection once every three days. The frequency of CD244+ NK cells (CD3-NK1.1+) in the blood was assayed on days 8 (before the first dose), 14 (24 hours after the third dose), and 20 (24 hours after the fifth dose). Immune cells from the blood were assayed by flow cytometry. Statistical significance was determined by two-way ANOVA with Bonferroni's multiple comparison test. N=5 / group. [Figure 10]

[0053] Figure 1 shows the effect of IL-15 blockade on the efficacy of surface-functionalized particles, CNP-301, in a B16F10 tumor model. C57BL / 6 mice were injected subcutaneously with B16F10 tumor cells. After the formation of palpable tumors (50 mm), animals were treated with saline or CNP-301, with or without anti-IL-15 antibody. Isotype IgG antibody was used as a control. Saline / CNP-301 was administered intravenously. Isotype / anti-IL-15 antibody was administered intraperitoneally. All treatments were performed once every three days. Isotype / anti-IL15 treatment began one day before the initiation of saline / CNP-301. Tumor volumes are shown for mice treated with saline or CNP-301 in the presence (IgG) or absence (anti-IL-15) of IL-15. Statistical significance was determined by two-way ANOVA with Bonferroni's multiple comparison test. N=5 / group. [Figure 11]

[0054] Figure 1 shows the effect of NK cell depletion on the efficacy of surface-functionalized particles, CNP-301, in a B16F10 tumor model. C57BL / 6 mice were injected subcutaneously with B16F10 tumor cells. After the formation of palpable tumors (50 mm), animals were treated with saline or CNP-301, with or without anti-NK1.1 antibody. Isotype antibody was used as a control. Saline / CNP-301 was administered by intravenous injection. Isotype / anti-NK1.1 antibody was administered by intraperitoneal injection. All treatments were performed once every three days. Tumor volumes are shown for mice treated with saline or CNP-301 in the presence (IgG) or absence (anti-NK1.1) of NK cells. Statistical significance was determined by two-way ANOVA with Bonferroni's multiple comparison test. N=5 per group. [Figure 12]

[0055] Figure 1 shows the effect of NK cell depletion on the efficacy of surface-functionalized particles, CNP-301, in the MC38 tumor model. C57BL / 6 mice were injected subcutaneously with MC38 tumor cells. After the formation of palpable tumors (50 mm), animals were treated with saline or CNP-301, with or without anti-NK1.1 antibody. Isotype antibody was used as a control. Saline / CNP-301 was administered intravenously. Isotype / anti-NK1.1 antibody was administered intraperitoneally. All treatments were performed once every three days. Tumor volumes are shown for mice treated with saline or CNP-301 in the presence (IgG) or absence (anti-NK1.1) of NK cells. Statistical significance was determined by two-way ANOVA with Bonferroni's multiple comparison test. N=5 per group. [Figure 13A]

[0056] This figure shows the effect of surface-functionalized particles, CNP-301, on bone marrow-derived cells in the blood and lungs. Orthotopic 4T1 mammary tumors were established in BALB / c mice. Three days after tumor inoculation, mice were administered a single dose of saline or CNP-301. Blood was collected from the mice 12 hours after treatment, and the frequencies of macrophages (CD11b+ / F4 / 80+), monocytes (CD11b+Ly6C+), MDSCs (CD11b+ / Ly6Clo / - / Ly6G+), and dendritic cells (CD11c+) were assessed by flow cytometry (n=4). Two-way ANOVA with Tukey's multiple comparison test was performed. Statistical significance was defined as ***p<0.001 and ****p<0.0001. [Figure 13B]

[0056] The effect of surface-functionalized particles, CNP-301, on bone marrow-derived cells in blood and lungs is shown. Orthotopic 4T1 mammary tumors were established in BALB / c mice. Three days after tumor inoculation, mice were administered saline or CNP-301 for six consecutive days. Ten days after tumor inoculation, lungs were harvested, and the frequencies of macrophages (CD11b+ / F4 / 80+), monocytes (CD11b+Ly6C+), MDSCs (CD11b+ / Ly6Clo / - / Ly6G+), and dendritic cells (CD11c+) were assessed by flow cytometry (n=4). Two-way ANOVA with Tukey's multiple comparison test was performed. Statistical significance was defined as ***p<0.001 and ****p<0.0001. [Figure 14A]

[0057] Figure 1 shows the results of an assay of cellular uptake of surface-functionalized particles, CNP-301, in LLC tumor-bearing mice. LLC tumors were established in C57BL / 6 mice. After palpable tumor formation, animals were intravenously administered saline (control) or CNP-301 encapsulating fluorescently labeled (Alexa-Fluor 647) OVA. Two hours after intravenous injection, mice were sacrificed, and CNP-301 uptake was assayed by flow cytometry. Figure 2 shows flow cytometry plots showing the frequencies of CNP-301-positive (APC-CNP-301) TAM (CD11b+F4 / 80+), M-MDSC (CD11b+Ly6C+Ly6G-), PMN-MDSC (CD11b+Ly6C-Ly6G+), and fibroblasts (CD45-CD140a+) in LLC tumors. [Figure 14B]

[0057] This figure shows the results of an assay of cellular uptake of surface-functionalized particles, CNP-301, in LLC tumor-bearing mice. LLC tumors were established in C57BL / 6 mice. After palpable tumor formation, animals were intravenously administered saline (control) or CNP-301 encapsulating fluorescently labeled (Alexa-Fluor 647) OVA. Mice were sacrificed 2 hours after intravenous injection, and CNP-301 uptake was assayed by flow cytometry. The frequencies of CNP-301-positive (APC-CNP-301) TAM (CD11b+F4 / 80+), M-MDSC (CD11b+Ly6C+Ly6G-), PMN-MDSC (CD11b+Ly6C-Ly6G+), and fibroblast (CD45-CD140a+) in LLC tumors. [Figure 14C]

[0057] Figure 1 shows the results of an assay of cellular uptake of surface-functionalized particles, CNP-301, in LLC tumor-bearing mice. LLC tumors were established in C57BL / 6 mice. After palpable tumor formation, animals were administered saline (control) or CNP-301 encapsulating fluorescently labeled (Alexa-Fluor 647) OVA via intravenous injection. Mice were sacrificed 2 hours after intravenous injection, and CNP-301 uptake was assayed by flow cytometry. Figure 2 shows flow cytometry plots showing the frequencies of CNP-301-positive (APC-CNP-301) macrophages (CD11b+F4 / 80+), M-MDSCs (CD11b+Ly6C+Ly6G-), and PMN-MDSCs (CD11b+Ly6C-Ly6G+) in the spleens of LLC tumor-bearing mice. [Figure 14D]

[0057] Figure 1 shows the results of an assay of cellular uptake of surface-functionalized particles, CNP-301, in LLC tumor-bearing mice. LLC tumors were established in C57BL / 6 mice. After palpable tumor formation, animals were intravenously administered saline (control) or CNP-301 encapsulating fluorescently labeled (Alexa-Fluor 647) OVA. Mice were sacrificed 2 hours after intravenous injection, and CNP-301 uptake was assayed by flow cytometry. The frequencies of CNP-301-positive (APC-CNP-301) macrophages (CD11b+F4 / 80+), M-MDSCs (CD11b+Ly6C+Ly6G-), and PMN-MDSCs (CD11b+Ly6C-Ly6G+) in the spleens of LLC tumor-bearing mice. [Figure 15A]

[0058] This figure shows the effect of surface-functionalized particles, CNP-301, on gene expression in tumor-associated macrophages and fibroblasts in LLC tumors. LLC tumors were established in C57BL / 6 mice. After the formation of palpable tumors, animals were administered saline (control) or CNP-301 by intravenous injection twice a week for 2 weeks. At the end of treatment, mice were sacrificed and tumors were harvested. Expression of the indicated genes was assessed by qPCR in TAMs (CD11b+F4 / 80+) isolated from LLC tumors. [Figure 15B]

[0058] This figure shows the effect of surface-functionalized particles, CNP-301, on gene expression in tumor-associated macrophages and fibroblasts in LLC tumors. LLC tumors were established in C57BL / 6 mice. After the formation of palpable tumors, animals were administered saline (control) or CNP-301 by intravenous injection twice a week for 2 weeks. At the end of treatment, mice were sacrificed and tumors were harvested. Expression of the indicated genes was assessed by qPCR in fibroblasts (CD45-CD140a+) isolated from LLC tumors. DETAILED DESCRIPTION OF THE INVENTION

[0045] Detailed Description

[0059] The present disclosure demonstrates that the surface-functionalized particles described herein can reduce tumor growth in vivo to a greater extent than traditional immunotherapy with checkpoint inhibitors in tumors that are (i) immune-evasive, (ii) immunologically "cold," (iii) immunologically protected, (iv) microsatellite stable, (iv) microsatellite-low instability, (v) contain a low immune infiltrate, (vi) contain a low tumor mutational burden, and / or (vii) exhibit heterogeneity. The ability of surface-functionalized particles alone to have such an effect is surprising and unexpected, and supports the use of surface-functionalized particles to treat subjects with cancer, particularly cancers that may be non-responsive or refractory to immunotherapeutic agents.

[0046] definition

[0060] Each publication, patent application, patent, and other reference cited herein The references are incorporated by reference in their entirety to the extent not inconsistent with this disclosure.

[0047]

[0061] As used in this specification and claims, the singular forms "a," "an," It is noted herein that " and "the" include plural references unless the context clearly dictates otherwise. It is intended.

[0048]

[0062] As used herein, "particle" refers to any non-tissue-derived composition of matter, which may be a spherical or sphere-like entity, a bead, or a liposome. The terms "particle," "immunomodulated particle," and "bead" may be used interchangeably depending on the context. Furthermore, the term "particle" may be used to encompass beads and spheres.

[0049]

[0063] As used herein, "surface functionalization" refers to particles having one or more functional groups on their surface. In some embodiments, surface functionalization occurs by introducing one or more functional groups onto the surface of the particle. "Surface-functionalized particle" (SFP) refers to a particle described herein that includes functional groups on the particle surface. In embodiments, surface functionalization can be achieved by carboxylation (i.e., the addition of one or more carboxyl groups to the particle surface) or the addition of other chemical groups (e.g., other chemical groups that impart a negative surface charge). Because the surface functional groups provide sites for the conjugation of ligands, surface-functionalized particles can also include targeting agents such as polypeptides, antibodies, nucleic acids, lipids, small molecules, carbohydrates, and surfactants. Methods for producing surface-functionalized nanoparticles are described, for example, in Froimowicz et al., Curr Org. Chem 17:900-912, 2013. In various embodiments, the surface Functionalized particles are contemplated to include negatively charged particles that do not contain a therapeutic agent, for example, that do not contain an attached peptide or antigenic moiety or other bioactive agent.

[0050]

[0064] As used herein, "negatively charged particles" refers to particles that have been modified to have a net surface charge less than zero. In embodiments, the negatively charged particles are surface-functionalized particles in which the particles are carboxylated, thereby having a negative surface charge.

[0051]

[0065] Zeta potential is the charge generated at the interface between a solid surface and its liquid medium. "Negative zeta potential" refers to particles having a zeta potential at their surface, expressed in millivolts (mV), measured by instruments known in the art for calculating zeta potential, such as a NanoBrook ZetaPlus zeta potential analyzer or a Malvern Zetasizer.

[0052]

[0066] "Carboxylated particles" or "carboxylated beads" or "carboxylated spheres" include any particles that have been modified or surface-functionalized to add one or more carboxyl groups to the particle surface. In some embodiments, the addition of carboxyl groups enhances phagocytic / monocyte uptake of the particles from the circulation, for example, by interaction with scavenger receptors such as MARCO. Particle carboxylation can be achieved using any compound that adds carboxyl groups, including, but not limited to, poly(ethylene-maleic anhydride) (PEMA). Carboxylation can also be achieved by forming particles using a polymer with negative carboxyl groups (e.g., PLGA), where the manufacturing process results in carboxyl groups located on the surface of the particles.

[0053]

[0067] As used herein, "biodegradable" refers to particles comprising polymers that may undergo degradation, for example, as a result of functional groups reacting with water in solution. As used herein, the term "degradation" refers to becoming soluble either by a decrease in molecular weight or by converting hydrophobic groups to hydrophilic groups. Biodegradable particles do not persist in the body for extended periods of time, and the time for complete degradation can be controlled. Biocompatible biodegradable polymers useful in the present invention include caprolactones, carbonates, amides, amino acids, orthoesters, acetals, silsesquioxanes, and the like. Included are polymers or copolymers of anoacrylates and degradable urethanes, as well as copolymers thereof with linear or branched, substituted or unsubstituted alkanyl, haloalkyl, thioalkyl, aminoalkyl, alkenyl, or aromatic hydroxy- or dicarboxylic acids. Additionally, biologically important amino acids with reactive side groups, such as lysine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine, and cysteine, or their enantiomers, can be incorporated into copolymers with any of the above materials to provide reactive groups or conjugate moieties for conjugation to antigenic peptides and proteins. Biodegradable materials suitable for the present invention include diamond, PLA, PGA, polypropylene sulfide, and PLGA polymers, as well as metals such as iron (Fe), zinc (Zn), cadmium (Cd), gold, or silver. Biocompatible but non-biodegradable materials can also be used in the particles described herein. For example, non-biodegradable polymers of acrylates, ethylene-vinyl acetate, acyl-substituted cellulose acetate, non-degradable urethanes, styrene, vinyl chloride, vinyl fluoride, vinyl imidazole, chlorosulfonated olefins, ethylene oxide, vinyl alcohol, TEFLON® (DuPont, Wilmington, Del.), and nylon may be used.

[0054]

[0068] As used herein, the term "tumor microenvironment" (TME) refers to the cells, molecules, and blood vessels that surround and nourish tumor cells (National Cancer Institute Dictionary of Cancer Terms). The tumor microenvironment includes immune cells, such as myeloid-derived inflammatory cells, myelomonocytic cells, myeloid-derived suppressor cells, tumor-associated macrophages, and lymphocytes, fibroblasts, signaling molecules, and the extracellular matrix (ECM) (Joyce et al., Science 348:74-80, 2015).

[0055]

[0069] As used herein, the term "hot tumor" refers to a tumor that exhibits a higher degree of immune cell infiltration, for example, in the TME or tumor site, and that generally responds well to immunotherapy.

[0056]

[0070] As used herein, the term "cold tumor" encompasses tumors that exhibit low levels of immune infiltrate, respond poorly to immunotherapy, have low tumor mutational burden, and are microsatellite stable or microsatellite instability (with respect to DNA mismatch repair), and / or exhibit tumor heterogeneity. Cold tumors are also said to be immune-evasive or immunologically protected. For further description of the characterization of hot and cold tumors, see, e.g., References 9, 16, 17, 23, and 24.

[0057]

[0071] As used herein, the term "subject" refers to a human or non-human animal, including a mammal or primate, to which the particles described herein are administered. Subjects can include animals such as dogs, cats, rats, mice, rabbits, horses, pigs, sheep, cows, and humans and other primates.

[0058]

[0072] The term "therapeutic agent" refers to a moiety that, when administered in a therapeutically effective amount, can ameliorate or alleviate one or more symptoms or signs of the disease or disorder being treated. Non-limiting examples of therapeutic agents include peptide, protein, or other cancer therapeutic agents, including small molecule therapeutic agents.

[0059]

[0073] The term "therapeutically effective amount" is used herein to indicate an amount of a target-specific composition of the present disclosure that is effective in ameliorating or alleviating the symptoms or signs of the disease or disorder being treated.

[0060]

[0074] The terms "treat," "treated," "treating," and "treatment" As used in connection with the methods herein, refers to the temporary or permanent, partial or complete elimination, reduction, inhibition, or amelioration of one or more clinical symptoms, signs, or progression of an event, disease, or condition. Such treatment need not be absolute to be beneficial.

[0061] surface functionalized particles

[0075] The present disclosure provides for the use of surface-functionalized particles in the methods of treatment described herein.

[0062]

[0076] Surface-functionalized particles can be formed from a wide variety of materials. The particles are preferably composed of materials suitable for biological use (e.g., pharmaceutically acceptable materials). For example, the particles can be composed of glass, silica, polyesters of hydroxycarboxylic acids, polyanhydrides of dicarboxylic acids, or copolymers of hydroxycarboxylic acids and dicarboxylic acids, and biocompatible metals. In various embodiments, the particles can be composed of polyesters of linear or branched, substituted or unsubstituted, saturated or unsaturated, linear or bridged alkanyl, haloalkyl, thioalkyl, aminoalkyl, aryl, aralkyl, alkenyl, aralkenyl, heteroaryl, or alkoxy hydroxy acids, or linear or branched, substituted or unsubstituted, saturated or unsaturated, linear or bridged alkanyl, haloalkyl, thioalkyl, aminoalkyl, aryl, aralkyl, alkenyl, aralkenyl, heteroaryl, or alkoxy dicarboxylic acid polyanhydrides. Furthermore, the particles may be or may consist of quantum dots, such as quantum dot polystyrene particles (Joumaa et al. (2006) Langmuir 22: 1810-6). Particles containing a mixture of ester and anhydride linkages (e.g., copolymers of glycolic acid and sebacic acid) may also be used. For example, the particles may comprise materials including polyglycolic acid polymer (PGA), polylactic acid polymer (PLA), polysebacic acid polymer (PSA), poly(lactic acid-co-glycolic acid) copolymer (PLGA or PLG; these terms are interchangeable), [rho]oly(lactic acid-co-sebacic acid) copolymer (PLSA), poly(glycolic acid-co-sebacic acid) copolymer (PGSA), polypropylene sulfide polymer, poly(caprolactone), chitosan, and the like. Other biocompatible, biodegradable polymers useful in the present invention include polymers or copolymers of caprolactone, carbonate, amide, amino acid, orthoester, acetal, cyanoacrylate, and degradable urethanes, and copolymers thereof with linear or branched, substituted or unsubstituted alkanyl, haloalkyl, thioalkyl, aminoalkyl, alkenyl, or aromatic hydroxy- or dicarboxylic acids.Additionally, biologically important amino acids with reactive side groups, such as lysine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine, and cysteine, or their enantiomers, can be included in copolymers with any of the above materials to provide reactive groups or conjugation moieties for conjugation to antigenic peptides and proteins.

[0063]

[0077] In embodiments, the surface-functionalized particles comprise one or more biodegradable polymers or materials. Biodegradable materials suitable for the present invention include diamond, PLA, PGA, polypropylene sulfide, and PLGA polymers, and metals such as iron (Fe), zinc (Zn), cadmium (Cd), gold (Au), or silver (Ag).

[0064]

[0078] Biocompatible but non-biodegradable materials can also be used in the particles described herein, such as non-biodegradable polymers of acrylate, ethylene-vinyl acetate, acyl-substituted cellulose acetate, non-degradable urethane, styrene, vinyl chloride, vinyl fluoride, vinyl imidazole, chlorosulfonated olefins, ethylene oxide, vinyl alcohol, TEFLON® (DuPont, Wilmington, Del.), and nylon.

[0065]

[0079] In various embodiments, the particles comprise polymers, copolymers, dendrimers, diamond nanoparticles, polystyrene nanoparticles, or metals. In various embodiments, it is contemplated that the particles comprise polyglycolic acid polymers (PGA), polylactic acid (PLA), polystyrene, copolymers of PLG and PLA (poly(lactide-co-glycolide), PLGA), diamond, liposomes, PEG, cyclodextran, or metals such as iron (Fe), zinc (Zn), cadmium (Cd), gold (Au), or silver (Ag), or combinations thereof.

[0066]

[0080] The surface-functionalized particles of the present disclosure can be produced by any means known in the art. Exemplary methods for producing particles include, but are not limited to, microemulsion polymerization, interfacial polymerization, precipitation polymerization, emulsion evaporation, emulsion diffusion, solvent displacement, and salting out (Astete and Sabliov, J. Biomater. Sci. Polymer Edn., 17:247-289(2006)). The method for producing surface-functionalized particles contemplated herein is disclosed in U.S. Patent No. 9,616,113 and International Publication No. 2017 / 143346. By manipulating the production process of PLGA particles, particle properties (e.g., size, size distribution, zeta potential, morphology, hydrophobicity / hydrophilicity, polypeptide encapsulation, etc.) can be controlled. The size of the surface-functionalized particles is influenced by several factors, including, but not limited to, the concentration of the polymer, e.g., PLGA, the solvent used in the preparation of the particles, the nature of the organic phase, the surfactants used in the preparation, the viscosity of the continuous and discontinuous phases, the nature of the solvent used, the temperature of the water used, sonication, evaporation rate, additives, shear stress, sterilization, and the nature of any encapsulated antigens or polypeptides.

[0067]

[0081] In various embodiments, the surface-functionalized particles are polylactic acid:polyglycolic acid, or copolymers having a molar ratio of about 50:50 or about 80:20 to about 99:1 polyglycolic acid:polylactic acid of about 50:50 or about 80:20 to about 99:1. In some embodiments, the surface-functionalized particles are poly(lactic-co-glycolic acid) particles. In various embodiments, the surface-functionalized particles comprise 50:50 polylactic acid:polyglycolic acid. In various embodiments, the surface-functionalized particles comprise polylactic acid:polyglycolic acid in a ratio of about 99:1 to about 1:99, e.g., about 99:1, about 95:5, about 90:10, about 85:15, about 80:20, about 75:25, about 70:30, about 65:35, about 60:40, about 55:45, about 50:50, about 45:55, about 40:60, about 35:65, about 30:70, about 25:75, about 20:80, about 15:85, about 10:90, about 5:95, and about 1:99, including all values ​​and ranges therebetween.

[0068]

[0082] In some embodiments, the zeta potential of the surface-functionalized particles is about -100 mV to about -1 mV. In some embodiments, the zeta potential of the surface-functionalized particles is about -100 mV to about -40 mV, about -80 mV to about -30 mV, about -75 mV to about -40 mV, about -70 mV to about -30 mV, about -60 mV to about -35 mV, or about -50 mV to about -40 mV. In various embodiments, the zeta potential is about -30 mV, -35 mV, -40 mV, -45 mV, -50 mV, -55 mV, -60 mV, -65 mV, -70 mV, -75 mV, -80 mV, -85 mV, -90 mV, -95 mV, or -100 mV (including all values ​​and subranges therebetween).

[0069]

[0083] In some embodiments, the surface-functionalized particles have an average diameter between about 0.1 μm and about 10 μm. In some embodiments, the surface-functionalized particles have an average diameter between 0.2 μm and about 2 μm. In some embodiments, the surface-functionalized particles have a diameter between about 0.3 μm and about 5 μm. In some embodiments, the surface-functionalized particles have a diameter between about 0.5 μm and about 3 μm. In some embodiments, the surface-functionalized particles have a diameter between about 0.5 μm and about 1 μm. In some embodiments, the surface-functionalized particles have a diameter between about 100-1500 nm, about 200-2000 nm, about 100 The diameter is about 10,000 nm, about 300 to 1,000 nm, about 400 to 800 nm, or about 200 to 700 nm (including all values ​​and subranges therebetween).

[0070]

[0084] To administer the surface-functionalized particles described herein to humans or other mammals, the particles can be formulated in a sterile composition containing one or more sterile, pharmaceutically acceptable carriers. The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce allergic or other adverse reactions when administered using routes well known in the art, as described below. "Pharmaceutically acceptable carriers" include any and all clinically useful solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc.

[0071]

[0085] The pharmaceutical compositions of the present disclosure containing the surface-functionalized particles herein may contain sterile, pharmaceutically acceptable carriers or additives depending on the route of administration. Examples of such carriers or additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymers, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, casein, gelatin, agar, diglycerin, glycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), mannitol, sorbitol, lactose, and pharmaceutically acceptable surfactants. The additives used may be selected from the above or combinations thereof as needed depending on the dosage form of the present invention, but are not limited to these. In the case of solutions or emulsions, suitable carriers include, for example, aqueous or alcoholic / aqueous solutions, emulsions, or suspensions (including saline and buffered media). Parenteral vehicles can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles can include various additives, preservatives, or fluid, nutrient, or electrolyte replenishers. Various aqueous carriers are appropriate, such as sterile phosphate-buffered saline, bacteriostatic water, water, buffered water, 0.4% saline, 0.3% glycine, and the like, and can include other proteins for stability enhancement, such as albumin, lipoproteins, globulins, or the like, which are mildly chemically modified.

[0072]

[0086] It is contemplated that the surface-functionalized particles may further comprise a surfactant. The surfactant may be anionic, cationic, or nonionic. Poloxamer and poloxamine families of surfactants are commonly used in particle synthesis. Surfactants that can be used include, but are not limited to, PEG, Tween-80, gelatin, dextran, Pluronic L-63, PVA, methylcellulose, lecithin, DMAB, and PEMA. Additionally, biodegradable and biocompatible surfactants include vitamin E. The surfactants include, but are not limited to, TPGS (D-α-tocopheryl polyethylene glycol 1000 succinate).In certain embodiments, two surfactants are used.For example, when particles are produced by double emulsion method, the two surfactants can include a hydrophobic surfactant for the first emulsion and a hydrophobic surfactant for the second emulsion.

[0073]

[0087] Therapeutic formulations of surface-functionalized particles are prepared for storage in the form of a lyophilized formulation or aqueous solution by mixing particles having the desired degree of purity with optional physiologically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citric acid, succinic acid, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride); benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; or metal complexes (e.g., Zn-protein complexes).

[0074]

[0088] Particle preparations can be stabilized by lyophilization. The addition of a cryoprotectant such as trehalose can reduce particle aggregation during lyophilization. Any suitable lyophilization and reconstitution technique can be used. Those skilled in the art will recognize that lyophilization and reconstitution can lead to varying degrees of loss of antibody activity, and that use levels may have to be adjusted to compensate.

[0075] How to use

[0089] In one aspect, it is hypothesized herein that SFPs promote the conversion of immunologically cold tumors (immune evasive) to immunologically hot tumors (immunogenic), which in turn may enable the treatment of immunologically protected and often refractory tumors. Furthermore, SFPs can enhance the efficacy of other cancer treatments when administered in combination to a subject. It is suggested herein that treatment using SFPs alone or in combination with cancer therapeutic agents can alter the tumor immune infiltrate, which is composed of T cells, B cells, APCs, monocytes, MDSCs, TAMs, neutrophils, other monocyte-derived cells, tumor-associated stroma, cancer stem cells, and mesenchymal stem cells, resulting in enhanced anti-tumor therapeutic effects.

[0076] Identification of tumor type

[0090] In various embodiments, the present disclosure provides methods of treating cancer in a subject comprising administering to the subject surface-functionalized particles, alone or in combination with a cancer therapeutic, the subject having one or more tumors characterized as being immunoevasive, immunologically protected, immunologically "cold," microsatellite stable, microsatellite low instability, containing a low immune infiltrate, containing a low tumor mutational burden, and / or exhibiting heterogeneity.

[0077]

[0091] In various embodiments, the present disclosure provides methods for treating a tumor characterized as being immunoevasive, immunologically protected, immunologically "cold," microsatellite stable, microsatellite low instability, containing a low immune infiltrate, containing a low tumor mutational burden, and / or exhibiting heterogeneity in a subject, the methods comprising: (i) diagnosing the subject as having an immune evasive tumor, an immunologically protected tumor, an immunologically "cold" tumor, a microsatellite stable tumor, a microsatellite low instability tumor, containing a low immune infiltrate, containing a low tumor mutational burden, and / or exhibiting heterogeneity; and (ii) administering to the subject surface-functionalized particles, alone or in combination with a cancer therapeutic agent. In various embodiments, diagnosing comprises assaying biomarkers / features associated with tumors characterized as being immunoevasive, immunologically protected, immunologically "cold," microsatellite stable, microsatellite low instability, containing a low immune infiltrate, containing a low tumor mutational burden, and / or exhibiting heterogeneity. In various embodiments, the method further comprises (iii) determining whether the subject's tumor becomes immunoresponsive (e.g., immunogenic), and then (iv) administering immunotherapy, optionally in combination with surface-functionalized particles.

[0078]

[0092] Also provided herein are methods for determining whether a subject can / will respond to treatment with the surface-functionalized particles described herein, alone or in combination with a cancer therapeutic, and treating the subject accordingly. In various embodiments, a patient diagnosed with cancer is tested to identify the tumor as a cold tumor, for example, using the methods described herein and other methods described in the art. The present disclosure provides methods for treating a subject with cancer, e.g., an immune-evasive tumor, using surface-functionalized particles, alone or in combination with a cancer therapeutic, the methods including obtaining a tumor sample from the subject, performing an assay to determine whether the tumor is a cold tumor, and, if the tumor is identified as a cold tumor, treating the subject with surface-functionalized particles, alone or in combination with a cancer therapeutic. Assays for determining whether a tumor is a cold tumor include tumor mutation burden analysis, microsatellite instability (MSI) testing, immune cell (e.g., CD4 + T cells, CD8 + T cells, NK1.1 + The tumor infiltration rate of immune cells (NK cells, APCs, monocytes, and neutrophils), immune cell phenotype (e.g., PD-1 + , PD-L1 + , and PD-L2 + ), immune cell function (e.g., expression of IFN-γ, IL-12, IL-15, and MHCII), and the ratio of pro- to anti-inflammatory mediators in the tumor microenvironment (TME).

[0079]

[0093] Several diagnostic tools designed to characterize tumors at the cellular and molecular level are FDA approved and commercially available. Examples of approved diagnostics include FOUNDATIONONE® CDX, FOUNDATIONONE® LIQUID, FOUNDATIONONE® HEME, BRACAnalysis CDx, therascreen EGFR RGQ PCR kit, cobase EGFR Mutation Test v2, PD-L1 IHC 22C3 pharmDx, Abbott Real Time IDH1, MRDx BCR-ABL test, and VENTANA ALK. (D5F3) CDx Assay, Abbott RealTime IDH2, Praxis Extended RAS Panel, Oncomine Dx Target Test, LeukoStrat CDx FLT3 Mutation Assay, FoundationFocus CDxBRCA Assay, Vysis CLL FISH Probe Kit, KIT D816V Mutation Detection, PDGFRB FISH, cobas KRAS Mutation Test, therascreen KRAS RGQ PCR Kit, FerriScan, Dako c-KIT pharmDx, INFORM Her-2 / neu, PathVysion HER-2 DNA Probe Kit, SPOT-LIGHT HER2 CISH Kit, Bond Oracle HER2 IHC System, HER2 CISH pharmDx Kit, INFORM HER2 DUAL ISH DNA Probe Cocktail, HercepTest, HER2 FISH pharmDx Kit, THXID BRAF Kit, Vysis ALK Break Apart FISH Probe Kit, cobas 4800 These include the BRAF V600 Mutation Test, the VENTANA PD-L1 (SP142) Assay, therascreen FGFR RGQ RT-PCR Kit, and therascreen PIK3CA RGQ PCR Kit.

[0080]

[0094] In various embodiments, subjects are screened for eligibility for treatment with one or more immunotherapies described herein. In various embodiments, subjects who are not eligible for treatment with such immunotherapies may first be treated with surface-functionalized particles according to the methods described herein. Non-limiting examples of immunotherapies include pembrolizumab (KEYTRUDA®, Merck Sharp & Dohme Corp), nivolumab (OPDIVO®, Bristol-Myers Squibb), atezolizumab (TECENTRIQ®), avelumab (BAVENCIO®), and durvalumab (IMFINZI®). Eligibility criteria for these immunotherapies are known in the art. For example, without limitation, pembrolizumab (KEYTRUDA®), nivolumab (OPDIVO®), and atezolizumab (TECENTRIQ®) have eligibility criteria based on PD-L1 expression levels. PD-L1 expression criteria and methods for measuring it are available at https: / / www.keytrudahcp.com / biomarker-testing / pd-l1-expression-testing / (pembrolizumab). tetanib (KEYTRUDA®), or pembrolizumab (KEYTRUDA®, revised 1 / 2020), atezolizumab (e.g., TECENTRIQ®, revised 5 / 2 The present disclosure can be found in the FDA-approved prescribing information for nivolumab (e.g., OPDIVO®, 6 / 2020 Revision) and nivolumab (e.g., OPDIVO®, 6 / 2020 Revision). Each of these publications is incorporated herein by reference in its entirety for all purposes. As described, treatment of such patients with surface-functionalized particles may facilitate the conversion of tumors ineligible for immunotherapy treatment into immunogenic tumors, which may then allow such tumors to be treated with immunotherapy. In various embodiments, the subject's tumor ineligible for immunotherapy can be monitored throughout the course of treatment with surface-functionalized particles (e.g., based on PD-l1 expression levels, as described herein) to determine when the tumor becomes eligible for immunotherapy treatment. Once the tumor becomes eligible for immunotherapy treatment, immunotherapy can be administered to the subject, either alone or in combination with surface-functionalized particles.

[0081]

[0095] In various embodiments, the present disclosure provides a method of treating cancer in a subject comprising administering surface-functionalized particles, alone or in combination with a cancer therapeutic, to the subject, wherein the subject has one or more tumors with a low immune infiltrate. In various embodiments, administration to a subject with one or more tumors with a low immune infiltrate alters the tumor immune infiltrate. In various embodiments, the tumor immune infiltrate comprises antigen-presenting cells, myeloid cells, and lymphoid cells. In various embodiments, the antigen-presenting cells in the tumor immune infiltrate comprise macrophages and / or dendritic cells. In various embodiments, the myeloid cells in the tumor immune infiltrate comprise monocytes, neutrophils, myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages (TAMs). In various embodiments, the TAMs in the tumor immune infiltrate comprise M1 macrophages, M2 macrophages, and MARCO macrophages. + In various embodiments, lymphoid cells in the tumor immune infiltrate include T cells, B cells, NKT cells, and NK cells.

[0082]

[0096] Qualitative and quantitative methods have been described for characterizing tumor immune infiltrates, including, but not limited to, microscopic analysis, histological assays, cytological assays, flow cytometry, polymerase chain reaction (PCR), quantitative polymerase chain reaction (qPCR), RNA sequencing (RNA-seq), single-cell RNA sequencing (scRNA-seq), next-generation sequencing, whole-exome sequencing, epigenetic sequencing, ATAC-seq, microarray analysis, and mass cytometry or CyTOF. Biomarkers can be used alone or in combination to assess immune cells, including cell surface markers and secreted proteins. Exemplary biomarkers for characterizing tumor immune infiltrates include CD45, CD3, CD4, CD8, CD25, CD44, CD134, CD252, CD137, CD79, CD39, FOXP3, PD-1, LAG-3, TIM-1, IFN-γ, granzyme, perforin, CD11b, CD11c, Ly6C, Ly6G, CD14, CD16, CD80, MARCO, CD68, CD115, CD206, CD163, CD103c, F4 / 80, PD-L1, PD-L2, arginase, iNOS, ROS, TNFR2, and TNFR2. These include, but are not limited to, F-α, TGF-β, MHC-I, MHC-II, NK1.1, NKG2D, CD244, Ki67, CD19, CD20, CCR2, CXCR3, CCR4, CCR5, CCR6, CCR7, CCR10, CCL2, CCL5, Cx3CR1, CCL10, ICOS, CD40, CD40L, IL1α, IL1β, IL2, IL4, IL5, IL6, IL8, IL12, IL15, IL17, IL21, IL22, TCRγ / δ, TCRα / β, STAT3, ROR1c, and RORγt.

[0083]

[0097] Cancer stem cells (CSCs) have been described as a subset of cells found in solid and hematological tumors that are tumorigenic and capable of self-renewal and differentiation. Several reports have documented the importance of CSCs in the pathogenesis of various tumors, tumor recurrence after treatment, and the development of therapeutic resistance. Several cell surface markers can be used to distinguish CSCs in solid and hematological tumors. CSC markers include CD19, CD20, CD24, CD34, CD38, CD44, CD90, CD133, aldehyde dehydrogenase 1, CEACAM-6 / CD66c, BMI-1, connexin 43 / GJA1, and D These include, but are not limited to, LL4, EpCAM / TROP1, GLI-1, GLI-2, integrin, PON1, PTEN, ALCAM / CD166, DPPIV / CD26, Lgr5, Musashi-1, A20, ABCG2, CD15, fractalkine, HIF-2α, L1CAM, c-MAF, nestin, podoplanin, SOX2, CD96, CD117, FLT3, AFP, CD13, CD90, NF2 / merlin, ABCB5, NGFR, syndecan-1, endoglin, STRO-1, and PON1.

[0084]

[0098] In various embodiments, the present disclosure provides a method of treating cancer in a subject, comprising administering to the subject surface-functionalized particles, alone or in combination with a cancer therapeutic, the subject having one or more immune-evasive tumors. In various embodiments, the subject has one or more immunologically protected tumors. In various embodiments, the subject has one or more microsatellite-stable tumors. In various embodiments, the subject has one or more microsatellite-low instability tumors. In various embodiments, the subject has one or more tumors with intermediate microsatellite instability. In various embodiments, the subject has one or more tumors with low tumor mutation burden. In various embodiments, the subject has one or more tumors with intermediate tumor mutation burden. In various embodiments, the subject has one or more tumors that are resistant to treatment. In various embodiments, the subject has one or more immunologically heterogeneous tumors. In various embodiments, the subject has genetically heterogeneous tumors. In various embodiments, the subject has one or more refractory tumors. In one or more embodiments, the subject has a tumor that develops resistance to therapy during the course of treatment.

[0085]

[0099] In various embodiments, tumor characteristics are determined from one or more biological samples from a subject suffering from cancer. In various embodiments, tumor characteristics are determined by comparing one or more biological samples from a subject suffering from cancer with one or more biological samples from one or more healthy subjects. In various embodiments, tumor characteristics are determined from one or more biological samples selected from the group consisting of blood, cerebrospinal fluid, urine, stool, buccal swab, nasal swab, lavage, tissue biopsy, bone marrow biopsy, and tumor biopsy. In various embodiments, tumor characteristics are determined from analysis of cells, proteins, and / or nucleic acids in one or more biological samples from a subject suffering from cancer. In various embodiments, tumor characteristics are determined by comparing analysis of cells, proteins, and / or nucleic acids in one or more biological samples from a subject suffering from cancer with analysis of one or more biological samples from one or more healthy subjects. In various embodiments, tumor characteristics are determined by comparing analysis of cells, proteins, and / or nucleic acids in one or more biological samples from subjects with cancer with analysis of one or more biological samples from one or more subjects with cancer who are responsive to treatment. In various embodiments, the cells are selected from the group consisting of leukocytes, epithelial cells, mesenchymal cells, mesenchymal stem cells, stromal cells, endothelial cells, fibroblasts, cancer-associated fibroblasts (CAFs), pericytes, adipocytes, cancer stem cells, circulating tumor cells (CTCs), hematopoietic stem cells, and hematopoietic progenitor cells. In various embodiments, the proteins are selected from the group consisting of cytokines, chemokines, growth factors, signaling proteins, enzymes, proteases, and nucleases. In various embodiments, the nucleic acids are selected from the group consisting of DNA, ssDNA, circulating tumor DNA (ctDNA), RNA, mRNA, dsRNA, siRNA, miRNA, and lncRNA. In various embodiments, nucleic acid analysis is performed by PCR, RT-PCR, qRT-PCR, next generation sequencing (NGS), RNA-seq, ATAC-seq, exome sequencing, Southern blot, microarray analysis, and / or single cell sequencing.

[0086]

[0100] In various embodiments, the characteristics of a tumor in a subject suffering from cancer are determined from analysis of one or more blood samples taken from the subject. Tumor characteristics of a subject suffering from cancer are determined from analysis of cells, proteins, and / or nucleic acids in one or more blood samples taken from the subject. In various embodiments, tumor characteristics of a subject suffering from cancer are determined by comparing analysis of cells, proteins, and / or nucleic acids in one or more blood samples from the subject suffering from cancer with analysis of one or more blood samples from one or more healthy subjects. In various embodiments, the cells analyzed in the one or more blood samples are leukocytes, epithelial cells, mesenchymal cells, mesenchymal stem cells, stromal cells, endothelial cells, fibroblasts, cancer-associated fibroblasts (CAFs), pericytes, adipocytes, cancer stem cells, circulating tumor cells (CTCs), hematopoietic stem cells, and hematopoietic progenitor cells. In various embodiments, the leukocytes are myeloid and lymphoid cells. In various embodiments, the myeloid cells are monocytes, macrophages, neutrophils, granulocytes, dendritic cells, mast cells, eosinophils, and basophils. In various embodiments, the lymphoid cell is a T cell, a B cell, an NK cell, an NK-T cell, or an iNK cell.

[0087]

[0101] In various embodiments, analysis of cells from one or more blood samples taken from a subject suffering from cancer demonstrates elevated levels of immunosuppressive cells compared to analysis of cells from one or more healthy subjects and / or one or more subjects suffering from cancer and responding to treatment. In various embodiments, the immunosuppressive cells include myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), neutrophils, T cells, or the like. reg cells, and B regIn various embodiments, the MDSCs are monocytic MDSCs (M-MDSCs) and polymorphonuclear MDSCs (PMN-MDSCs). In various embodiments, the TAMs are M2TAMs. In various embodiments, the immunosuppressive cells are CAFs. In various embodiments, the level of immunosuppressive cells in one or more blood samples from a subject having one or more tumors characterized as immunoevasive, immunologically protected, and / or immunologically "cold" is about 5 to 100% (e.g., about 5 to 100%) lower than one or more blood samples taken from one or more healthy subjects and / or one or more subjects suffering from cancer and responding to treatment. %, 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%, or about 100%, including all values ​​and ranges therebetween), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50% increase. In various embodiments, the level of immunosuppressive cells in one or more blood samples from subjects having one or more tumors characterized as immunoevasive, immunologically protected, and / or immunologically "cold" is elevated by about 2-100 fold (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold, including all values ​​and ranges therebetween) compared to one or more blood samples taken from one or more healthy subjects and / or one or more subjects afflicted with cancer and responsive to treatment. In various embodiments, the immunosuppressive cells are identified by assaying cell surface protein expression.In various embodiments, analysis of cells from one or more blood samples taken from a subject having one or more tumors characterized as being immunoevasive, immunologically protected, and / or immunologically "cold" demonstrates reduced levels or absence of activated pro-inflammatory immune cells (e.g., reduced by 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%, or about 100% (including all values ​​and ranges therebetween) compared to a healthy subject or a subject having cancer and responding to treatment).

[0088]

[0102] In various embodiments, analysis of cells from one or more blood samples taken from a subject with one or more tumors characterized as immunoevasive, immunologically protected, and / or immunologically "cold" reveals activated pro-inflammatory immune cells. The subject demonstrates reduced levels or absence of immune cells (e.g., reduced by about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100-fold (including all values ​​and ranges therebetween) compared to a healthy subject or a subject with cancer and responding to treatment). In various embodiments, the activated pro-inflammatory cells are dendritic cells (DCs), macrophages, M1 macrophages, T cells, B cells, NK cells, NK-T cells, and iNK cells. In various embodiments, the frequency of pro-inflammatory immune cells is 10% or less (e.g., about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1%) of all leukocytes analyzed from one or more blood samples taken from the subject. In various embodiments, activated pro-inflammatory immune cells are identified by assaying cell surface protein expression.

[0089]

[0103] In various embodiments, the analysis of cells in one or more blood samples from a subject suffering from cancer is performed by assaying cell surface proteins, such as receptor tyrosine kinase (RTK), CD1c, CD2, CD3, CD4, CD5, CD8, CD9, CD10, CD11b, CD11c, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD24, TACI, CD25, CD27, CD28, CD30, CD30L, CD31, CD32, CD32b, CD34, CD33, CD38, CD39, CD40, CD40-L, CD41b, CD42a, CD42b, CD43, CD44, and CD45. , CD47, CD45RA, CD45RO, CD48, CD52, CD55, CD56, CD58, CD61, CD66b, CD70, CD72, CD79, CD68, CD84, CD86, CD93, CD94, CD95, CRACC, BLAME, BCMA, CD103 , CD107, CD112, CD120a, CD120b, CD123, CD125, CD134, CD135, CD140a, CD141, CD154, CD155, CD160, CD163, CD172a, XCR1, CD203c, CD204, CD206, CD207CD226, CD244, CD267, CD268, CD269, CD355, CD358, NKG2A, NKG2B, NKG2C, NK G2D、NKG2E、NKG2F、NKG2H、KIR2DL1、KIR2DL2、KIR2DL3、KIR2DL5A、KIR2DL5 B、KIR3DL1、KIR3DL2、KIR3DL3、KIR3DL4、KIR2DS1、KIR2DS2、KIR2DS3、KIR2 DS4, KIR2DS5, DAP12, KIR3DS, NKp44, NKp46, TCR, BCR, FcβεRI, MHC -I, MHC-II, IL-1R, IL-2Rα, IL-2Rβ, IL-2Rγ, IL-3Rα, CSF2RB, IL-4R, and IL-5R α, CSF2RB, IL-6Rα, gp130, IL-7Rα, IL-9R, IL-12Rβ1, IL-12Rβ2, IL-13Rα1. IL-13Rα2, IL-15Rα, IL-21R, IL23R, IL-27Rα, IL-31Rα, OSMR, and CSF-1R IL-15, IL-10Rα, IL-10Rβ, IL-20Rα, IL-20Rβ, IL-22Rα1, IL-22Rα2, IL-22R β, IL-28RA, PD-1, PD-1H, BTLA, CTLA-4, PD-L1, PD-L2, 2B4, B7-1, B7-2, B7- H1, B7-H4, B7-DC, DR3, LIGHT, LAIR, LTα1β2, LTβR, TIM-1, TIM-3, TIM-4, TI GIT, LAG-3, ICOS, ICOS-L, SLAM, SLAMF2, OX-40, OX-40L, GITR, GITRL, TL1A 41-BB 41BB-L TL-1A TRAF1 TRAF2 TRAF3 TRAF5 BAFF BAFF-R A PRIL, TRAIL, RANK, AITR, TRAMP, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7 CCR8, CCR9, CCR10, CCR11, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR 7. CLECL9a, DC-SIGN, IGSF4A, SIGLEC, EGFR, PDGFR, VEGFR, FAP, α-SMA, ビメンFAS, FAS-L, Fc, ICAM-1, ICAM-2, ICAM-3, ICAM-4, ICAM-5, PECAM- 1, MICA, MICB, UL16, ULBP1, ULBP2, ILBP3, ULBP4, ULBP5, ULBP6, MULT1, RAE1α, β, γ, δ, and ε, A1R, A 2A R.A. 2B In various embodiments, the integrin is selected from the group consisting of αR, αR, A3R, H60a, H60b, and H60c. In various embodiments, the integrin is selected from the group consisting of α1, α2, αIIb, α3, α4, α5, α6, α7, α8, α9, α10, α11, αD, αE, αL, αM, αV, αX, β1, β2, β3, β4, β5, β6, β7, β8, and / or combinations thereof. In various embodiments, the TCR is selected from the group consisting of α, β, γ, δ, ε, and ζ TCR. Several methods for assaying cell surface protein expression have been described in the literature, including flow cytometry and mass cytometry (CyTOF). The presence or abundance of one or more of these cell surface proteins indicates that the patient is suitable for treatment with the methods disclosed herein.

[0090]

[0104] In various embodiments, analysis of cells from one or more blood samples taken from subjects having one or more tumors characterized as being immunoevasive, immunologically protected, and / or immunologically "cold" demonstrates a high neutrophil-to-lymphocyte ratio (NLR). In various embodiments, analysis of cells from one or more blood samples taken from subjects having one or more tumors characterized as being immunoevasive, immunologically protected, and / or immunologically "cold" demonstrates an NLR of > 2. In various embodiments, analysis of cells from one or more blood samples taken from subjects having one or more tumors characterized as being immunoevasive, immunologically protected, and / or immunologically "cold" demonstrates an NLR of between 2 and 10 (e.g., an NLR of 2, 3, 4, 5, 6, 7, 8, 9, and 10, including all values ​​and ranges therebetween). In various embodiments, the NLR is used to determine the prognosis of a subject suffering from cancer and having one or more tumors characterized as immunoevasive, immunologically protected, and / or immunologically "cold." In various embodiments, an NLR of ≧2 determines a poor prognosis.

[0091]

[0105] In various embodiments, the cells analyzed from one or more blood samples taken from a subject suffering from cancer are circulating tumor cells (CTCs). In various embodiments, assay of one or more blood samples taken from a subject suffering from cancer demonstrates an increased frequency of CTCs compared to analysis of one or more blood samples taken from one or more healthy subjects and / or one or more subjects suffering from cancer and responding to treatment. In various embodiments, the frequency of circulating tumor cells in one or more blood samples from a subject having one or more tumors characterized as immunoevasive, immunologically protected, and / or immunologically "cold" is 3 or more CTCs per 7.5 mL of blood.

[0092]

[0106] In various embodiments, tumor characteristics of a subject suffering from cancer are determined from an analysis of proteins in one or more blood samples from the subject. In various embodiments, tumor characteristics of a subject suffering from cancer are determined by comparing an analysis of proteins in one or more blood samples from the subject suffering from cancer with an analysis of one or more blood samples from one or more healthy subjects and / or one or more subjects suffering from cancer and responding to treatment. In various embodiments, the protein is an intracellular protein or a secreted protein. In various embodiments, the protein is selected from the group consisting of cytokines, chemokines, growth factors, enzymes, proteases, and nucleases. In various embodiments, the cytokines and chemokines include IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL -13, IL-14, IL-15, IL-16, IL-17, IL-17, IL-18, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35, IL-36, CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CC L14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1, CXCL2(M CP-1), CXCL3(MIP-1α), CXCL4(MIP-1β), CXCL5(RANTES), CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, C XCL14, CXCL15, CXCL16, CXCL17, IFN-α, IFN-β, IFN-γ, グランザイム-B, パーフォリン, TNF-α, TGF-β1, TGF-β2, and びTGF-β3 are selected by the group. Erythropieitn ), TPO, BMP, HGF, GDF, neurotrophins, MSF, SGF, GDF, G-CSF, and GM-CSF. In various embodiments, the protein is a protease selected from the group consisting of aspartic acid proteases, cysteine ​​proteases, metalloproteases, serine proteases, and / or threonine proteases. In some embodiments, the protein is a protease selected from the group consisting of ADAM1, ADAM2, ADAM7, ADAM8, ADAM9, ADAM10, ADAM11, ADAM12, ADAM15, ADAM17, ADAM18, ADAM19, ADAAM20, ADAM21, ADAM22, ADAM23, ADAM28, ADAM29, ADAM30, ADAM33, MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP23A, MMP23B, MMP24, MMP25, MMP26, MMP27, and MMP28. In various embodiments, the protein is an enzyme selected from the group consisting of arginase, asparaginase, kynureninase, indoleamine 2,3 dioxygenase (IDO1 and IDO2), tryptophan 2,3 dioxygenase (TDO), and IL4I1. In various embodiments, the protein is associated with apoptosis. In various embodiments, the protein associated with apoptosis is selected from the group consisting of P53, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11, caspase 12, caspase 13, caspase 14, BCL-2, BCL-XL, MCL-1, CED-9, A1, BFL1, BAX, BAK, DIVA, BCL-XS, BIK, BIM, BAD, BID, and EGL-1. Several methods for assaying proteins from blood samples have been described in the literature, including Western blot and ELISA.

[0093]

[0107] In various embodiments, analysis of proteins from one or more blood samples of subjects having one or more tumors characterized as immunoevasive, immunologically protected, and / or immunologically "cold" demonstrates elevated levels of tumor-promoting, anti-inflammatory, and / or immunosuppressive proteins. In various embodiments, the tumor-promoting, anti-inflammatory, and / or immunosuppressive proteins are cell surface proteins, intracellular proteins, or secreted proteins. In various embodiments, the tumor-promoting, anti-inflammatory, and / or immunosuppressive protein is selected from the group consisting of CD39, CD79, MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP23A, MMP23B, MMP24, MMP25, MMP26, MMP27, and MMP28, CXCL12, GM-CSF, G-CSF, TGF-β1, TGF-β2, and TGF-β3, arginase, asparaginase, kyneurinase, indoleamine 2,3 dioxygenase (IDO1 and IDO), and / or IL-16. 2), tryptophan 2,3 dioxygenase (TDO), myeloperoxidase (MPO), neutrophil elastase (NE), and IL4I1. In various embodiments, the level of a tumor-promoting, anti-inflammatory, and / or immunosuppressive protein in one or more blood samples from a subject having one or more tumors characterized as being immunoevasive, immunologically protected, and / or immunologically "cold" is increased by 5-100% (e.g., 5-100%) compared to one or more blood samples taken from one or more healthy subjects and / or one or more subjects suffering from cancer and responding to treatment. For example, an increase of 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%, or about 100% (including all values ​​and ranges therebetween), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, 50%, or 100%. In various embodiments, the level of tumor-promoting, anti-inflammatory, and / or immunosuppressive proteins in one or more blood samples of subjects having one or more tumors characterized as being immunoevasive, immunologically protected, and / or immunologically "cold" is elevated by 2 to 100 fold (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold, including all values ​​and ranges therebetween) compared to one or more blood samples taken from one or more healthy subjects and / or one or more subjects afflicted with cancer and responsive to the treatment. In various embodiments, analysis of proteins from one or more blood samples of subjects having one or more tumors characterized as being immunoevasive, immunologically protected, and / or immunologically "cold" demonstrates reduced, low, and / or absent levels of tumor-inhibitory, anti-tumor, and / or pro-inflammatory proteins.In various embodiments, the tumor inhibitory, anti-tumor, and / or pro-inflammatory protein is IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9 , IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-17, IL-18, IL-20, IL-21, IL-22, IL-23, IL-24, I L-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35, IL-36, cell surface IL-15, CXCL2 (MCP-1), CXCL 3 (MIP-1α), CXCL4 (MIP-1β), CXCL5 (RANTES), IFN-α, IFN-β, IFN-γ, granzyme-B, perforin, and TNF-α. In various embodiments, the levels of tumor inhibitory, anti-tumor, and / or pro-inflammatory proteins in one or more blood samples from subjects having one or more tumors that are characterized as being immunoevasive, immunologically protected, and / or immunologically "cold" are increased by 5-100% (e.g., from healthy subjects or from subjects having cancer) compared to one or more blood samples taken from one or more healthy subjects and / or one or more subjects having cancer and responding to treatment. and is reduced by 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%, or about 100% (including all values ​​and ranges therebetween), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, 50%, or 100% compared to a subject who is resistant to treatment and who responds to treatment.In various embodiments, the level of tumor inhibitory, anti-tumor, and / or pro-inflammatory proteins in one or more blood samples from a subject having one or more tumors characterized as being immunoevasive, immunologically protected, and / or immunologically "cold" is increased by 2-100 fold (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold) compared to one or more blood samples taken from one or more healthy subjects, or subjects suffering from cancer and responding to treatment. (including all values ​​and ranges therebetween.) Several methods for assaying proteins from blood samples have been described in the literature, including Western blot and ELISA.

[0094]

[0108] In various embodiments, analysis of one or more blood samples from subjects having one or more tumors characterized as being immunoevasive, immunologically protected, and / or immunologically "cold" demonstrates elevated levels of neutrophil extracellular traps (NETs). In various embodiments, analysis of one or more blood samples from subjects having one or more tumors characterized as being immunoevasive, immunologically protected, and / or immunologically "cold" demonstrates elevated levels of neutrophil extracellular traps (NETs) compared to analysis of one or more blood samples from one or more healthy subjects and / or one or more subjects afflicted with cancer and responsive to treatment. In various embodiments, the level of NETs in one or more blood samples from subjects with one or more tumors characterized as being immunoevasive, immunologically protected, and / or immunologically "cold" is increased by 5-100% (e.g., about 5%, about 10%, about 20%) compared to one or more blood samples taken from one or more healthy subjects and / or one or more subjects with cancer and responding to treatment. The amount of the increase may be increased by 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%, or about 100% (including all values ​​and ranges therebetween), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, 50%, or 100%. In various embodiments, the level of NETs in one or more blood samples from a subject having one or more tumors characterized as being immunoevasive, immunologically protected, and / or immunologically "cold" is elevated by 2-100 fold (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold, including all values ​​and ranges therebetween) compared to one or more blood samples taken from one or more healthy subjects and / or one or more subjects afflicted with cancer and responsive to treatment.Several methods for assaying NETs from blood samples have been described in the literature, including Western blot, ELISA, and flow cytometry.

[0095]

[0109] In various embodiments, tumor characteristics of a subject suffering from cancer are determined from analysis of nucleic acids in one or more blood samples from the subject. In various embodiments, tumor characteristics of a subject suffering from cancer are determined by comparing analysis of nucleic acids in one or more blood samples from the subject suffering from cancer with analysis of one or more blood samples from one or more healthy subjects and / or one or more subjects suffering from cancer and responding to treatment. In various embodiments, the nucleic acid is selected from the group including DNA, ssDNA, circulating tumor DNA (ctDNA), RNA, mRNA, dsRNA, siRNA, miRNA, and lncRNA. In various embodiments, analysis of ctDNA from one or more blood samples from a subject suffering from cancer with one or more tumors characterized as immune-evasive, immunologically protected, or immunologically "cold" demonstrates low levels and / or absence of one or more tumor mutations, tumor antigens, and / or neoantigens. In various embodiments, analysis of ctDNA from one or more blood samples from a subject suffering from cancer demonstrates low or no tumor mutational burden. In various embodiments, analysis of ctDNA from one or more blood samples of subjects with one or more tumors characterized as immune-evasive, immunologically protected, or immunologically "cold" reveals a somatic mutation count between 5 and 0.001 (e.g., about 5, about 4, about 3, about 2, about 1, about 0.9, about 0.8, about 0.7, about 0.6, about 0.5, about 0.4, about 0.3, about 0.2, about 0.1, about 0.09, about 0.08, about 0.07, about 0.08, about 0.09, about 0.10, about 0.11, about 0.12, about 0.13, about 0.14, about 0.15, about 0.16, about 0.17, about 0.18, about 0.19, about 0.20, about 0.21, about 0.22, about 0.23, about 0.24, about 0.25, about 0.26, about 0.27, about 0.28, about 0.29, about 0.30, about 0.31, about 0.32, about 0.33, about 0.34, about 0.35, about 0.36, about 0.37, about 0.38, about 0.39, about 0.40, about 0.41, about 0.42, about 0.43, about 0.44, about 0.45, about 0.46, about 0.47, about 0.48, about 0.49 ... In various embodiments, the nucleic acid analysis is performed by PCR, RT-PCR, qRT-PCR, next generation sequencing (NGS), RNA-seq, ATAC-seq, exome sequencing, Southern blot, microarray analysis, and / or single cell sequencing.

[0096]

[0110] In various embodiments, the characteristics of a subject's tumor are determined from gene expression analysis from nucleic acids in one or more blood samples from subjects suffering from cancer. In various embodiments, gene expression analysis from nucleic acids in one or more blood samples from subjects having one or more tumors characterized as being immunoevasive, immunologically protected, or immunologically "cold" demonstrates increased expression of tumor-promoting, tumor-permissive, and / or immunosuppressive genes compared to analysis of one or more blood samples from one or more healthy subjects and / or one or more subjects suffering from cancer and responding to treatment. In various embodiments, the expression of tumor-promoting, tumor-permissive, and / or immunosuppressive genes in one or more blood samples from subjects having one or more tumors characterized as being immunoevasive, immunologically protected, or immunologically "cold" is increased by 5-100% (e.g., 100-150%) compared to one or more blood samples taken from one or more healthy subjects and / or one or more subjects suffering from cancer and responding to treatment. , 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%, or about 100% (including all values ​​and ranges therebetween), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50% increased. In various embodiments, expression of tumor-promoting, tumor-permissive, and / or immunosuppressive genes in one or more blood samples from subjects having one or more tumors characterized as being immunoevasive, immunologically protected, or immunologically "cold" is increased by 2 to 100 fold (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold, including all values ​​and ranges therebetween) compared to one or more blood samples taken from one or more healthy subjects and / or one or more subjects afflicted with cancer and responsive to treatment.In various embodiments, analysis of nucleic acids in one or more blood samples from subjects having one or more tumors characterized as being immunoevasive, immunologically protected, or immunologically "cold" demonstrates reduced expression of tumor-inhibitory, anti-tumor, and / or pro-inflammatory genes compared to analysis of one or more blood samples from one or more healthy subjects and / or one or more subjects suffering from cancer and responding to treatment. In various embodiments, analysis of nucleic acids in one or more blood samples from subjects having one or more tumors characterized as being immunoevasive, immunologically protected, or immunologically "cold" demonstrates reduced or no expression of tumor-inhibitory, anti-tumor, and / or anti-inflammatory genes compared to analysis of one or more blood samples from one or more healthy subjects and / or one or more subjects suffering from cancer and responding to treatment. In various embodiments, expression of tumor inhibitory, anti-tumor, and / or pro-inflammatory genes in one or more blood samples of a subject having one or more tumors characterized as being immunoevasive, immunologically protected, or immunologically "cold" is reduced by 5-100% (e.g., 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%, or about 100% (including all values ​​and ranges therebetween)), 10-95%, 15-90%, 20-85%, 25-75%, 30-75%, 40-85%, 45-95%, 50-95%, 55-90%, 60-85%, 65-75%, 70-85%, 75-95%, 80-85%, 85-95%, 90 ... In various embodiments, the expression of tumor inhibitory, anti-tumor, and / or pro-inflammatory genes in one or more blood samples of a subject having one or more tumors characterized as being immunoevasive, immunologically protected, or immunologically "cold" is reduced by 2-100 fold (e.g., reduced by about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold (including all values ​​and ranges therebetween) relative to a healthy subject, or a subject having cancer and responding to treatment). In various embodiments, gene expression analysis is performed by PCR, RT-PCR, qRT-PCR, next generation sequencing (NGS), RNA-seq, ATAC-seq, exome sequencing, Southern blot, microarray analysis, and / or single cell sequencing.

[0097]

[0111] In various embodiments, tumor characteristics of a subject suffering from cancer are determined from analysis of one or more tumor samples taken from the subject. In various embodiments, the tumor samples are biopsies. In various embodiments, tumor characteristics of a subject suffering from cancer are determined from analysis of cells, proteins, and / or nucleic acids in one or more tumor samples taken from the subject. In various embodiments, tumor characteristics of a subject suffering from cancer are determined by comparing analysis of cells, proteins, and / or nucleic acids in one or more tumor samples from a subject suffering from cancer with analysis of tissue samples from one or more healthy subjects and / or one or more subjects suffering from cancer and responding to treatment. In various embodiments, the cells analyzed in the one or more tumor samples are leukocytes, epithelial cells, mesenchymal cells, mesenchymal stem cells, stromal cells, endothelial cells, fibroblasts, pericytes, adipocytes, and cancer stem cells. In various embodiments, the leukocytes are myeloid cells and lymphoid cells. In various embodiments, the myeloid cells are monocytes, macrophages, neutrophils, granulocytes, dendritic cells, mast cells, eosinophils, and basophils. In various embodiments, the lymphoid cells are T cells, B cells, NK cells, NK-T cells, or iNK cells.

[0098]

[0112] In various embodiments, analysis of cells from one or more tumor samples taken from a subject having one or more tumors characterized as being immunoevasive, immunologically protected, or immunologically "cold" demonstrates the presence of immunosuppressive cells. In various embodiments, analysis of one or more tumor samples taken from a subject having one or more tumors characterized as being immunoevasive, immunologically protected, or immunologically "cold" demonstrates the presence of immunosuppressive cells in the tumor core. In various embodiments, analysis of cells from one or more tumor samples taken from a subject having one or more tumors characterized as being immunoevasive, immunologically protected, or immunologically "cold" demonstrates elevated levels of immunosuppressive cells. In various embodiments, analysis of one or more tumor samples demonstrates elevated levels of immunosuppressive cells in the tumor core. In various embodiments, immunosuppressive cells include myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), neutrophils, T reg cells, and B reg In various embodiments, the MDSCs are monocytic MDSCs (M-MDSCs) and polymorphonuclear MDSCs (PMN-MDSCs). In various embodiments, the TAMs are M2TAMs. In various embodiments, the immunosuppressive cells are CAFs. In various embodiments, the level of immunosuppressive cells in one or more tumor samples from a subject having one or more tumors characterized as being immunoevasive, immunologically protected, or immunologically "cold" is increased by 5-100% (e.g., 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%, or about 100% (including all values ​​and ranges therebetween) compared to tissue samples from one or more healthy subjects and / or one or more subjects afflicted with cancer and responsive to treatment). The range of increase is 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%. In various embodiments, the level of immunosuppressive cells in one or more tumor samples from a subject having one or more tumors characterized as being immunoevasive, immunologically protected, or immunologically "cold" is elevated by 2 to 100 fold compared to one or more tissue samples from one or more healthy subjects, or one or more subjects suffering from cancer and responding to treatment (e.g., elevated by about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold (including all values ​​and ranges therebetween) compared to healthy subjects and / or subjects suffering from cancer and responding to treatment).

[0099]

[0113] In various embodiments, analysis of cells from one or more tumor samples taken from subjects having one or more tumors characterized as being immunoevasive, immunologically protected, or immunologically "cold" demonstrates an absence of leukocytes. In various embodiments, analysis of cells from one or more tumor samples taken from subjects having one or more tumors characterized as being immunoevasive, immunologically protected, or immunologically "cold" demonstrates reduced or low levels of leukocytes. In various embodiments, the frequency of leukocytes is 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, or 5% or less of all cells analyzed (including all values ​​and ranges therebetween).

[0100]

[0114] In various embodiments, analysis of cells from one or more tumor samples taken from subjects having one or more tumors characterized as being immunoevasive, immunologically protected, or immunologically "cold" demonstrates an absence of activated pro-inflammatory immune cells. In various embodiments, analysis of cells from one or more tumor samples taken from subjects having one or more tumors characterized as being immunoevasive, immunologically protected, or immunologically "cold" demonstrates an absence of activated pro-inflammatory immune cells from the tumor core. In various embodiments, analysis of cells from one or more tumor samples taken from subjects having one or more tumors characterized as being immunoevasive, immunologically protected, or immunologically "cold" demonstrates low or reduced levels of activated pro-inflammatory immune cells. In various embodiments, analysis of cells from one or more tumor samples taken from subjects having one or more tumors characterized as being immunoevasive, immunologically protected, or immunologically "cold" demonstrates low or reduced levels of activated pro-inflammatory immune cells in the tumor core. In various embodiments, the activated pro-inflammatory cells are dendritic cells (DCs), macrophages, M1 macrophages, T cells, B cells, NK cells, NK-T cells, and iNK cells. In various embodiments, the frequency of pro-inflammatory immune cells is 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, or 5% or less of all cells analyzed (including all values ​​and ranges therebetween).

[0101]

[0115] In various embodiments, characteristics of a tumor of a subject suffering from cancer are determined from an analysis of the location of immune cells in one or more tumor samples from the subject. In various embodiments, immune cells in one or more tumor samples from one or more immunoevasive, immunologically protected, and / or immunologically "cold" subjects are located at the tumor penumbra. In various embodiments, immune cells in one or more tumor samples from one or more immunoevasive, immunologically protected, and / or immunologically "cold" subjects are absent from the tumor core. In various embodiments, immune cells in one or more tumor samples from one or more immunoevasive, immunologically protected, and / or immunologically "cold" subjects are reduced in the tumor core. In various embodiments, immune cells in the tumor core are localized to one or more healthy subjects and / or is reduced by 5-100% (e.g., 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%, or about 100%, including all values ​​and ranges therebetween), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%, compared to one or more samples from one or more subjects having cancer and responding to the treatment.

[0102]

[0116] In various embodiments, tumor characteristics of a subject suffering from cancer are determined from an analysis of the location of stromal cells in one or more tumor samples from the subject. In various embodiments, the stromal cells are CAFs, pericytes, adipocytes, and endothelial cells. In various embodiments, CAFs in one or more tumor samples from a subject having one or more tumors that are immunoevasive, immunologically protected, and / or immunologically "cold" are enriched in the tumor margin. In various embodiments, CAFs in one or more tumor samples from a subject having one or more tumors that are immunoevasive, immunologically protected, and / or immunologically "cold" are enriched in the tumor core. In various embodiments, the frequency of CAFs at the tumor margin is increased by 5-100% (e.g., 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%, or about 100% (including all values ​​and ranges therebetween)), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, 50%, or 100% compared to one or more tissue samples from one or more healthy subjects and / or subjects afflicted with cancer and responsive to treatment. In various embodiments, the frequency of CAFs at the tumor margin is increased by 2 to 100 fold (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 fold (including all values ​​and ranges therebetween)) compared to one or more tissue samples from one or more healthy subjects and / or one or more subjects afflicted with cancer and responding to treatment.In various embodiments, the frequency of CAFs in the tumor core is increased by 5-100% (e.g., 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%, or about 100% (including all values ​​and ranges therebetween)), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, 50%, or 100% compared to one or more tissue samples from one or more healthy subjects and / or one or more subjects afflicted with cancer and responsive to treatment. In various embodiments, the frequency of CAFs in the tumor core is increased by 2 to 100 times compared to one or more healthy tissue samples (e.g., increased by about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 times (including all values ​​and ranges therebetween) compared to healthy subjects and / or subjects with cancer and who respond to treatment).

[0103]

[0117] In various embodiments, the analysis of cells in one or more tumor samples from a subject suffering from cancer is performed by assaying cell surface proteins, such as receptor tyrosine kinase (RTK), CD1c, CD2, CD3, CD4, CD5, CD8, CD9, CD10, CD11b, CD11c, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD24, TACI, CD25, CD27, CD28, CD30, CD30L, CD31, CD32, CD32b, CD34, CD33, CD38, CD39, CD40, CD40-L, CD41b, CD42a, CD42b, CD43, CD44, and CD45. , CD47, CD45RA, CD45RO, CD48, CD52, CD55, CD56, CD58, CD61, CD66b, CD70, CD72, CD79, CD68, CD84, CD86, CD93, CD94, CD95, CRACC, BLAME, BCMA, CD103, CD107, CD112, CD120a, CD120b, CD123, CD125, CD134, CD135, CD140a, CD141, CD154, CD155, CD160, CD163, CD172a, XCR1, CD203c, CD204, CD206, CD207 CD226, CD244, CD267, CD268, CD269, CD355, CD358, NKG2A, NKG2B, NKG2C, NKG2D, NKG2E, NKG2F, NKG2H, KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, KIR3DL4, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, DAP12, KIR3DS, NKp44, NKp46, TCR, BCR, Integrin, FcβεRI, MHC-I, MHC-II, IL-1R, IL-2Rα, IL-2Rβ, IL-2Rγ, IL-3Rα, CSF2RB, IL-4R, IL-5Rα, CSF2RB, IL-6Rα, gp130, IL-7Rα, IL-9R, IL-12Rβ1, IL-12Rβ2, IL-13Rα1, IL-13Rα2, IL-15Rα, IL-21R, IL23R, IL-27Rα, IL-31Rα, OSMR, CSF-1R, Cell surface IL-15, IL-10Rα, IL-10Rβ, IL-20Rα, IL-20Rβ, IL-22Rα1, IL-22Rα2, IL-22Rβ, IL-28RA, PD-1, PD-1H, BTLA, CTLA-4, PD-L1, PD-L2, 2B4, B7-1, B7-2, B7-H1, B7-H4, B7-DC, DR3, LIGHT, LAIR, LTα1β2, LTβR, TIM-1, TIM-3, TIM-4, TIGIT, LAG-3, ICOS, ICOS-L, SLAM, SLAMF2, OX-40, OX-40L, GITR, GITRL, TL1A, HVEM, 41-BB, 41BB-L, TL-1A, TRAF1, TRAF2, TRAF3, TRAF5, BAFF, BAFF-R, APRIL,TRAIL, RANK, AITR, TRAMP, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, CX CR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CLECL9a, DC-SIGN, IGSF4A, SIGLEC, EGFR, PDGFR, VEGFR, FAP, α-SMA, vimentin, laminin, FAS, FAS-L, Fc, ICAM-1, ICAM-2, ICAM-3, ICAM-4, ICAM-5, PECAM- 1, MICA, MICB, UL16, ULBP1, ULBP2, ILBP3, ULBP4, ULBP5, ULBP6, MULT1, RAE1α, β, γ, δ, and ε, A1R, A, 2A R.A. 2B In various embodiments, the integrin is selected from the group consisting of αR, αR, A3R, H60a, H60b, and H60c. In various embodiments, the integrin is selected from the group consisting of α1, α2, αIIb, α3, α4, α5, α6, α7, α8, α9, α10, α11, αD, αE, αL, αM, αV, αX, β1, β2, β3, β4, β5, β6, β7, β8, and / or combinations thereof. In various embodiments, the TCR is selected from the group consisting of α, β, γ, δ, ε, and ζ TCR. Several methods for assaying cell surface protein expression from tumor samples have been described in the literature, including immunohistochemistry, immunofluorescence, Western blot, flow cytometry, and mass cytometry (CyTOF).

[0104]

[0118] The tumor core is generally defined as the densely packed, central, bulk-forming, and differentiated tumor region. In contrast, the tumor margin is generally defined as the invasive edge of the tumor that interacts with the surrounding stroma and parenchyma [35, 36].

[0105]

[0119] In various embodiments, the characteristics of a tumor in a subject suffering from cancer are determined from an analysis of proteins in one or more tumor samples from the subject. The level of a cancer-related protein is determined by comparing an analysis of the protein in the sample with an analysis of one or more tissues from one or more healthy subjects and / or one or more subjects suffering from cancer and responding to treatment. In various embodiments, the protein is intracellular or extracellular. In various embodiments, the protein is selected from the group consisting of cytokines, chemokines, growth factors, enzymes, proteases, and nucleases. In various embodiments, the cytokines and chemokines include IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, I L-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-17, IL-18, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL- 35, IL-36, CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL1 4, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1, CXCL2 (MCP-1), CXCL3 (MIP-1α), CXCL4 (MIP-1β), CXCL5 (RANTES), CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, IFN-α, IFN-β, IFN-γ, granzyme-B, perforin, TNF-α, TGF-β1, TGF-β2, and TGF-β3. In various embodiments, the growth factor is selected from the group consisting of EGF, FGF, NGF, PDGF, VEGF, IGF, GMCSF, GCSF, TGF, erythropoietin, TPO, BMP, HGF, In various embodiments, the protein is selected from the group consisting of aspartic acid proteases, cysteine ​​proteases, metalloproteases, serine proteases, and / or threonine proteases. In some embodiments, the protein is a protease selected from the group consisting of ADAM1, ADAM2, ADAM7, ADAM8, ADAM9, ADAM10, ADAM11, ADAM12, ADAM15, ADAM17, ADAM18, ADAM19, ADAAM20, ADAM21, ADAM22, ADAM23, ADAM28, ADAM29, ADAM30, ADAM33, MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP23A, MMP23B, MMP24, MMP25, MMP26, MMP27, and MMP28. In various embodiments, the protein is an enzyme selected from the group consisting of arginase, asparaginase, kynureninase, indoleamine 2,3 dioxygenase (IDO1 and IDO2), tryptophan 2,3 dioxygenase (TDO), and IL4I1. In various embodiments, the protein is associated with apoptosis. In various embodiments, the protein associated with apoptosis is selected from the group consisting of P53, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11, caspase 12, caspase 13, caspase 14, BCL-2, BCL-XL, MCL-1, CED-9, A1, BFL1, BAX, BAK, DIVA, BCL-XS, BIK, BIM, BAD, BID, and EGL-1. Several methods for assaying proteins from tumor samples have been described in the literature, including immunohistochemistry, immunofluorescence, Western blot, and ELISA.

[0106]

[0120] In various embodiments, analysis of proteins from one or more tumor samples from a subject having one or more tumors characterized as being immunoevasive, immunologically protected, and / or immunologically "cold" can provide insight into tumor progression, anti-inflammatory activity, and In various embodiments, the proteins associated with tumor progression, anti-inflammatory activity, and / or immunosuppression are cell surface proteins, intracellular proteins, or secreted proteins. In various embodiments, the proteins associated with tumor progression, anti-inflammatory activity, and / or immunosuppression are CD39, CD47, CD79, CD140a, CD163, CD206, FOXP3, FAP, PD-1, PD-L1, PD-L2, CSF-1R, A1R, A2R, A3R, A4R, A5R, A6R, A7R, A8R, A9R, A1 ... 2A R.A. 2B R and A3R, TIM-1, TIM-3, TIM-4, TIGIT, CSFR, SIGLEC, MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP23A, MMP23B, MMP24, MMP25, MMP26, MMP27, and MMP28, CXCL12, GM-CSF, G-CSF, FAP, TGF-β1, TGF-β2, and TGF-β3, arginase, asparaginase, kyneurinase, indoleamine 2,3 dioxygenase (IDO1 and IDO2), tryptophan 2,3 dioxygenase (TDO), myeloperoxidase (MPO), neutrophil elastase (NE), and IL4I1. In various embodiments, the levels of proteins associated with tumor progression, anti-inflammatory activity, and / or immunosuppression in one or more tumor samples from a subject having one or more tumors characterized as being immunoevasive, immunologically protected, and / or immunologically "cold" are increased by 5-100% (e.g., 5-100%) compared to one or more tissue samples from one or more healthy subjects, and / or one or more subjects afflicted with cancer and responding to treatment. For example, an increase of 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%, or about 100% (including all values ​​and ranges therebetween), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, 50%, or 100%. In various embodiments, the level of a protein associated with tumor progression, anti-inflammatory activity, and / or immunosuppression in one or more tumor samples from a subject having one or more tumors characterized as being immunoevasive, immunologically protected, and / or immunologically "cold" is elevated 2-100 fold compared to one or more tissue samples from one or more healthy subjects and / or subjects with cancer and responding to treatment (e.g., elevated by about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 fold (including all values ​​and ranges therebetween) compared to healthy subjects and / or subjects with cancer and responding to treatment).

[0107]

[0121] In various embodiments, analysis of proteins from one or more tumor samples from subjects having one or more tumors characterized as being immunoevasive, immunologically protected, and / or immunologically "cold" demonstrates reduced levels, low levels, and / or absence of proteins associated with tumor growth inhibition, anti-tumor activity, and / or pro-inflammatory activity. In various embodiments, proteins associated with tumor growth inhibition, anti-tumor activity, and / or pro-inflammatory activity include CD44, CD56, CD103c, CD69, KG2A, NKG2B, NKG2C, NKG2D, NKG2E, NKG2F, NKG2H, ICOS, ICOS-L, SLAM, SLAMF2, OX-40, OX-40L, GITR, GITRL, TL1A, HVEM, 41-BB, 41BB-L, TL-1A, TRAF1, TRAF2, TRAF3, TRAF5, BAF F, BAFF-R, APRIL, TRAIL, RANK, AITR, TRAMP, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, I L-13, IL-14, IL-15, IL-16, IL-17, IL-17, IL-18, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, I In various embodiments, the level of a protein associated with tumor growth inhibition, anti-tumor activity, and / or pro-inflammatory activity is increased by 5-100% (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 210%, about 220%, about 230%, about 240%, about 250%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 460%, about 470%, about 480%, about 490%, about 500%, about 510%, about 520%, about 530%, about 540%, about 550%, about 560%, about 570%, about 580%, about 590%, about 600%, about 610%, about 620%, about 630%, about 640%, about 650%, about 660%, about 670%, about 680%, about 690%, about 700%, about 710%, about 720%, about 730%, about 740%, about 750%, about 760%, about 770%, about 780%, about 790%, about 800%, about 810%, about 820%, about 830%, The protein may be reduced by 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%, or about 100% (including all values ​​and ranges between these values), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, 50%, or 100%. Several methods for assaying proteins from tumor samples have been described, including immunohistochemistry, immunofluorescence, Western blot, intracellular flow cytometry, and ELISA.

[0108]

[0122] In various embodiments, tumor characteristics of a subject suffering from cancer are determined from a Tumor Proportion Score (TPS) for PD-L1 expression in one or more tumor samples from the subject. In various embodiments, a subject having one or more tumors characterized as being immunoevasive, immunologically protected, and / or immunologically "cold" has a TPS of between 1 and 50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, including all ranges between these values). In various embodiments, subjects with one or more tumors characterized as immunoevasive, immunologically protected, and / or immunologically "cold" have a TPS of less than or equal to 1. The TPS for PD-L1 expression is defined as the proportion of viable tumor cells that demonstrate partial or complete membrane staining by immunohistochemical analysis.

[0109]

[0123] In various embodiments, the tumor characteristics of a subject suffering from cancer are determined by a Combined Positivity Score (CPS) for PD-L1 expression in one or more tumor samples from the subject. In various embodiments, a subject with one or more tumors characterized as immunoevasive, immunologically protected, and / or immunologically "cold" has a CPS of 10 or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, including all ranges between these values). In various embodiments, the CPS is 1 or less. The CPS of PD-L1 expression is determined from immunohistochemical determination of the number of viable tumor cells, lymphocytes, and macrophages that are positive for PD-L1 as a percentage of all viable tumor cells.

[0110]

[0124] In various embodiments, a tumor characteristic of a subject suffering from cancer is determined from microsatellite instability testing of one or more tumor samples from the subject. In various embodiments, a tumor characteristic of a subject suffering from cancer is determined by comparing microsatellite instability testing of one or more tumor samples with microsatellite stability testing from one or more healthy tissues of the subject. In various embodiments, the microsatellite instability testing is an assay of a microsatellite marker. In various embodiments, the microsatellite instability testing is an assay of a mismatch repair marker. In various embodiments, the microsatellite marker is selected from the group consisting of BAT25, BAT26, D2S123, D5S346, and D17S250. In various embodiments, the mismatch repair marker is selected from the group consisting of MLH1, MSH2, MLH6, and PMS2. In various embodiments, the subject has one or more tumors that are immunoevasive, immunologically protected, and / or immunologically "cold," which are determined to be microsatellite-low instability. In various embodiments, the subject has one or more tumors that are immunoevasive, immunologically protected, and / or immunologically "cold," which are determined to be microsatellite-stable. In various embodiments, the subject has one or more tumors that are immunoevasive, immunologically protected, and / or immunologically "cold," which are determined to be mismatch repair competent.

[0111]

[0125] In various embodiments, analysis of one or more tumor samples from subjects having one or more tumors characterized as being immunoevasive, immunologically protected, and / or immunologically "cold" demonstrates elevated levels of neutrophil extracellular traps (NETs). In various embodiments, analysis of one or more tumor samples from subjects having one or more tumors characterized as being immunoevasive, immunologically protected, and / or immunologically "cold" demonstrates elevated levels of neutrophil extracellular traps (NETs) compared to analysis of one or more tumor samples from one or more healthy subjects. In various embodiments, the level of NETs in one or more tumor samples from a subject suffering from cancer is increased by 5-100% (e.g., 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%, or about 100% (including all values ​​and ranges therebetween)), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, 50%, or 100% compared to one or more tissue samples from one or more healthy subjects and / or subjects suffering from cancer and responding to treatment. In various embodiments, the level of NETs in one or more tumor samples from subjects suffering from cancer is elevated 2-100 fold compared to one or more tissue samples from one or more healthy subjects and / or subjects suffering from cancer and responding to treatment (e.g., elevated by about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 fold (including all values ​​and ranges therebetween) compared to healthy subjects and / or subjects suffering from cancer and responding to treatment). Several methods for assaying NETs have been described in the literature, including Western blot, ELISA, and flow cytometry.

[0112]

[0126] In various embodiments, tumor characteristics of a subject suffering from cancer are determined from analysis of nucleic acids in one or more tumor samples from the subject. In various embodiments, tumor characteristics of a subject suffering from cancer are determined by comparing analysis of nucleic acids in one or more tumor samples from the subject suffering from cancer with analysis of one or more tissue samples from one or more healthy subjects and / or subjects suffering from cancer and responding to treatment. In various embodiments, the nucleic acid is selected from the group including DNA, ssDNA, RNA, mRNA, dsRNA, siRNA, miRNA, and lncRNA. In various embodiments, nucleic acid analysis is performed by PCR, RT-PCR, qRT-PCR, next-generation sequencing (NGS), RNA-seq, ATAC-seq, exome sequencing, Southern blot, microarray analysis, and / or single-cell sequencing.

[0113]

[0127] In various embodiments, analysis of nucleic acids from one or more tumor samples from subjects suffering from cancer is used to determine tumor mutational burden. In various embodiments, analysis of nucleic acids from one or more tumor samples from subjects having one or more tumors characterized as being immunoevasive, immunologically protected, and / or immunologically "cold" demonstrates low tumor mutational burden. Analysis of nucleic acids from one or more tumor samples from subjects having one or more tumors characterized as immune-evasive, immunologically protected, and / or immunologically "cold" reveals between 5 and 0.001 somatic mutations per megabase pair (e.g., about 5, about 4, about 3, about 2, about 1, about 0.9, about 0.8, about 0.7, about 0.6, about 0.5, about 0.4, about 0.6, about 0.8, about 0.9 ... In various embodiments, the nucleic acid analysis is performed by PCR, RT-PCR, qRT-PCR, next generation sequencing (NGS), RNA-seq, ATAC-seq, exome sequencing, Southern blot, microarray analysis, and / or single cell sequencing.

[0114]

[0128] In various embodiments, analysis of nucleic acids in one or more tumor samples from subjects having one or more tumors characterized as being immunoevasive, immunologically protected, and / or immunologically "cold" demonstrates increased expression of genes associated with tumor-promoting, tumor-permissive, anti-inflammatory, and / or immunosuppressive activity compared to analysis of one or more tissue samples from one or more healthy subjects and / or subjects afflicted with cancer and responsive to treatment. In various embodiments, genes associated with tumor-promoting, tumor-permissive, anti-inflammatory, and / or immunosuppressive activity include CD39, CD47, CD79, CD140a, CD163, CD206, FOXP3, FAP, PD-1, PD-L1, PD-L2, CSF-1R, A1R, A2R, A3R, A4R, A5R, A6R, A7R, A8R, A9R, A1 ... 2A R.A. 2BR and A3R, TIM-1, TIM-3, TIM-4, TIGIT, CSFR, SIGLEC, MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP23A, MMP23B, MMP24, MMP25, MMP26, MMP27, and MMP28, CXCL12, GM-CSF, G-CSF, FAP, TGF-β1, TGF-β2, and TGF-β3, arginase, asparaginase, kyneurinase, indoleamine 2,3 dioxygenase (IDO1 and IDO2), DO2), tryptophan 2,3 dioxygenase (TDO), myeloperoxidase (MPO), neutrophil elastase (NE), and IL4I1. In various embodiments, expression of genes associated with tumor-promoting, tumor-permissive, anti-inflammatory, and / or immunosuppressive activity is increased by 5-100% (e.g., increased by 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%, or about 100% (including all values ​​and ranges therebetween)), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, 50%, or 100% compared to one or more tissue samples from one or more healthy subjects and / or subjects afflicted with cancer and responsive to treatment. In various embodiments, gene expression analysis is performed by PCR, RT-PCR, qRT-PCR, next generation sequencing (NGS), RNA-seq, ATAC-seq, exome sequencing, Southern blot, microarray analysis, and / or single cell sequencing.

[0115]

[0129] In various embodiments, analysis of nucleic acids in one or more tumor samples from subjects having one or more tumors characterized as being immunoevasive, immunologically protected, and / or immunologically "cold" demonstrates low or reduced expression of genes associated with tumor inhibitory, anti-tumor, and / or pro-inflammatory activity. In various embodiments, analysis of nucleic acids in one or more tumor samples from subjects suffering from cancer demonstrates absence of expression of genes associated with tumor inhibitory, anti-tumor, and / or pro-inflammatory activity. In various embodiments, genes associated with tumor inhibitory, anti-tumor, and / or pro-inflammatory activity include CD44, CD56, CD103c, CD69, KG2A, NKG2B, NKG2C, NKG2D, NKG2E, NKG2F, NKG2H, ICOS, ICOS-L, SLAM, SLAMF2, OX-40, OX-40L, GITR, GITRL, TL1A, HVEM, 41-BB, 41BB-L, TL-1A, TRAF1, TRAF2, TRAF3, TRAF5, BAFF, BAFF-R, APRIL, TRAIL, RANK, AITR, TRAMP, cell surface IL-15, IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-1 7, IL-18, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-3 0, IL-31, IL-32, IL-33, IL-35, IL-36, CXCL2 (MCP-1), CXCL3 (MIP-1α), CXCL4 (MIP-1β), CXCL5 (RANTES), IFN-α, IFN-β, IFN-γ, granzyme-B, perforin, TNF-α, and p53. In various embodiments, expressed genes associated with tumor inhibitory, anti-tumor, and / or pro-inflammatory activity are reduced by 5-100% (e.g., reduced by 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%, or about 100% (including all values ​​and ranges therebetween)), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, 50% compared to a healthy subject or a subject suffering from cancer that responds to the treatment. In various embodiments, gene expression analysis is performed by PCR, RT-PCR, qRT-PCR, next generation sequencing (NGS), RNA-seq, ATAC-seq, exome sequencing, Southern blot, microarray analysis, and / or single cell sequencing.

[0116]

[0130] In various embodiments, the present disclosure provides a method of treating cancer in a subject, comprising administering to the subject surface-functionalized particles, alone or in combination with a cancer therapeutic, the subject having one or more tumors that are resistant and / or non-responsive to the treatment. In various embodiments, the subject has one or more tumors that are resistant and / or non-responsive to one or more treatments selected from the group consisting of surgery, radiation, chemotherapy, biologic agents, small molecules, cell-based therapies, hormone therapy, and immunotherapy. In various embodiments, the treatment is standard therapy, first-line therapy, second-line therapy, and / or third-line therapy. In various embodiments, the subject has one or more tumors that have progressed during one or more treatments, the treatment being standard therapy, first-line therapy, second-line therapy, and / or third-line therapy.

[0117]

[0131] A primary treatment is defined as a treatment administered to a subject with cancer who has not received any prior treatment. A secondary treatment is defined as a treatment administered to a subject with cancer who has received a prior primary treatment but has experienced disease progression during the primary treatment. A tertiary treatment is defined as a treatment administered to a subject with cancer who has received prior primary and secondary treatments but has experienced disease progression during the secondary treatment. Definitions of primary, secondary, and tertiary treatments can be found in the National Cancer Institute's (NCI) Dictionary of Cancer Terms (https: / / www.cancer.gov / publications / dictionaries). Certain types of cancer may be classified as having a primary, secondary, or tertiary treatment. These include first-line, second-line, and third-line treatments, respectively. First-line, second-line, and third-line treatments for specific forms of cancer or tumor types are known in the art. In addition, FDA-approved drug labels may indicate whether a particular drug is approved as a first-line, second-line, or third-line treatment.

[0118]

[0132] Several criteria and definitions published in the literature have been used to assess the risk of cancer in subjects with cancer. Based on these criteria, tumors are defined as "responsive," "stable," or "progressive" if they improve, remain stable, or worsen during the course of treatment. Examples of the most commonly used criteria published in the literature include the Response Evaluation Criteria in Solid Tumors (RECIST), Modified Response Evaluation Criteria in Solid Tumors (mRECIST), PET Response Criteria in Solid Tumors (PERCIST), Choi Criteria, Lugano Response Criteria, European Association for the Study of the Liver (EASL) Criteria, and Response Evaluation Criteria in the Cancer of the Liver (RECICL), and WHO Criteria in Tumor Response. 30-32].

[0119]

[0133] In various embodiments, the present disclosure provides methods of treating cancer in a subject comprising administering surface-functionalized particles, alone or in combination with a cancer therapeutic agent, to a subject, where the subject is unable to tolerate standard, first-line, second-line, and / or third-line therapy. In various embodiments, the present disclosure provides methods of treating cancer in a subject comprising administering surface-functionalized particles, alone or in combination with a cancer therapeutic agent, to a subject, where the subject has experienced tumor recurrence after surgical resection of the primary tumor. In various embodiments, the present disclosure provides methods of treating cancer in a subject comprising administering surface-functionalized particles, alone or in combination with a cancer therapeutic agent, to a subject, where the subject has a tumor that cannot be surgically removed. In various embodiments, the present disclosure provides methods of treating cancer in a subject comprising administering surface-functionalized particles, alone or in combination with a cancer therapeutic agent, to a subject, where the subject has no available treatment options.

[0120]

[0134] Some treatments used in cancer treatment (e.g., chemotherapy) are cytotoxic and associated with significant side effects and toxicity, which are associated with poor outcomes and poor response to treatment. Before administering such treatment, clinicians rely on several assessment tools to help determine the risk of a cancer patient experiencing treatment-related toxicity and adverse events. Based on the results of these assessments, a cancer patient is considered intolerant to treatment if they are determined to be at high risk of experiencing treatment-related toxicity and adverse events that result in poor outcomes. Examples of commonly used assessment tools used in determining treatment intolerance include the Karnofsky Performance Status (KPS), Eastern Cooperative Oncology Group Performance Status (ECOG PS), Timed Get Up and Go (TUG), Short Physical Performance Battery (SPPB), Comprehensive Geriatric Assessment (CGA), Cancer Aging Research Group (CARG) Score, and Chemotherapy Risk Assessment Scale for High-Age Patients (CRASH) [33, 34].

[0121]

[0135] Exemplary diseases, conditions, or disorders that can be treated using the methods herein include cancer, e.g., esophageal cancer, pancreatic cancer, metastatic pancreatic cancer, metastatic pancreatic adenocarcinoma, bladder cancer, gastric cancer, fibrotic cancer, glioma, malignant glioma, diffuse intrinsic pontine glioma, recurrent glioma, esophageal cancer, pancreatic cancer ... esophageal cancer, pancreatic cancer, metastatic pancreatic cancer, esophageal cancer, pancreatic cancer, esophageal cancer, pancreatic cancer, esophageal cancer, pancreatic cancer, esophageal cancer, Pediatric brain neoplasms, renal cell carcinoma, metastatic clear cell renal carcinoma, renal carcinoma, prostate cancer, metastatic castration-resistant prostate cancer, stage IV prostate cancer, metastatic melanoma, melanoma, malignant melanoma, recurrent cutaneous melanoma, melanoma brain metastasis, stage IIIA cutaneous melanoma; stage IIIB cutaneous melanoma; stage IIIC cutaneous melanoma; stage IV cutaneous melanoma, malignant melanoma of the head and neck, lung cancer, non-small cell lung cancer (NSCLC), squamous cell non-small cell lung cancer, breast cancer, recurrent Metastatic breast cancer, hepatocellular carcinoma, Hodgkin's lymphoma, follicular lymphoma, non-Hodgkin's lymphoma, aggressive B-cell NHL, HL including diffuse large B-cell lymphoma (DLBCL), multiple myeloma, chronic myeloid leukemia, adult acute myeloid leukemia in remission; adult acute myeloid leukemia with Inv(16)(p13.1q22);CBFB-MYH11;t(16;16)(p13.1;q22);CBFB-MYH11;t( Adult acute myeloid leukemia with t(8;21)(q22;q22); RUNX1-RUNX1T1; adult acute myeloid leukemia with t(9;11)(p22;q23); MLLT3-MLL; adult acute promyelocytic leukemia with t(15;17)(q22;q12); PML-RARA; alkylating agent-associated acute myeloid leukemia, chronic lymphocytic leukemia, Richter's syndrome; Waldenström's macroglobulinemia, adult glioblastoma; adult gliosarcoma, recurrent glioblastoma, recurrent childhood rhabdomyosarcoma, recurrent Ewing's sarcoma / peripheral primitive neuroectodermal tumor, recurrent neuroblastoma; recurrent osteosarcoma, colorectal cancer, MSI-positive colorectal cancer; MSI-negative These include colorectal cancer, nasopharyngeal nonkeratinizing carcinoma, recurrent nasopharyngeal undifferentiated carcinoma, cervical adenocarcinoma, cervical adenosquamous carcinoma, cervical squamous cell carcinoma, recurrent cervical cancer, stage IVA cervical cancer, stage IVB cervical cancer, anal canal squamous cell carcinoma, metastatic anal canal cancer, recurrent anal canal cancer, recurrent head and neck cancer, carcinoma, head and neck squamous cell carcinoma, head and neck squamous cell carcinoma (HNSCC), ovarian cancer, colon cancer, gastric cancer, advanced GI cancer, gastric adenocarcinoma, gastroesophageal junction adenocarcinoma, bone neoplasms, soft tissue sarcoma, osteosarcoma, thymic carcinoma, urothelial carcinoma, recurrent Merkel cell carcinoma, stage III Merkel cell carcinoma, stage IV Merkel cell carcinoma, myelodysplastic syndrome and recurrent mycosis fungoides, and Sézary syndrome. In various embodiments, the cancer is selected from brain cancer, skin cancer, eye cancer, breast cancer, prostate cancer, lung cancer, esophageal cancer, head and neck cancer, cervical cancer, liver cancer, colon cancer, bone cancer, uterine cancer, ovarian cancer, bladder cancer, stomach cancer, oral cancer, thyroid cancer, kidney cancer, testicular cancer, leukemia, lymphoma, and mesothelioma.

[0122]

[0136] Non-limiting examples of cancers or tumors that are typically immunologically "cold," immunoevasive, immunoprotected, immunologically "cold," microsatellite stable, microsatellite low instability, have a low immune infiltrate, have a low tumor mutational burden, and / or exhibit heterogeneity include Merkel cell carcinoma (MCC), renal cell carcinoma (RCC), ovarian cancer, MSS colorectal cancer, pancreatic cancer, glioblastoma, and the like. , neuroblastoma, and prostate cancer.

[0123] Treatment outcomes and clinical endpoints

[0137] In various embodiments, the present disclosure provides methods of treating cancer in a subject, comprising administering surface-functionalized particles, alone or in combination with a cancer therapeutic, to the subject, the subject having one or more immune-evasive tumors. In various embodiments, the administration alters the tumor immune infiltrate. In various embodiments, the administration alters the anti-tumor immune response. In various embodiments, the administration alters the tumor microenvironment, including tumor cells, immune cells, cancer stem cells, and stroma. In various embodiments, the administration converts an immunologically cold tumor to an immunologically hot tumor. In various embodiments, the administration reduces tumor size and / or inhibits tumor growth. In various embodiments, the administration induces tumor cell death, apoptosis, and / or necrosis through direct particle uptake by tumor cells.

[0124]

[0138] In various embodiments, the present disclosure provides methods of treating cancer in a subject comprising administering to the subject surface-functionalized particles, alone or in combination with a cancer therapeutic, the subject having one or more tumors characterized as immunologically protected and / or immune evasive. In various embodiments, the administration alters tumor-associated stroma, including fibroblasts, cancer-associated fibroblasts, adipocytes, pericytes, endothelium, vasculature, lymphatic vessels, tumor-associated vasculature, mesenchymal stromal cells, mesenchymal stem cells, and extracellular matrix.

[0125]

[0139] The methods herein are contemplated to reduce tumor size or tumor burden in a subject and / or reduce metastasis in a subject. In various embodiments, the methods reduce tumor size by 10%, 20%, 30% or more. In various embodiments, the methods reduce tumor size by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% (or all values ​​and ranges therebetween).

[0126]

[0140] Certain biomarkers decrease in abundance when tumors become immune evasive. It is contemplated herein that following treatment with the surface-functionalized particles described herein, optionally in combination with a cancer therapeutic agent, the level of one or more biomarkers increases by an amount ranging from about 1.1-fold to about 10-fold, e.g., about 1.1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, or about 10-fold. Similarly, certain biomarkers increase in abundance when tumors become immune evasive. Following treatment with the surface-functionalized particles described herein, the levels of one or more of such biomarkers are reduced by an amount ranging from about 1.1-fold to about 10-fold, e.g., about 1.1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, or about 10-fold. In various embodiments, administering the surface-functionalized particles, alone or in combination with a cancer therapeutic agent, to a subject having one or more tumors characterized as being immunoevasive, immunologically protected, and / or immunologically "cold" reduces the levels of immunosuppressive cells in the blood. In various embodiments, the suppressive cells include myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), neutrophils, T cells, and the like. reg cells, and B regIn various embodiments, the MDSCs are monocytic MDSCs (M-MDSCs) and polymorphonuclear MDSCs (PMN-MDSCs). In various embodiments, the TAMs are M2TAMs. In various embodiments, the immunosuppressive cells are CAFs. In various embodiments, the level of immunosuppressive cells is reduced by about 5-100% (e.g., reduced by 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%, or about 100% (including all values ​​and ranges therebetween)), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50% compared to one or more blood samples taken from the subject prior to treatment. In various embodiments, the level of immunosuppressive cells is reduced by about 2-100 fold (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold, including all values ​​and ranges therebetween) compared to one or more blood samples taken from the subject prior to treatment. In various embodiments, the immunosuppressive cells are identified by assaying cell surface protein expression.

[0127]

[0141] In various embodiments, administering surface-functionalized particles, alone or in combination with a cancer therapeutic, to a subject having one or more tumors characterized as being immunoevasive, immunologically protected, and / or immunologically "cold" reduces the level of activated pro-inflammatory immune cells by 5-100% (e.g., about 5%, about 10%, about 15%) compared to one or more blood samples taken from the subject prior to treatment. %, 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%, or about 100% (including all values ​​and ranges therebetween), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50% increase. In various embodiments, administering surface-functionalized particles to a subject having one or more tumors characterized as immunoevasive, immunologically protected, and / or immunologically "cold" increases the level of activated pro-inflammatory immune cells by 2-100 fold (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold, including all values ​​and ranges therebetween), compared to one or more blood samples taken from the subject prior to treatment. In various embodiments, the activated pro-inflammatory cells are dendritic cells (DCs), macrophages, M1 macrophages, T cells, B cells, NK cells, NK-T cells, and iNK cells. In various embodiments, the pro-inflammatory immune cells are The frequency of activated pro-inflammatory immune cells is increased to 10-50% (e.g., about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%, including all values ​​and ranges therebetween) of all leukocytes analyzed from one or more blood samples taken from the subject. In various embodiments, activated pro-inflammatory immune cells are identified by assaying cell surface protein expression.

[0128]

[0142] In various embodiments, the analysis of cells in one or more blood samples from a subject suffering from cancer is performed by assaying cell surface proteins, such as receptor tyrosine kinase (RTK), CD1c, CD2, CD3, CD4, CD5, CD8, CD9, CD10, CD11b, CD11c, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD24, TACI, CD25, CD27, CD28, CD30, CD30L, CD31, CD32, CD32b, CD34, CD33, CD38, CD39, CD40, CD40-L, CD41b, CD42a, CD42b, CD43, CD44, and CD45. , CD47, CD45RA, CD45RO, CD48, CD52, CD55, CD56, CD58, CD61, CD66b, CD70, CD72, CD79, CD68, CD84, CD86, CD93, CD94, CD95, CRACC, BLAME, BCMA, CD103 , CD107, CD112, CD120a, CD120b, CD123, CD125, CD134, CD135, CD140a, CD141, CD154, CD155, CD160, CD163, CD172a, XCR1, CD203c, CD204, CD206, CD207 CD226, CD244, CD267, CD268, CD269, CD355, CD358, NKG2A, NKG2B, NKG2C, NKG2D, NKG2E, NKG2F, NKG2H, KIR2DL1, KIR2DL2, KIR2DL3 , KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, KIR3DL4, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, DAP12, KIR3DS, NKp4 4, NKp46, TCR, BCR, integrin, FcβεRI, MHC-I, MHC-II, IL-1R, IL-2Rα, IL-2Rβ, IL-2Rγ, IL-3Rα, CSF2RB, IL-4R, IL-5Rα, CSF2RB, IL- 6Rα, gp130, IL-7Rα, IL-9R, IL-12Rβ1, IL-12Rβ2, IL-13Rα1, IL-13Rα2, IL-15Rα, IL-21R, IL23R, IL-27Rα, IL-31Rα, OSMR, CSF-1R,Precursors IL-15, IL-10Rα, IL-10Rβ, IL-20Rα, IL-20Rβ, IL-22Rα1, IL-22Rα2, IL-22Rβ, IL-28RA, PD-1 PD-1H, BTLA, CTLA-4, PD-L1, PD-L2, 2B4, B7-1, B7-2, B7-H1, B7-H4, B7-DC, DR3, LIGHT, LAIR, LT α1β2, LTβR, TIM-1, TIM-3, TIM-4, TIGIT, LAG-3, ICOS, ICOS-L, SLAM, SLAMF2, OX-40, OX-40L, GIT R, GITRL, TL1A, WHO, 41-BB, 41BB-L, TL-1A, TRAF1, TRAF2, TRAF3, TRAF5, BAFF, BAFF-R, APRIL, TR AIL, RANK, AITR, TRAMP, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, CXC R1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CLECL9a, DC-SIGN, IGSF4A, SIGLEC, EGFR, PDGFR EGFR, FAP, α-SMA, FAS, FAS-L, Fc, ICAM-1, ICAM-2, ICAM-3, ICAM-4, ICAM-5, PECAM- 1, MICA, MICB, UL16, ULBP1, ILBP3, ULBP4, ULBP5, ULBP6, MULT1, RAE1α, β, γ, δ, and ϵε, A1R, A. 2A R、A 2B R, A3R, H60a, H60b, and H60c The scent is very smooth and smooth α1, α2, αIIb, α3, α4, α5, α6, α7, α8, α9, α10, α11, αD, αE, αL, αM, αV. In various embodiments, the TCR is selected from the group consisting of αX, β1, β2, β3, β4, β5, β6, β7, β8, and / or combinations thereof. In various embodiments, the TCR is selected from the group consisting of α, β, γ, δ, ε, and ζ TCRs. Several methods for assaying cell surface protein expression have been described in the literature, including flow cytometry and mass cytometry (CyTOF). The presence or abundance of one or more of these cell surface proteins indicates that the patient will be responsive to treatment by the methods disclosed herein.

[0129]

[0143] In various embodiments, administering surface-functionalized particles, alone or in combination with a cancer therapeutic agent, to a subject having one or more tumors characterized as immunoevasive, immunologically protected, and / or immunologically "cold" reduces the neutrophil-to-lymphocyte (NLR) in one or more blood samples from high to moderate, or from high to low. In various embodiments, analysis of cells from one or more blood samples taken from a subject having one or more tumors characterized as immunoevasive, immunologically protected, and / or immunologically "cold" reduces the NLR to between 1 and 2 (e.g., 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, and 2, including all values ​​and ranges therebetween). In various embodiments, the NLR is reduced after administration of the surface-functionalized particles. In various embodiments, the NLR is less than 2 after administration of the surface-functionalized particles.

[0130]

[0144] In various embodiments, administering surface-functionalized particles, alone or in combination with a cancer therapeutic, to a subject having one or more tumors characterized as immunoevasive, immunologically protected, and / or immunologically "cold" reduces the level of CTCs in one or more blood samples. In various embodiments, the level of CTCs in the blood is reduced to 5 or less, 4 or less, 3 or less, 2 or less, 1 or less, or 0 (including all values ​​and ranges therebetween) per 7.5 mL of blood.

[0131]

[0145] In various embodiments, administering the surface-functionalized particles, alone or in combination with a cancer therapeutic agent, to a subject having one or more tumors characterized as being immunoevasive, immunologically protected, and / or immunologically "cold" reduces the levels of tumor-promoting, anti-inflammatory, and / or immunosuppressive proteins in one or more blood samples from the subject. In various embodiments, the tumor-promoting, anti-inflammatory, and / or immunosuppressive protein is selected from the group consisting of CD39, CD79, MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP23A, MMP23B, MMP24, MMP25, MMP26, MMP27, and MMP28, CXCL12, GM-CSF, G-CSF, TGF-β1, TGF-β2, and TGF-β3, arginase, asparaginase, kynureninase, ), indoleamine 2,3 dioxygenase (IDO1 and IDO2), tryptophan 2,3 dioxygenase (TDO), myeloperoxidase (MPO), neutrophil elastase (NE), and IL4I1. In various embodiments, the level of a tumor-promoting, anti-inflammatory, and / or immunosuppressive protein in one or more blood samples from the subject is reduced by 5-100% (e.g., reduced by 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%, or about 100% (including all values ​​and ranges therebetween)), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, 50%, or 100% compared to one or more blood samples taken before treatment. In various embodiments, the level of tumor-promoting, anti-inflammatory, and / or immunosuppressive proteins in one or more blood samples from the subject is reduced by 2-100 fold (e.g., by 2-100 fold) compared to one or more blood samples taken from the subject prior to treatment. For example, reduced by about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 times (including all values ​​and ranges therebetween) compared to one or more samples taken before treatment.

[0132]

[0146] In various embodiments, administering the surface-functionalized particles, alone or in combination with a cancer therapeutic agent, to a subject having one or more tumors characterized as being immunoevasive, immunologically protected, and / or immunologically "cold" increases the level of tumor-inhibitory, anti-tumor, and / or pro-inflammatory proteins in one or more blood samples taken from the subject. In various embodiments, the tumor-inhibitory, anti-tumor, and / or pro-inflammatory proteins include IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-20, IL-21, IL-22, IL-23, IL-24, IL-16, IL-18, IL-19, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, IL-39, IL-40, IL-41, IL-42, IL-43, IL-44, IL-45, IL-46, IL-47, IL-48, IL-49, IL-50, IL-51, IL-52, IL-53, IL-54, IL-55, IL-56, IL-57, IL-58, IL-59, IL-60, IL-61, IL-62, IL-63, IL-64, IL-65, L-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35, IL-36, cell surface IL-15, CXCL2 (MCP-1), CXCL 3 (MIP-1α), CXCL4 (MIP-1β), CXCL5 (RANTES), IFN-α, IFN-β, IFN-γ, granzyme-B, perforin, and TNF-α. In various embodiments, the level of an anti-tumor and / or pro-inflammatory protein is increased by 5-100% (e.g., 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%, or about 100% (including all values ​​and ranges therebetween)), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, 50%, or 100% compared to one or more blood samples taken before treatment.In various embodiments, the level of the anti-tumor and / or pro-inflammatory protein is elevated 2-100 fold compared to one or more blood samples taken from the subject prior to treatment (e.g., elevated by about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold (including all values ​​and ranges therebetween) compared to one or more samples taken prior to treatment). Several methods for assaying proteins from blood samples have been described in the literature, including Western blot and ELISA.

[0133]

[0147] In various embodiments, administering the surface-functionalized particles, alone or in combination with a cancer therapeutic agent, to a subject having one or more tumors characterized as being immunoevasive, immunologically protected, and / or immunologically "cold" reduces the level of neutrophil extracellular traps (NETs) in one or more blood samples taken from the subject. In various embodiments, the level of NETs in one or more blood samples is reduced by 5-100% (e.g., 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%, or about 100% (including all values ​​and ranges therebetween)), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, 50%, or 100% compared to one or more blood samples taken before treatment. In various embodiments, the level of NETs in one or more blood samples is reduced by 2-100 fold (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold, including all values ​​and ranges therebetween) compared to one or more blood samples taken from the subject prior to treatment. Western blot, ELISA, and flow cytometry Several methods have been described in the literature for assaying NETs from blood samples, including:

[0134]

[0148] In various embodiments, administering the surface-functionalized particles, alone or in combination with a cancer therapeutic agent, to a subject having one or more tumors characterized as being immunoevasive, immunologically protected, and / or immunologically "cold" reduces the expression of tumor-promoting, tumor-permissive, and / or immunosuppressive genes in one or more blood samples from the subject. In one or more embodiments, expression of tumor-promoting, tumor-permissive, and / or immunosuppressive genes is reduced by 5-100% compared to one or more blood samples taken before treatment (e.g., reduced by 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%, or about 100% (including all values ​​and ranges therebetween) compared to levels in one or more blood samples taken before treatment), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, 50%, or 100% compared to levels in one or more blood samples taken before treatment). In one or more embodiments, expression of tumor-promoting, tumor-permissive, and / or immunosuppressive genes is reduced by 2 to 100 fold (e.g., reduced by about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold (including all values ​​and ranges therebetween)) compared to one or more blood samples taken from the subject prior to treatment.

[0135]

[0149] In various embodiments, administering the surface-functionalized particles, alone or in combination with a cancer therapeutic agent, to a subject having one or more tumors that are characterized as immunoevasive, immunologically protected, and / or immunologically "cold" increases the expression of tumor-inhibitory, anti-tumor, and / or pro-inflammatory genes in one or more samples taken from the subject. In one or more embodiments, expression of tumor inhibitory, anti-tumor, and / or pro-inflammatory genes is increased by 5-100% (e.g., 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%, or about 100% (including all values ​​and ranges therebetween)), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, 50%, or 100% compared to one or more blood samples taken before treatment. In various embodiments, expression of tumor inhibitory, anti-tumor, and / or pro-inflammatory genes is increased 2-100 fold compared to one or more blood samples taken from the subject prior to treatment (e.g., increased by about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold (including all values ​​and ranges therebetween) compared to one or more samples taken prior to treatment). In various embodiments, gene expression analysis is performed by PCR, RT-PCR, qRT-PCR, next generation sequencing (NGS), RNA-seq, ATAC-seq, exome sequencing, Southern blot, microarray analysis, and / or single cell sequencing.

[0136]

[0150] In various embodiments, administering surface-functionalized particles, alone or in combination with a cancer therapeutic, to a subject having one or more tumors characterized as immunoevasive, immunologically protected, and / or immunologically "cold" increases the level of leukocytes in the tumor. In various embodiments, the level of leukocytes is increased in the tumor core and / or tumor margin. In various embodiments, the level of leukocytes is increased by 5-100% (e.g., 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%) compared to one or more tumor samples taken from the subject prior to treatment. %, about 90%, about 95%, or about 100% (including all values ​​and ranges therebetween), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50%. In various embodiments, the level of white blood cells is increased by 2-100 fold (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold, including all values ​​and ranges therebetween) compared to one or more tumor samples taken from the subject prior to treatment. In various embodiments, the frequency of leukocytes in the tumor core and / or tumor margin is 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, or 50% or more of all cells analyzed (including all values ​​and ranges therebetween).

[0137]

[0151] In various embodiments, administering surface-functionalized particles, alone or in combination with a cancer therapeutic, to a subject having one or more tumors characterized as immunoevasive, immunologically protected, and / or immunologically "cold" reduces the level of immunosuppressive cells in the tumor. In various embodiments, the level of immunosuppressive cells is reduced in the tumor core and / or tumor margin. In various embodiments, the suppressive cells include myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), neutrophils, T cells, and the like. reg cells, and B reg In various embodiments, the MDSCs are monocytic MDSCs (M-MDSCs) and polymorphonuclear MDSCs (PMN-MDSCs). In various embodiments, the TAMs are M2TAMs. In various embodiments, the immunosuppressive cells are CAFs. In various embodiments, the level of immunosuppressive cells is reduced by about 5-100% (e.g., reduced by 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%, or about 100% (including all values ​​and ranges therebetween)), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50% compared to one or more tumor samples taken from the subject prior to treatment. In various embodiments, the level of immunosuppressive cells is reduced by about 2-100-fold (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100-fold, including all values ​​and ranges therebetween) compared to one or more tumor samples taken from the subject prior to treatment. In various embodiments, immunosuppressive cells are identified by assays of cell surface protein expression. The level of leukocytes in tumor samples can be assessed by several methods, including flow cytometry and immunohistochemistry.

[0138]

[0152] In various embodiments, administering surface-functionalized particles, alone or in combination with a cancer therapeutic, to a subject having one or more tumors characterized as immunoevasive, immunologically protected, and / or immunologically "cold" increases the levels of activated pro-inflammatory immune cells in the tumor, hi various embodiments, the levels of activated pro-inflammatory cells are increased in the tumor core and / or tumor margin.

[0139]

[0153] In various embodiments, administering the surface-functionalized particles, alone or in combination with a cancer therapeutic agent, to a subject having one or more tumors characterized as being immunoevasive, immunologically protected, and / or immunologically "cold" results in an increase in the level of activated pro-inflammatory immune cells in the tumor of 5-100% (e.g., an increase of 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%, or about 100% (including all values ​​and ranges therebetween)), 10-95%, 15-90%, 16-18%, 18-20%, 20-25%, 25-30%, 25-30%, 35-40%, 35-40%, 45-50%, 55-60%, 65-60%, 70-75%, 80-85%, 90-95%, 95-100%, or 100% (including all values ​​and ranges therebetween) compared to one or more tumor samples taken from the subject prior to treatment. 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, or 50% increase. In various embodiments, administering the surface-functionalized particles to a subject having one or more tumors characterized as immunoevasive, immunologically protected, and / or immunologically "cold" increases the level of activated pro-inflammatory immune cells by 2-100 fold (e.g., about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold, including all values ​​and ranges therebetween) compared to one or more tumor samples taken from the subject prior to treatment. In various embodiments, the activated pro-inflammatory cells are dendritic cells (DCs), macrophages, M1 macrophages, T cells, B cells, NK cells, NK-T cells, and iNK cells. In various embodiments, the frequency of pro-inflammatory immune cells is between about 10-50% (e.g., about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%, including all values ​​and ranges therebetween) of all leukocytes analyzed from one or more tumor samples obtained from a subject. In various embodiments, activated pro-inflammatory immune cells are identified by assaying cell surface protein expression.

[0140]

[0154] In various embodiments, the analysis of cells in one or more tumor samples from a subject suffering from cancer is performed by assaying cell surface proteins, such as receptor tyrosine kinase (RTK), CD1c, CD2, CD3, CD4, CD5, CD8, CD9, CD10, CD11b, CD11c, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD24, TACI, CD25, CD27, CD28, CD30, CD30L, CD31, CD32, CD32b, CD34, CD33, CD38, CD39, CD40, CD40-L, CD41b, CD42a, CD42b, CD43, CD44, and CD45. , CD47, CD45RA, CD45RO, CD48, CD52, CD55, CD56, CD58, CD61, CD66b, CD70, CD72, CD79, CD68, CD84, CD86, CD93, CD94, CD95, CRACC, BLAME, BCMA, CD103 , CD107, CD112, CD120a, CD120b, CD123, CD125, CD134, CD135, CD140a, CD141, CD154, CD155, CD160, CD163, CD172a, XCR1, CD203c, CD204, CD206, CD207CD226、CD244、CD267、CD268、CD269、CD355、CD358、NKG2A、NKG2B、NKG2C、NKG2D、NKG2E、NKG2F、NKG2H、KIR2DL1、KIR2DL2 、KIR2DL3、KIR2DL5A、KIR2DL5B、KIR3DL1、KIR3DL2、KIR3DL3、KIR3DL4、KIR2DS1、KIR2DS2、KIR2DS3、KIR2DS4、KIR2DS5、 DAP12、KIR3DS、NKp44、NKp46、TCR、BCR、インテグリン、FcβεRI、MHC-I、MHC-II、IL-1R、IL-2Rα、IL-2Rβ、IL-2Rγ、IL-3Rα、CSF2R B、IL-4R、IL-5Rα、CSF2RB、IL-6Rα、gp130、IL-7Rα、IL-9R、IL-12Rβ1、IL-12Rβ2、IL-13Rα1、IL-13Rα2、IL-15Rα、IL-21R、 IL23R, IL-27Rα, IL-31Rα, OSMR, CSF-1R, cell surface IL-15, IL-10Rα, IL-10Rβ, IL-20Rα, IL-20Rβ, IL-22Rα1, IL-22Rα2, IL-22Rβ, IL-28RA, PD-1, PD-1H, BTLA, CTLA-4, PD-L1, PD-L2, 2B4, B7-1, B7-2, B7-H1, B7-H4, B7-DC, DR3, LIGHT, LAIR, LTα1β2 、LTβR、TIM-1、TIM-3、TIM-4、TIGIT、LAG-3、ICOS、ICOS-L、SLAM、SLAMF2、OX-40、OX-40L、GITR、GITRL、TL1A、HVEM、41-BB 、41BB-L、TL-1A、TRAF1、TRAF2、TRAF3、TRAF5、BAFF、BAFF-R、APRIL、TRAIL、RANK、AITR、TRAMP、CCR1、CCR2、CCR3、CCR4、C CR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CLECL9a, DC-SIGN, IGSF4A, SIGLEC, EGFR, PDGFR, VEGFR, FAP, α-SMA, Vimentin , laminin, FAS, FAS-L, Fc, ICAM-1, ICAM-2, ICAM-3, ICAM-4, ICAM-5, PECAM-1, MICA, MICB, UL16, ULBP1, ULBP2, ILBP3, ULBP4, ULBP5, ULBP6, MULT1, RAE1α, β, γ, δ, and ε, A1R, A 2A R.A. 2B In various embodiments, the integrin is selected from the group consisting of αR, αR, A3R, H60a, H60b, and H60c. In various embodiments, the integrin is selected from the group consisting of α1, α2, αIIb, α3, α4, α5, α6, α7, α8, α9, α10, α11, αD, αE, αL, αM, αV, αX, β1, β2, β3, β4, β5, β6, β7, β8, and / or combinations thereof. In various embodiments, the TCR is selected from the group consisting of α, β, γ, δ, ε, and ζ TCRs. Several methods for assaying cell surface protein expression have been described in the literature, including flow cytometry and mass cytometry (CyTOF). The presence or abundance of one or more of these cell surface proteins indicates that the patient will be responsive to treatment by the methods disclosed herein.

[0141]

[0155] In various embodiments, administering the surface-functionalized particles, alone or in combination with a cancer therapeutic agent, to a subject having one or more tumors characterized as being immunoevasive, immunologically protected, and / or immunologically "cold" reduces the levels of tumor-promoting, anti-inflammatory, and / or immunosuppressive proteins in one or more tumor samples from the subject. In various embodiments, the tumor-promoting, anti-inflammatory, and / or immunosuppressive protein is selected from the group consisting of CD39, CD79, MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP23A, MMP23B, MMP24, MMP25, MMP26, MMP27, and MMP28, CXCL12, GM-CSF, G-CSF, TGF-β1, TGF-β2, and TGF-β3, arginase, asparaginase, kynureninase, ), indoleamine 2,3 dioxygenase (IDO1 and IDO2), tryptophan 2,3 dioxygenase (TDO), myeloperoxidase (MPO), neutrophil elastase (NE), and IL4I1. In various embodiments, the level of a tumor-promoting, anti-inflammatory, and / or immunosuppressive protein in one or more tumor samples from the subject is reduced by 5-100% (e.g., by 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%, or about 100% (including all values ​​and ranges therebetween)), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, 50%, or 100% compared to one or more tumor samples taken before treatment. In various embodiments, the level of tumor-promoting, anti-inflammatory, and / or immunosuppressive proteins in one or more tumor samples from the subject is reduced by 2 to 100 fold compared to one or more tumor samples taken from the subject prior to treatment (e.g., reduced by about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold (including all values ​​and ranges therebetween) compared to one or more samples taken from the subject prior to treatment).

[0142]

[0156] In various embodiments, administering the surface-functionalized particles, alone or in combination with a cancer therapeutic agent, to a subject having one or more tumors characterized as being immunoevasive, immunologically protected, and / or immunologically "cold" increases the levels of proteins associated with tumor growth inhibition, anti-tumor activity, and / or pro-inflammatory activity. The proteins include CD44, CD56, CD103c, CD69, KG2A, NKG2B, NKG2C, NKG2D, NKG2E, NKG2F, NKG2H, ICOS, ICOS-L, SLAM, SLAMF2, OX-40, OX-40L, GITR, GITRL, TL1A, HVEM, 41- BB, 41BB-L, TL-1A, TRAF1, TRAF2, TRAF3, TRAF5, BAFF, BAFF-R, APRIL, TRAIL, RANK, AITR, TRAMP, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-17, IL-18, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, selected from the group consisting of IL-32, IL-33, IL-35, IL-36, CXCL2 (MCP-1), CXCL3 (MIP-1α), CXCL4 (MIP-1β), CXCL5 (RANTES), IFN-α, IFN-β, IFN-γ, granzyme-B, perforin, and TNF-α. In various embodiments, the level of a protein associated with tumor growth inhibition, anti-tumor activity, and / or pro-inflammatory activity is increased by 5-100% (e.g., 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%, or about 100% (including all values ​​and ranges therebetween)), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, 50%, or 100% compared to one or more tumor samples taken prior to treatment.In various embodiments, the level of a protein associated with tumor growth inhibition, anti-tumor activity, and / or pro-inflammatory activity is elevated 2-100 fold compared to one or more tumor samples taken from the subject prior to treatment (e.g., elevated by about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold (including all values ​​and ranges therebetween) compared to one or more samples taken prior to treatment). Several methods for assaying proteins from tumor samples have been described in the literature, including Western blot and ELISA.

[0143]

[0157] In various embodiments, administering the surface-functionalized particles, alone or in combination with a cancer therapeutic agent, to a subject having one or more tumors characterized as being immunoevasive, immunologically protected, and / or immunologically "cold" reduces the level of neutrophil extracellular traps (NETs) in one or more tumor samples taken from the subject. In various embodiments, the level of NETs in one or more tumor samples is reduced by 5-100% (e.g., by 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%, or about 100% (including all values ​​and ranges therebetween)), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, 50%, or 100% compared to one or more tumor samples taken before treatment. In various embodiments, the level of NETs in one or more tumor samples is reduced 2-100 fold compared to one or more tumor samples taken from the subject prior to treatment (e.g., reduced by about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold (including all values ​​and ranges therebetween) compared to one or more samples taken prior to treatment). Several methods for assaying NETs from tumor samples have been described in the literature, including Western blot, ELISA, and flow cytometry.

[0144]

[0158] In various embodiments, subjects with one or more tumors that are characterized as immunoevasive, immunologically protected, and / or immunologically "cold" are Administering the surface-functionalized particles, alone or in combination with a cancer therapeutic agent, to an elephant reduces the expression of tumor-promoting, tumor-permissive, and / or immunosuppressive genes in one or more tumor samples from the subject. In one or more embodiments, expression of tumor-promoting, tumor-permissive, and / or immunosuppressive genes is reduced by 5-100% (e.g., 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%, or about 100% (including all values ​​and ranges therebetween)), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, 50%, or 100% compared to one or more tumor samples taken before treatment. In one or more embodiments, expression of tumor-promoting, tumor-permissive, and / or immunosuppressive genes is reduced by 2-100 fold compared to one or more tumor samples taken from the subject prior to treatment (e.g., reduced by about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold (including all values ​​and ranges therebetween) compared to one or more samples taken prior to treatment).

[0145]

[0159] In various embodiments, administering the surface-functionalized particles, alone or in combination with a cancer therapeutic agent, to a subject having one or more tumors that are characterized as immunoevasive, immunologically protected, and / or immunologically "cold" increases the expression of tumor-inhibitory, anti-tumor, and / or pro-inflammatory genes in one or more samples taken from the subject. In one or more embodiments, expression of tumor inhibitory, anti-tumor, and / or pro-inflammatory genes is increased by 5-100% (e.g., 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%, or about 100% (including all values ​​and ranges therebetween)), 10-95%, 15-90%, 20-85%, 25-75%, 30-70%, 35-65%, 40-60%, 45-55%, 50%, or 100% compared to one or more tumor samples taken before treatment. In various embodiments, expression of tumor inhibitory, anti-tumor, and / or pro-inflammatory genes is increased 2-100 fold (e.g., increased by about 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 fold (including all values ​​and ranges therebetween)) compared to one or more tumor samples taken from the subject prior to treatment. In various embodiments, gene expression analysis is performed by PCR, RT-PCR, qRT-PCR, next-generation sequencing (NGS), RNA-seq, ATAC-seq, exome sequencing, Southern blot, microarray analysis, and / or single-cell sequencing.

[0146]

[0160] In various embodiments, treatment of a subject with cancer with surface-functionalized particles alone or in combination with a cancer therapeutic agent converts a cold tumor to a hot tumor. Such conversion can be detected using methods described herein and known in the art. Once a subject is diagnosed with a tumor that has converted from a cold tumor to a hot tumor, treatment by administering surface-functionalized particles alone or in combination with a cancer therapeutic agent can be continued, where the cancer therapeutic agent is useful for treating hot tumors, or tumors that are rich in immune cells or immunogenic. In other embodiments, once the tumor has converted from a cold tumor to a hot tumor, the patient stops treatment with the surface-functionalized particles, and the patient begins treatment with a cancer therapeutic agent that is useful for treating hot tumors, or tumors that are rich in immune cells or immunogenic. Such cancer therapeutic agents can include chemotherapeutic agents, cytokines, angiogenesis inhibitors, enzymes, immune checkpoint modulators and monoclonal antibodies, hormone therapy, one or more cell-based therapies, such as adoptive cell transfer, tumor-infiltrating leukocyte therapy, chimeric antigen receptor T-cell therapy (CAR-T), NK cell therapy, and stem cell therapy. This includes cell therapy, or oncolytic viruses or bacteria.

[0147]

[0161] In various embodiments, the immune checkpoint modulator targets programmed cell death protein 1 (PD1), programmed cell death protein ligand-1 (PD-L1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), T-cell immunoglobulin and mucin domain-containing 3 (TIM-3), lymphocyte-activation gene 3 (LAG-3), and / or TIGIT (T-cell immunoreceptor with Ig and ITIM domains). In various embodiments, the immune checkpoint modulator is an antibody selected from the group consisting of ipilimumab, tremelimumab, pembrolizumab, nivolumab, atezolizumab, avelumab, cemiplimab, and durvalumab.

[0148]

[0162] In various embodiments, subjects diagnosed with cold tumors and receiving treatment with surface-functionalized particles alone or in combination with a cancer therapeutic agent are monitored periodically to determine whether the tumor has transformed into a hot tumor. Monitoring can be performed, for example, monthly, every two months, every three months, every six months, or yearly, as determined necessary by a physician.

[0149]

[0163] In various embodiments, the subject has previously been treated with immunotherapy but has developed resistance to immunotherapy or has converted from a hot tumor to a cold tumor. Also provided are methods of treating a subject with cancer that has developed resistance to immunotherapy or has developed a cold tumor, comprising administering to the subject surface-functionalized particles, alone or in combination with a cancer therapeutic agent.

[0150] Administration and Dosage

[0164] Contemplated herein is a method for treating a subject suffering from cancer, comprising administering a composition comprising the negatively charged particles described herein in combination with a cancer therapeutic agent.

[0151]

[0165] The methods of the present disclosure are carried out using any medically acceptable means for directly or indirectly introducing a therapeutic agent into a mammalian subject, including, but not limited to, injection, oral ingestion, intranasal administration, topical administration, transdermal administration, parenteral administration, inhalation spray, vaginal administration, or rectal administration. As used herein, the term parenteral includes subcutaneous, intravenous, intramuscular, intraarticular, intraperitoneal, intrathecal, and intracisternal injection, as well as catheter or infusion techniques. In various embodiments, the particles are administered intravenously, but may also be administered by other routes of administration, including, but not limited to, intradermal, subcutaneous, epicutaneous, oral, intraarticular, and intrathecal. In various embodiments, the composition is administered at the tumor site.

[0152]

[0166] In various embodiments, the surface-functionalized particles are administered at a dose of about 0.1 to about 24 mg / kg. In various embodiments, the particles are administered at a dose of about 0.1 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 3.0 mg / kg, 4.0 mg / kg, 5.0 mg / kg, 6.0 mg / kg, 7.0 mg / kg, 8.0 mg / kg, 9.0 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 21 mg / kg, 22 mg / kg, 23 mg / kg, or 24 mg / kg (including all values ​​and ranges therebetween). In various embodiments, the particles are administered at a dose ranging from about 8.0 mg to about 1920 mg. In various embodiments, the particles are administered in a dose of about 8.0 mg, 80 mg, 320 mg, 640 mg, 800 mg, 960 mg, 1120 mg, 1280 mg, 1440 mg, 1600 mg, 1760 mg, or 1920 mg. Values ​​within and between the listed dose endpoints are also contemplated. These concentrations can be administered in a single dosage form or as multiple doses.

[0153]

[0167] It is contemplated that the cancer therapeutic, if any, will be administered as directed by the manufacturer and treating physician. If the particles and cancer therapeutic are administered in the same formulation, they may be combined as described herein.

[0154]

[0168] The amount of immunomodulatory agent or biologic cancer therapeutic agent in a given dosage may vary according to the size of the individual to whom the treatment is administered and the characteristics of the disorder being treated. In an exemplary treatment, it may be necessary to administer approximately 1 mg / day, 5 mg / day, 10 mg / day, 20 mg / day, 50 mg / day, 75 mg / day, 100 mg / day, 150 mg / day, 200 mg / day, 250 mg / day, 500 mg / day, or 1000 mg / day (including all values ​​and ranges therebetween). Standard dose-response studies, first in animal models and then in clinical trials, will reveal optimal dosages for specific disease conditions and patient populations.

[0155]

[0169] Conditions treatable by the methods of the present disclosure preferably occur in mammals, including, for example, humans and other primates, as well as pet or companion animals such as dogs and cats, laboratory animals such as rats, mice, and rabbits, and livestock animals such as horses, pigs, sheep, and cows. In various embodiments, the subject is a human.

[0156]

[0170] In various embodiments, the particles are administered daily, every other day, twice a day, three times a day, seven times a week, six times a week, five times a week, four times a week, three times a week, twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once every two months, once every three months, once every six months, or once a year.

[0157]

[0171] The present disclosure further contemplates a sterile pharmaceutical composition comprising a particle described herein, a cancer therapeutic agent, and a pharmaceutically acceptable carrier.

[0158]

[0172] The present disclosure further contemplates a sterile pharmaceutical composition comprising the particles described separately herein and a pharmaceutically acceptable carrier.

[0159]

[0173] The present disclosure further contemplates a sterile pharmaceutical composition comprising a separate cancer therapeutic agent and a pharmaceutically acceptable carrier.

[0160]

[0174] Syringes, e.g., disposable or pre-filled syringes, sterile, sealed containers, e.g., vials, bottles, vessels, and / or kits or packages, containing any of the above antibodies or compositions, optionally with suitable instructions for use, are also contemplated.

[0161] Combination Therapy

[0175] It is contemplated that the particles described herein are administered in combination with a cancer therapeutic to treat the proliferative disorder cancer, In various embodiments, the cancer therapeutic is a chemotherapeutic agent, a biologic agent, a cell-based therapy, a hormone therapy, an antibody-drug conjugate, an oncolytic virus, or a cancer vaccine.

[0162]

[0176] Hormone therapy includes tamoxifen for breast cancer, Zoladex for breast and prostate cancer, aromatase inhibitors (e.g., anastrozole), retroviral therapy (e.g., cyclosporine), and steroids (e.g., cyclosporine). Antibody-drug conjugates include brentuximab vedotin (anti-CD30 mAB + monomethyl auristatin E) for lymphoma, ado-trastuzumab emtansine (anti-Her2 / Neu) for breast cancer, and fluoxetine (anti-TNF-α) for breast cancer. Oncolytic viruses include ImmuRidge and Inotuzumab Ozagamicin (anti-CD22 + maytansinoid) for ALL and Inotuzumab Ozagamicin (anti-CD22 + calicheamicin) for ALL. Cancer vaccines include sipuleucel-T for prostate cancer and flu vaccine (Amgen®). Several cancer vaccines are under development, including, but not limited to, protein, polypeptide, and nucleic acid vaccines.

[0163]

[0177] In various embodiments, the cancer therapeutic agent is a chemotherapeutic agent selected from the group consisting of a growth inhibitor, a cytotoxic agent, a DNA replication inhibitor, a kinase inhibitor, a signal transduction cascade inhibitor, an angiogenesis inhibitor, a metabolism inhibitor, an amino acid synthesis inhibitor, a selective inhibitor of an oncogenic protein, a metastasis inhibitor, an inhibitor of an anti-apoptotic factor, an apoptosis inducer, a nucleoside signaling inhibitor, an enzyme inhibitor, and a DNA damaging agent.

[0164]

[0178] A cytotoxic agent refers to a substance that inhibits or prevents the function of cells and / or causes destruction of cells. The term is intended to include radioactive isotopes (e.g., I131, I125, Y90, and Re186), chemotherapeutic agents, and toxins, such as enzymatically active toxins of bacterial, fungal, plant, or animal origin, or synthetic toxins, or fragments thereof. A non-cytotoxic agent refers to a substance that does not inhibit or prevent the function of cells and / or does not cause destruction of cells. Non-cytotoxic agents can include agents that can be activated to become cytotoxic.

[0165]

[0179] Chemotherapeutic agents contemplated for use in the methods of the present disclosure include, but are not limited to, those set forth in Tables 1-3.

[0166] [Table 1]

[0167] [Table 2]

[0168] [Table 3]

[0169]

[0180] It is also contemplated that the cancer therapeutic agent may include one or more biologic agents, such as cytokines, angiogenesis inhibitors, immune checkpoint modulators, and monoclonal antibodies.

[0170]

[0181] Cytokines include interferons (IFNs) and interleukins (ILs), such as IFN-alpha, IFN-beta, IFN-gamma, IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-11, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, members of the transforming growth factor beta superfamily (including TGF-β1, TGF-β2, and TGF-β3), tumor necrosis factor alpha, granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0171]

[0182] In various embodiments, the cancer therapeutic agent comprises an enzyme. In various embodiments, the cancer therapeutic agent comprises an enzyme that targets T cells, B cells, APCs, monocytes, MDSCs, TAMs, neutrophils, other monocyte-derived cells, tumor-associated stroma, cancer stem cells, mesenchymal stem cells, extracellular matrix, and amino acids. In various embodiments, the cancer therapeutic agent comprises an enzyme selected from the group including asparaginase, kynureninase, L-arginine deiminase, L-methionine-γ-lyase, one or more amino acid-degrading enzymes, and one or more nucleoside-degrading enzymes.

[0172]

[0183] Biologic agents, such as immune checkpoint modulators, target PD1, PD-L1, CTLA-4, TIMP-3, LAG-3, and / or TIGIT (T cell immunoreceptor with Ig and ITIM domains). In various embodiments, the immune checkpoint modulator is an antibody specific for PD-1, PD-L1, or CTLA-4. Antibodies specific for checkpoint proteins include ipilimumab (YERVOY®, Bristol-Myers Squibb Company) and tremelimumab, which bind to CTLA-4; antibodies against PD-1, such as pembrolizumab (KEYTRUDA®, Merck Sharp & Dohme Co.); rp) and nivolumab (OPDIVO®, Bristol-Myers Squibb); and antibodies targeting PD-L1, such as atezolizumab (TECENTRIQ®), avelumab (BAVENCIO®), and durvalumab (IMFINZI®) (approved for the treatment of urothelial carcinoma and non-small cell lung cancer), cemiplimab (LIBTAYO®) (approved for the treatment of cutaneous (approved for squamous cell carcinoma).

[0173]

[0184] In various embodiments, the monoclonal antibody is a monospecific, bispecific, trispecific, or bispecific T cell engager (BiTE) antibody.

[0174]

[0185] In various embodiments, the monoclonal antibody is an immune cell costimulatory molecule agonist that induces an anti-tumor immune response. Exemplary costimulatory molecules include, but are not limited to, ICOS (inducible T cell costimulator) (CD278), OX40 (CD134), GITR (glucocorticoid-induced tumor necrosis factor receptor), CD40, and CD27.

[0175]

[0186] In various embodiments, the monoclonal antibody useful in the present methods is selected from the group including alemtuzumab, bevacizumab, brentuximab, cetuximab, denosumab, ibritumomab, trastuzumab, panitumumab, pertuzumab, and rituximab. In various embodiments, the monoclonal antibody useful in the present methods targets receptor tyrosine kinases, EGFR, VEGF, VEGFR, PDGF, PDGFR, TGF-β, TGF-β-LAP, SIRP-α, CD47, CD39, CD73, and fibroblast activation protein (FAP).

[0176]

[0187] Biologic agents include monoclonal antibodies that are monospecific, bispecific, trispecific, or bispecific T cell engagers (BiTEs). Monoclonal antibodies useful in the treatment of cancer include bevacizumab (AVASTIN), an antibody against VEGF-A. (Registered trademark), Genentech); Erlotinib, a tyrosine kinase inhibitor that acts on EGFR (TARCEVA®, Genentech and OSI Pharmaceuticals), the oral Bcr-Abl tyrosine kinase inhibitor dasatinib (SPRYCEL®, Bristol-Myers Squibb Company); IL-21; PEGylated IFN-α2b; tyrosine kinase inhibitors These include the anti-cancer drug axitinib (INLYTA®, Pfizer, Inc.); and the MEK inhibitor trametinib (MEKINIST®, GlaxoSmithKline) (Philips and Atkins, Int Immunol., 27(1):39-46 (2015), which is incorporated herein by reference). Bispecific antibodies useful for treating cancer include blinatumomab and catumaxomab, as described by Krishnamurthy et al., (Pharmacol Ther. 2018 May;185:122-134), and Yu et al. al., (J. Hematol Oncol 2017, 10:155).

[0177]

[0188] The method also provides that the cancer therapeutic agent comprises one or more cell-based therapies, including adoptive cell transfer, tumor-infiltrating leukocyte therapy, chimeric antigen receptor T-cell (CAR-T) therapy, NK cell therapy, and stem cell therapy.

[0178]

[0189] In various embodiments, the cell-based therapy is the adoptive transfer of autologous cells from the patient. In various embodiments, the cell-based therapy is the adoptive transfer of allogeneic cells from a donor.

[0179]

[0190] In various embodiments, the cell-based therapy is the transfer of cells derived from universal donors or induced pluripotent stem cells that are not patient-specific and are suitable for long-term storage, also referred to as "off-the-shelf" therapy.

[0180]

[0191] In various embodiments, the cancer treatment agent is a hormone therapy. In various embodiments, the cancer therapeutic comprises one or more antibody-drug conjugates. In various embodiments, the cancer therapeutic comprises one or more cancer vaccines. In various embodiments, the cancer vaccine is a protein, polypeptide, and / or nucleic acid vaccine.

[0181]

[0192] In various embodiments, the cancer therapeutic agent is an immunotherapy selected from the group including an oncolytic virus, a bacterium, an oncolytic bacterium or other bacterial consortium, Bacillus Calmette-Guerin (BCG), a microbiome modulating agent, and / or a Toll-like receptor (TLR) agonist. In various embodiments, the TLR agonist is a TLR3, TLR4, TLR5, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, and / or TLR13 agonist. In various embodiments, the TLR agonist is derived from a virus, a bacterium, and / or synthetically produced. In various embodiments, the immunotherapy is a STING pathway modulator.

[0182]

[0193] In various embodiments, the cancer therapeutic agent comprises a viral or bacterial vector. In various embodiments, the viral vector is selected from the group including adenovirus, adeno-associated virus (AAV), herpes simplex virus, lentivirus, retrovirus, alphavirus, flavivirus, rhabdovirus, measles virus, Newcastle disease virus, poxvirus, vaccinia virus, modified Ankara virus, vesicular stomatitis virus, picornavirus, tobacco mosaic virus, potato virus x, comovirus, or cucumber mosaic virus. In various embodiments, the virus is an oncolytic virus. In various embodiments, the virus is a chimeric virus, synthetic virus, mosaic virus, or pseudotyped virus.

[0183]

[0194] It is contemplated that the particles and the cancer therapeutic agent can be administered concurrently, simultaneously, or sequentially. Concurrent administration of two therapeutic agents does not require that the agents be administered at the same time or by the same route, so long as there is an overlap in the time periods during which the agents are exerting their therapeutic effect. Simultaneous or sequential administration is contemplated, as is administration on different days or weeks.

[0184]

[0195] It is contemplated that the particles and the cancer therapeutic agent can be administered simultaneously in the same formulation. It is also contemplated that the agents can be administered in separate formulations and administered concurrently, where concurrent refers to agents given within 30 minutes of each other.

[0185]

[0196] In another embodiment, the cancer therapeutic agent is administered before the administration of particle composition.Pre-administration refers to the administration of cancer therapeutic agent within the range of 1 week before particle treatment and 30 minutes before particle administration.Furthermore, it is intended that the cancer therapeutic agent is administered after the administration of particle composition.Post-administration is intended to describe the administration from 30 minutes after particle treatment to 1 week after administration.

[0186]

[0197] In various embodiments, the particles and / or cancer therapeutic agent are administered once a day, twice a day, three times a day, seven times a week, six times a week, five times a week, four times a week, three times a week, twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once every two months, once every three months, once every six months, or once a year.

[0187]

[0198] In various embodiments, the particles and / or cancer therapeutic agents are administered intravenously, orally, intranasally, intramuscularly, intraocularly, transdermally, or subcutaneously.

[0188]

[0199] In various embodiments, the subject is a mammal, hi various embodiments, the subject is a human.

[0189] kit

[0200] In an additional aspect, the present disclosure includes kits containing one or more compounds or compositions packaged in a manner that facilitates their use to practice the methods of the present disclosure. In one embodiment, such kits include a compound or composition described herein (e.g., particles, alone or in combination with a cancer therapeutic, or a composition thereof) packaged in a container such as a sealed bottle or vessel, with a label affixed to the container or included within the package that describes the use of the compound or composition in practicing the present invention. Preferably, the compound or composition is packaged in a unit dosage form. The kit may further include a device suitable for administering the composition according to a specific route of administration or for performing a screening assay. Preferably, the kit contains a label that describes the use of the inhibitor composition.

[0190] Embodiment

[0201] Embodiment 1. A method of treating cancer in a subject comprising administering a surface-functionalized particle alone or in combination with a cancer therapeutic, wherein the subject has one or more immune-evasive tumors, immunologically protected tumors, immunologically "cold" tumors, microsatellite-stable tumors, microsatellite-low instability tumors, tumors with low immune infiltrate, tumors with low tumor mutational burden, tumors exhibiting heterogeneity, or combinations thereof.

[0191]

[0202] Embodiment 2. A method for treating an immune evasive tumor in a subject, comprising: (i) diagnosing a subject with an immune evasive tumor; (ii) administering the surface-functionalized particles to a subject, alone or in combination with a cancer therapeutic agent; A method comprising:

[0192]

[0203] Embodiment 3. The method of embodiment 2, wherein diagnosing comprises assaying biomarkers / characteristics associated with immune evasive tumors, microsatellite stability / instability, tumor mutational burden, resistance to therapy, tumor heterogeneity, or a combination thereof.

[0193]

[0204] Embodiment 4. The method of embodiment 2 or 3, further comprising (iii) determining whether the subject's tumor becomes immunoresponsive, and then (iv) administering the surface-functionalized particles in combination with immunotherapy.

[0194]

[0205] Embodiment 5. A method for treating a subject having cancer and who has previously received immunotherapy or whose cancer is refractory to immunotherapy, comprising administering to the subject surface-functionalized particles alone or in combination with a cancer therapeutic agent.

[0195]

[0206] Embodiment 6. The method of any one of the preceding embodiments, wherein the particles comprise polyglycolic acid (PGA) polymer, polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), polystyrene, chitosan, polysaccharides, one or more lipids, diamond, or iron, zinc, cadmium, gold, or silver.

[0196]

[0207] Embodiment 7. The method of any one of the preceding embodiments, wherein the surface-functionalized particles are poly(lactic-co-glycolic acid) (PLGA) particles.

[0197]

[0208] Embodiment 8. The method of embodiment 7, wherein the particles comprise a ratio of polylactic acid:polyglycolic acid of about 1:99 to about 99:1.

[0198]

[0209] Embodiment 9. The method of embodiment 8, wherein the particles comprise about 50:50, about 80:20 to about 100:0 polylactic acid:polyglycolic acid, or about 50:50, about 80:20 to about 100:0 polyglycolic acid:polylactic acid.

[0199]

[0210] Embodiment 10. The method of any one of the preceding embodiments, wherein the particles comprise 50:50 polylactic acid:polyglycolic acid.

[0200]

[0211] Embodiment 11 The method of any one of the preceding embodiments, wherein the particles are surface functionalized by the addition of one or more carboxyl groups.

[0201]

[0212] Embodiment 12 The method of any one of the preceding embodiments, wherein the particles are negatively charged particles.

[0202]

[0213] Embodiment 13 The method of embodiment 12, wherein the particles do not comprise a therapeutic agent.

[0203]

[0214] Embodiment 14 The method of embodiment 12 or 13, wherein the particles do not contain attached peptide or antigenic moieties or other bioactive agents.

[0204]

[0215] Embodiment 15. The method of any one of the preceding embodiments, wherein the particles have a zeta potential between -100mV and -1mV.

[0205]

[0216] Embodiment 16 The method of any one of the preceding embodiments, wherein the particles have a zeta potential between -80mV and -30mV, or between -50mV and -40mV.

[0206]

[0217] Embodiment 17. The method of any one of the preceding embodiments, wherein the diameter of the surface-functionalized particles is between 0.1 μm and 10 μm.

[0207]

[0218] Embodiment 18. The method of any one of the preceding embodiments, wherein the diameter of the surface-functionalized particles is between 400 nm and 800 nm.

[0208]

[0219] Embodiment 19. The method of any one of embodiments 1 to 18, wherein the surface-functionalized particles and / or the cancer therapeutic agent are administered in a composition.

[0209]

[0220] Embodiment 20. The method of embodiment 19, wherein the composition comprises a pharmaceutically acceptable excipient, diluent, or carrier.

[0210]

[0221] Embodiment 21 The method of any one of the preceding embodiments, wherein the subject has one or more immunological cold tumors.

[0211]

[0222] Embodiment 22 The method of any one of the preceding embodiments, wherein the subject has one or more tumors with a low tumor mutational burden.

[0212]

[0223] Embodiment 23 The method of any one of the preceding embodiments, wherein the subject has one or more microsatellite-stable tumors.

[0213]

[0224] Embodiment 24 The method of any one of the preceding embodiments, wherein the subject has one or more tumors with low microsatellite instability.

[0214]

[0225] Embodiment 25 The method of any one of the preceding embodiments, wherein the subject has one or more tumors with a low tumor immune infiltrate.

[0215]

[0226] Embodiment 26 The method of any one of the preceding embodiments, wherein the administration alters the tumor immune infiltrate.

[0216]

[0227] Embodiment 27 The method of embodiment 25 or 26, wherein the tumor immune infiltrate comprises antigen-presenting cells, myeloid cells, and lymphoid cells.

[0217]

[0228] Embodiment 28 The method of embodiment 27, wherein the antigen-presenting cells comprise macrophages and / or dendritic cells.

[0218]

[0229] Embodiment 29. The method of embodiment 27, wherein the myeloid cells comprise monocytes, neutrophils, myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages (TAMs).

[0219]

[0230] Embodiment 30 The method of embodiment 29, wherein the tumor-associated macrophages comprise M1 macrophages, M2 macrophages, and / or MARCO+ macrophages.

[0220]

[0231] Embodiment 31 The method of embodiment 27, wherein the lymphoid cells include T cells, B cells, NKT cells, and NK cells.

[0221]

[0232] Embodiment 32 The method of any one of the preceding embodiments, wherein the administration alters an anti-tumor immune response.

[0222]

[0233] Embodiment 33 The method of any one of the preceding embodiments, wherein administering alters the tumor microenvironment.

[0223]

[0234] Embodiment 34 The method of embodiment 33, wherein the tumor microenvironment comprises tumor cells, cancer stem cells, immune cells, and stroma.

[0224]

[0235] Embodiment 35 The method of embodiment 34, wherein the stroma comprises fibroblasts, adipocytes, endothelium, vasculature, mesenchymal stromal cells, and / or extracellular matrix.

[0225]

[0236] Embodiment 36 The method of any one of the preceding embodiments, wherein the administering converts an immunologically cold tumor to an immunologically hot tumor.

[0226]

[0237] Embodiment 37 The method of any one of the preceding embodiments, wherein the administration reduces tumor size and / or inhibits tumor growth.

[0227]

[0238] Embodiment 38. The method of any one of the preceding embodiments, wherein the subject has a cancer selected from the group consisting of brain cancer, skin cancer, eye cancer, breast cancer, prostate cancer, pancreatic cancer, lung cancer, esophageal cancer, head and neck cancer, cervical cancer, liver cancer, colorectal cancer, bone cancer, uterine cancer, ovarian cancer, bladder cancer, endometrial cancer, stomach cancer, gastric cancer, oral cancer, thyroid cancer, kidney cancer, testicular cancer, leukemia, lymphoma, and mesothelioma.

[0228]

[0239] Embodiment 39. The method of any one of the preceding embodiments, wherein the cancer therapeutic agent administered in combination with the particles is a chemotherapeutic agent selected from the group consisting of a growth inhibitor, a DNA replication inhibitor, a kinase inhibitor, a receptor tyrosine kinase inhibitor, a signal transduction cascade inhibitor, an angiogenesis inhibitor, a metabolism inhibitor, an amino acid synthesis inhibitor, a selective inhibitor of an oncogenic protein, a metastasis inhibitor, an inhibitor of an anti-apoptotic factor, an apoptosis inducer, an enzyme inhibitor, a nucleoside signaling inhibitor, an antibody-drug conjugate, and a DNA damaging agent.

[0229]

[0240] Embodiment 40. The method of any one of embodiments 1-38, wherein the cancer therapeutic agent administered in combination with the particles comprises one or more biologic agents selected from the group consisting of cytokines, angiogenesis inhibitors, receptor tyrosine kinase inhibitors, immune checkpoint modulator enzymes, and monoclonal antibodies.

[0230]

[0241] Embodiment 41 The method of embodiment 40, wherein the cytokine is selected from the group consisting of transforming growth factors, tumor necrosis factors, interferons, and interleukins.

[0231]

[0242] Embodiment 42. The method of embodiment 40, wherein the immune checkpoint modulator targets programmed cell death protein 1 (PD1), programmed cell death protein ligand-1 (PD-L1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), T-cell immunoglobulin and mucin domain-containing-3 (TIM-3), lymphocyte-activation gene-3 (LAG-3), and / or TIGIT (T-cell immunoreceptor with Ig and ITIM domains).

[0232]

[0243] Embodiment 43. The method of embodiment 42, wherein the immune checkpoint modulator is an antibody selected from the group consisting of ipilimumab, tremelimumab, pembrolizumab, nivolumab, atezolizumab, avelumab, cemiplimab, and durvalumab.

[0233]

[0244] Embodiment 44 The method of embodiment 40, wherein the monoclonal antibody comprises a monospecific, bispecific, or trispecific antibody.

[0234]

[0245] Embodiment 45 The method of embodiment 40, wherein the monoclonal antibody comprises a bispecific T cell engager (BiTE).

[0235]

[0246] Embodiment 46. The method of embodiment 40, wherein the monoclonal antibody is selected from the group including alemtuzumab, bevacizumab, brentuximab, cetuximab, denosumab, ibritumomab, trastuzumab, panitumumab, pertuzumab, and rituximab.

[0236]

[0247] Embodiment 47. The method of embodiments 1-38, wherein the cancer therapeutic administered in combination with the particles comprises one or more cell-based therapies selected from the group consisting of adoptive cell transfer, tumor-infiltrating leukocyte therapy, chimeric antigen receptor T-cell therapy (CAR-T), NK cell therapy, and stem cell therapy.

[0237]

[0248] Embodiment 48 The method of any one of embodiments 1 to 38, wherein the cancer therapeutic agent administered in combination with the particles is a hormone therapy.

[0238]

[0249] Embodiment 49 The method of any one of embodiments 1 to 38, wherein the cancer therapeutic administered in combination with the particles comprises one or more cancer vaccines.

[0239]

[0250] Embodiment 50. The method of any one of embodiments 1-38, wherein the cancer therapeutic agent administered in combination with the particles is one or more immunotherapies including an oncolytic virus, an oncolytic bacterium or other bacterial composition, Bacillus Calmette-Guerin (BCG), a microbiome modulator, a STING pathway modulator, and / or a toll-like receptor (TLR) modulator.

[0240]

[0251] Embodiment 51. A method of treating cancer in a subject, comprising: a. determining the immunological status of the tumor and / or the tumor mutational burden and / or the microsatellite instability status of the tumor; b. immune-evasive and / or immunologically protected and / or immunologically cold and / or with low tumor immune infiltrate and / or with low tumor mutational burden and / or microsatellite stable and / or refractory and / or with low microsatellite instability and / or exhibit heterogeneity; diagnosing the combined tumor; c. administering the surface-functionalized particles alone or in combination with a cancer therapeutic agent; A method comprising:

[0241]

[0252] Embodiment 52. The method of any one of the preceding embodiments, wherein the particles and / or cancer medicament are administered once daily, twice daily, three times daily, seven times per week, six times per week, five times per week, four times per week, three times per week, twice per week, once per week, once per two weeks, once per three weeks, once per four weeks, once per two months, once per three months, once per six months, or once per year.

[0242]

[0253] Embodiment 53 The method of any one of the preceding embodiments, wherein the particles are administered intravenously, orally, intranasally, intramuscularly, intraocularly, transdermally, or subcutaneously.

[0243]

[0254] Embodiment 54 The method of any one of the preceding embodiments, wherein the subject is a human.

[0244]

[0255] Embodiment 55. The method of any one of the preceding embodiments, wherein administering ameliorates one or more symptoms of cancer.

[0245]

[0256] Embodiment 56 The method of any one of the preceding embodiments, wherein the surface-functionalized particles are negatively charged particles that do not contain attached peptide or antigenic moieties or other bioactive agents.

[0246]

[0257] Embodiment 57. The method of embodiment 56, wherein the particles are PLGA particles having a zeta potential between -80 and -30 mV and a diameter between 200 and 2000 nm.

[0247]

[0258] Embodiment 58 The method of any one of the preceding embodiments, wherein the tumor is an immune evasive tumor.

[0248]

[0259] Embodiment 59. The method of any of embodiments 1-57, wherein the tumor is an immunologically protected tumor.

[0249]

[0260] Embodiment 60. The method of any of embodiments 1-57, wherein the tumor is an immunologically "cold" tumor.

[0250]

[0261] Embodiment 61 The method of any of embodiments 1-57, wherein the tumor is a microsatellite-stable tumor.

[0251]

[0262] Embodiment 62. The method of any of embodiments 1-57, wherein the tumor is a microsatellite low instability tumor.

[0252]

[0263] Embodiment 63 The method of any of embodiments 1-57, wherein the tumor comprises a low immune infiltrate.

[0253]

[0264] Embodiment 64. The method of any one of embodiments 1 to 57, wherein the tumor comprises a low tumor mutational burden.

[0254]

[0265] Embodiment 65. The method of any of embodiments 1 to 57, wherein the tumor exhibits heterogeneity.

[0255]

[0266] Additional aspects and details of the present disclosure will be apparent from the following examples, which are intended to be illustrative rather than limiting. [Example]

[0256] Example Example 1

[0267] To determine the efficacy of surface-functionalized particles against immunologically "cold" tumors with low tumor mutation burden or immune-evasive tumors, a syngeneic tumor model was established using the murine B16F10 melanoma cell line. B16F10 cells have previously been shown to be immunologically "cold," have low TMB, and exhibit resistance to checkpoint inhibitor treatment (Song et al., Nat Commun. 9:2237, 2018). Briefly, 6-8 week-old C57BL / 6 mice were implanted with B16F10 cells via subcutaneous injection into the flank. Palpable tumors (approximately 100 mm) formed. 3 After 10 min (size of 1000 mg / kg), animals were randomized into one of four treatment groups as follows: Group 1: control treatment (n=15); Group 2: SFP (n=15); Group 3: anti-PD1 (n=15); Group 4: combination (SFP + anti-PD1) (n=15).

[0257] [Table 4]

[0258]

[0268] According to the following treatment schedule, SFP (1 mg) composed of PLGA and having a negative zeta potential (e.g., ranging between -100 mV and -1 mV, e.g., between -80 mV and -30 mV) was administered by intravenous (iv) injection, and anti-PD1 (100 μg) was administered by intraperitoneal (ip) injection.

[0259]

[0269] Tumor growth was assessed by measuring tumor size in two dimensions using calipers, using the formula V = 0.5 × a × b 2 The tumor volume was calculated using the formula (where a and b are the long and short diameters of the tumor, respectively). The tumor size was expressed in mm 3 It is expressed as:

[0260]

[0270] Five animals from each group were sacrificed 12 days after the start of treatment, and the frequency of MDSCs and TAMs in the tumors was assessed by flow cytometry. Cell viability was also assessed using live / dead staining. As shown in Figure 1A, 12 days of treatment with SFP alone or in combination with anti-PD1 resulted in a complete abrogation of cell viability in the tumors. Treatment with anti-PD1 alone also resulted in a moderate but significant decrease in cell viability.

[0261]

[0271] Consistent with its effect on cell viability, treatment with SFP but without anti-PD1 strongly inhibited tumor growth. The inhibition of tumor growth by combined SFP and anti-PD1 treatment was comparable to that by SFP alone, suggesting that tumor inhibition in the combination group was primarily driven by SFP and that B16F10 melanoma tumors were resistant to checkpoint blockade. These results suggest that SFP treatment results in a longer survival time (approximately 10 days) compared with control and anti-PD1 treatment, consistent with its effect on tumor growth. Mice treated with SFP alone eventually succumbed to the disease, demonstrating the highly aggressive disease course of B16F10 melanoma tumors. As expected, due to the resistance of B16F10 tumors to anti-PD1, the combination treatment did not demonstrate a synergistic effect, and survival in this group was comparable to that in the SFP-only treatment group (Figure 1C).

[0262]

[0272] As shown in Figures 1D–1F, treatment with anti-PD1 alone resulted in a significant decrease in the frequency of MDSCs (CD11b+Ly6G+) and TAMs (CD11b+F4 / 80+) in tumors. MDSC frequency tended to decrease after combined SFP and anti-PD1 treatment (p = 0.055), while TAM frequency was significantly reduced in this treatment group. Treatment with anti-PD1 alone resulted in a trend toward an increase in NK cell frequency in tumors, and the combination treatment resulted in a statistically significant increase in NK cell frequency (Figure 1F). Because the frequencies of MDSCs, TAMs, and NK cells in tumors were not affected by treatment with anti-PD1 alone, the effects of the combination treatment on these cells appeared to be driven by SFP.

[0263] Example 2: Treatment with surface-functionalized particles results in the reduction of primary orthotopic 4T1 breast tumors and inhibits their metastasis to the lung.

[0273] To determine the efficacy of the surface-functionalized particles described in Example 1, e.g., CNP-301, in inhibiting the growth and metastasis of immunologically "cold" breast tumors with low tumor mutation burden, a syngeneic orthotopic tumor model was established using the murine 4T1 breast tumor cell line. The 4T1 tumor cell line was derived from the mammary gland tissue of BALB / c mice. 4T1 cells are triple-negative for estrogen, progesterone, and HER2 receptors and are widely used as a model of stage IV human breast cancer. 4T1 tumors are highly immunogenic and invasive, mimicking human disease by spontaneously metastasizing to distant organs such as the lungs. Importantly, 4T1 tumors, like human triple-negative breast cancers, are resistant to anti-PD1 checkpoint inhibitor treatment. In this study, the efficacy of CNP-301 against orthotopic 4T1 breast tumors was compared with control (saline) and anti-PD1 monoclonal antibody treatment.

[0264]

[0274] 1 × 10 6- to 8-week-old BALB / c mice in the fourth mammary fat pad 5 The 4T1 tumor cells used in these experiments were engineered to express luciferase, allowing their detection by IVIS® bioluminescence imaging.

[0265]

[0275] Treatment was initiated at different time points after tumor injection as follows:

[0266] [Table 5]

[0267]

[0276] Each group consisted of 7-8 animals. Treatment was performed once every 3 days. CNP-301 was administered via tail vein injection at a dose of 1 mg / mouse. Anti-PD1 was administered via i.p. injection at a dose of 200 μg / mouse once every 3 days. Tumor growth was constantly monitored by measuring tumors using standard calipers. Tumor volume was calculated using the following formula: Tumor volume = 0.5 (length) x (width) 2

[0268]

[0277] Animals were euthanized 20 days after tumor inoculation and lung metastases were detected by IVIS® bioluminescence imaging.

[0269]

[0278] As shown in Figures 2A-2B, treatment with CNP-301 initiated on days 1 and 2 resulted in a significant reduction in the growth of primary orthotopic 4T1 tumors compared with control and anti-PD-1 treatment. As shown in Figures 3A-3B, IVIS® bioluminescence imaging of the lungs on day 20 revealed that treatment with CNP-301 significantly reduced lung metastasis of primary 4T1 tumors. While the majority of animals in the control and anti-PD-1 treatment groups developed metastases, treatment with CNP-301 initiated on day 1 completely abrogated lung metastasis. Mice treated with CNP-301 initiated on days 2, 3, and 5 developed lung metastases in certain animals (1 / 8, 2 / 8, and 3 / 8 mice, respectively); however, these metastatic lesions were significantly smaller in size compared with metastases in the control and anti-PD-1 treatment groups. Overall, these data demonstrate that CNP-301 treatment causes a reduction in the growth of primary orthotopic 4T1 tumors and inhibits their metastasis to the lung.

[0270] Example 3: Treatment with surface-functionalized particles inhibits the growth of pre-existing 4T1 metastatic lesions in the lung.

[0279] To determine the efficacy of surface-functionalized particles, e.g., CNP-301, in inhibiting existing metastatic lesions, a syngeneic orthotopic tumor resection model was established using the murine 4T1 breast tumor cell line. Briefly, 6-8 week-old BALB / c mice were inoculated with 1 × 10 CNP-301 in the fourth mammary fat pad. 54T1 tumor cells were injected into the mice. The 4T1 tumor cells used in these experiments were engineered to express luciferase, which allows their detection by IVIS® bioluminescence imaging. The primary 4T1 tumor was surgically removed 11 days after tumor injection, after the primary tumor had already begun to metastasize to the lungs. 12 days after tumor injection, the animals were treated with saline (control) or CNP-301. CNP-301 was administered via tail vein injection at a dose of 1 mg / mouse. 42 days after tumor injection, the animals were euthanized, and 4T1 primary tumor metastasis to the lungs was assessed by evaluating metastatic lesions in the lungs using IVIS® bioluminescence imaging. Study The design is depicted in Figure 4A.

[0271]

[0280] As shown in Figure 4B, treatment with CNP-301 completely inhibited the growth of 4T1 lung metastases, as none of the mice showed evidence of metastatic lesions as examined by IVIS® bioluminescence imaging. In contrast, in the control (saline) treatment group, 4 / 9 (44.44%) mice showed evidence of lung metastases. These observations are significant because the study design of the tumor resection model is similar to the current treatment paradigm for metastatic triple-negative breast cancer in the clinic, where the primary tumor is surgically removed followed by a neoadjuvant treatment regimen targeted at inhibiting the growth of metastatic lesions. These data indicate that CNP-301 may be effective for the treatment of human triple-negative breast cancer in the neoadjuvant setting.

[0272] Example 4: Treatment with surface-functionalized particles induces pro-inflammatory immunological changes in the blood and tumors of B16F10 tumor-bearing mice.

[0281] B16F10 murine melanoma tumors are considered immunologically "cold" with low tumor mutational burden. Furthermore, these tumors are resistant to treatment with immunotherapies (e.g., anti-PD1 checkpoint inhibitors) in part due to their immunological status, including low tumor immune infiltrate.

[0273]

[0282] We investigated the efficacy of CNP-301 in inducing pro-inflammatory antitumor immunological changes in B16F10 tumor-bearing mice. Briefly, 6- to 8-week-old C57BL / 6 mice were subcutaneously injected with B16F10 tumor cells. Palpable tumors (approximately 50 mm) were observed. 3 After the first 24 h, the animals were treated with saline (control) or CNP-301. The animals were treated once every three days. CNP-301 was administered via tail vein injection at a dose of 1 mg / mouse.

[0274]

[0283] The following parameters were assessed at different time points [day 8 (before the first dose), day 14 (24 hours after the third dose), and day 20 (24 hours after the fifth dose) after tumor injection]: pro-inflammatory cytokines / chemokines in blood by ELISA, myeloid cell phenotype in blood and tumor by flow cytometry, and lymphoid cell functional phenotype in blood and tumor by flow cytometry.

[0275]

[0284] As shown in Figures 5A-5E, compared with saline (control), CNP-301 treatment caused a statistically significant increase from baseline (day 8) in the levels of the pro-inflammatory cytokines / chemokines (A) MIP-1β, (B) TNF-α, and (C) RANTES on days 14 and 20. CNP-301 treatment also caused an increase from baseline (day 8) in the levels of (D) IFN-γ and (E) MCP-1 on day 14; however, this increase was not statistically significant.

[0276]

[0285] As shown in Figure 6, compared to saline (control), CNP-301 treatment significantly reduced blood PD-L1 levels on days 14 and 20. + induced a statistically significant increase from baseline (day 8) in the frequencies of monocytes (Figure 6A) and granulocytes (Figure 6B). +Expression is associated with immunoregulatory functions and is induced upon activation of these cells. Consistent with an activated phenotype, CNP-301 treatment caused a statistically significant increase from baseline (day 8) in the frequency of myeloid cells expressing IL-15 on their cell surface in the blood on days 14 and 20 (Fig. 6C). Cell surface IL-15 expression on myeloid cells is known to induce T cell and NK cell activation through the interaction between IL-15 on myeloid cells and its cognate receptors on T cells and NK cells in trans. In line with these observations, CNP-301 treatment increased the frequency of total NK cells in the blood (Fig. 6D) and activated (granzyme) NK cells on days 14 and 20. + , perforin + , and CD244 + ) caused a statistically significant increase from baseline in the frequency of NK cells (Figures 6E-6G).

[0277]

[0286] Similar to the observations in blood, examination of B16F10 tumors at different time points after treatment revealed that, compared with saline (control), treatment with CNP-301 significantly increased cell surface IL-15-expressing myeloid cells at day 20 (Fig. 7A) and activated CD244 expression at day 14 in tumors. + NK cells (Figure 7B), activated perforin on day 20 + NK cells (Fig. 7C), and activated granzymes at day 20 + It was found to result in a statistically significant increase from baseline (day 8) in the frequency of NK cells (FIG. 7D).

[0278]

[0287] Overall, these data demonstrate that treatment with CNP-301 induces pro-inflammatory immunological changes in the blood and tumors of B16F10 tumor-bearing mice.

[0279] Example 5: Treatment with surface-functionalized particles induces pro-inflammatory immunological changes in the blood and tumors of MC38 tumor-bearing mice.

[0288] The efficacy of CNP-301 in inducing pro-inflammatory anti-tumor immunological changes in MC38 tumor-bearing mice was also investigated. Briefly, 6- to 8-week-old C57BL / 6 mice were subcutaneously injected with B16F10 tumor cells. Palpable tumors (approximately 50 mm) were observed. 3 After the first 24 h, the animals were treated with saline (control) or CNP-301. The animals were treated once every three days. CNP-301 was administered via tail vein injection at a dose of 1 mg / mouse.

[0280]

[0289] The following parameters were assessed at different time points [day 7 (before the first dose), day 14 (24 hours after the third dose), and day 20 (24 hours after the fifth dose) after tumor injection]: pro-inflammatory cytokines / chemokines in blood by ELISA, myeloid cell phenotype in blood and tumor by flow cytometry, and lymphoid cell functional phenotype in blood and tumor by flow cytometry.

[0281]

[0290] As shown in Figure 8A, compared with saline (control), treatment with CNP-301 caused a significant decrease in tumor growth. CNP-301-mediated tumor growth inhibition was observed from day 14 after tumor injection, or after three doses, and continued to inhibit tumor growth until day 20.

[0282]

[0291] As shown in Figures 8B-8D, compared with saline (control), CNP-301 treatment caused a statistically significant increase from baseline (day 8) in the levels of the pro-inflammatory cytokines / chemokines MIP-1β, TNF-α, and RANTES on days 14 and 20. CNP-301 treatment also caused an increase from baseline (day 8) in the levels of IFN-γ and MCP-1 on day 14; however, this increase was not statistically significant.

[0283]

[0292] As shown in Figure 9, compared to saline (control), CNP-301 treatment significantly increased blood PD-L1 levels on days 14 and 20, respectively. +This resulted in a statistically significant increase from baseline (day 8) in the frequencies of monocytes (Figure 9A) and granulocytes (Figure 9B). + Expression is associated with immunoregulatory functions and is induced upon activation of these cells. Consistent with the activated phenotype, CNP-301 treatment caused a statistically significant increase from baseline (day 7) in the frequency of myeloid cells expressing IL-15 on their cell surface in the blood on day 20 (Fig. 9C). In line with the observed increase in IL-15, CNP-301 treatment increased the frequency of total NK cells in the blood (Fig. 9D) and activated (granzyme) cells on days 14 and 20. + , perforin + , and CD244 + )NK thin It also caused a statistically significant increase from baseline in the frequency of alveoli (Figures 9E-9G).

[0284]

[0293] Overall, these data demonstrate that treatment with CNP-301 induces pro-inflammatory immunological changes in the blood of MC38 tumor-bearing mice.

[0285] Example 6: The efficacy of surface-functionalized particles is dependent on the presence of IL-15 and NK cells in the B16F10 tumor model.

[0294] As shown in Examples 4 and 5, treatment with CNP-301 induced cell surface IL-15 expression in myeloid cells along with activation of NK cells. Next, we investigated whether the efficacy of CNP-301 in inhibiting tumor growth depends on the presence of IL-15 and NK cells.

[0286]

[0295] First, we evaluated the effect of anti-IL-15 antibody-mediated IL-15 blockade on the efficacy of CNP-301 in inhibiting tumor growth. Briefly, 6- to 8-week-old C57BL / 6 mice were subcutaneously injected with B16F10 tumor cells. Palpable tumors (approximately 50 mm in size) were observed. 3 ) animals were randomized into the following treatment groups: Saline (control) + isotype control antibody Saline (control) + anti-IL-15 CNP-301 + isotype control antibody · CNP-301+ anti-IL-15 antibody

[0287]

[0296] Animals received CNP-301 treatment once every three days. CNP-301 was administered via tail vein injection at a dose of 1 mg / mouse. Animals received anti-IL-15 or isotype control antibodies at a dose of 100 μg via intraperitoneal injection once every three days. Tumor growth was constantly monitored by measuring tumors using standard calipers. Tumor volume was calculated using the following formula: Tumor volume = 0.5 (length) x (width) 2

[0288]

[0297] As shown in Figure 10, compared with saline, CNP-301 treatment in the absence of IL-15 blockade (isotype control) inhibited B16F10 tumor growth. IL-15 blockade (anti-IL-15) reversed CNP-301 antitumor efficacy and exacerbated tumor growth in the saline-treated group. Overall, these data demonstrate that CNP-301 efficacy is dependent on the presence of IL-15.

[0289]

[0298] Next, the effect of anti-NK1.1 antibody-mediated NK cell depletion on the efficacy of CNP-301 in inhibiting tumor growth was examined in a B16F10 tumor model. Briefly, 6-8 week-old C57BL / 6 mice were subcutaneously injected with B16F10 tumor cells. Palpable tumors (approximately 50 mm) formed. 3 ) animals were randomized into the following treatment groups: Saline (control) + isotype control antibody Saline (control) + anti-NK1.1 CNP-301 + isotype control antibody · CNP-301+anti-NK1.1

[0290]

[0299] Animals received CNP-301 treatment once every three days. CNP-301 was administered via tail vein injection at a dose of 1 mg / mouse. Anti-NK1.1 / isotype antibody treatment was performed one day prior to saline / CNP-301 treatment. Animals received anti-NK1.1 or isotype control antibody at a dose of 100 μg via intraperitoneal injection once every three days. Tumor growth was constantly monitored by measuring tumors using standard calipers. Tumor volume was calculated using the following formula: Tumor volume = 0.5 (length) x (width) 2

[0291]

[0300] As shown in Figure 11, compared with saline, CNP-301 treatment in the absence of NK cell depletion (isotype control) inhibited B16F10 tumor growth. NK cell depletion (anti-NK1.1) reversed CNP-301 antitumor efficacy. Overall, these data demonstrate that CNP-301 efficacy is dependent on the presence of NK cells.

[0292] Example 7 The efficacy of surface-functionalized particles is dependent on the presence of NK cells in the MC38 tumor model.

[0301] The effect of anti-NK1.1 antibody-mediated NK cell depletion on the efficacy of surface-functionalized particles, such as CNP-301, in inhibiting tumor growth was investigated in the MC38 tumor model. Briefly, 6-8 week-old C57BL / 6 mice were subcutaneously injected with MC38 tumor cells. Palpable tumors (approximately 50 mm in size) were observed. 3 ) animals were randomized into the following treatment groups: Saline (control) + isotype control antibody Saline (control) + anti-NK1.1 CNP-301 + isotype control antibody · CNP-301+anti-NK1.1

[0293]

[0302] Animals received CNP-301 treatment once every three days. CNP-301 was administered via tail vein injection at a dose of 1 mg / mouse. Animals received anti-NK1.1 or isotype control antibody at a dose of 100 μg via intraperitoneal injection once every three days. Tumor growth was constantly monitored by measuring tumors using standard calipers. Tumor volume was calculated using the following formula: Tumor volume = 0.5 (length) x (width) 2

[0294]

[0303] As shown in Figure 12, compared with saline, CNP-301 treatment in the absence of NK cell depletion (isotype control) inhibited MC38 tumor growth. NK cell depletion (anti-NK1.1) reversed CNP-301 antitumor efficacy. Overall, these data demonstrate that CNP-301 efficacy is dependent on the presence of NK cells.

[0295] Example 8 The effect of surface-functionalized particles on myeloid-derived suppressor cells (MDSCs)

[0304] The effect of surface-functionalized particles on bone marrow-derived cells was evaluated in a murine 4T1 orthotopic breast cancer model. Briefly, orthotopic 4T1 tumors were established in BALB / c mice by injecting tumor cells into the fourth mammary fat pad. Three days after tumor injection, animals were randomized into one of two treatment groups: Saline (control) (n=4) CNP-301 (n=4)

[0296]

[0305] Treatment was performed via tail vein injection. CNP-301 was administered at a dose of 1 mg / mouse. Animals were given a single treatment with saline or CNP-301, and 12 hours after treatment, blood macrophages (CD11b + / F4 / 80 + ), monocytes (CD11b + Ly6C + ), MDSCs (CD11b + / Ly6C lo / - / Ly6G + ), and dendritic cells (CD11c+ The levels of MDSCs were assessed by flow cytometry. As shown in Figure 13A, treatment with CNP-301 resulted in a significant decrease in the frequency of MDSCs in the blood compared to the saline-treated group (p<0.001). Compared to saline, CNP-301 treatment did not change the levels of monocytes, macrophages, and dendritic cells in the blood after a single treatment.

[0297]

[0306] Next, CNP-30 was administered to bone marrow-derived cells in the lungs of 4T1 tumor-bearing mice. The effects of treatment with CNP-301 were evaluated. The lung is the predominant metastatic site of orthotopic 4T1 mammary tumors. Starting on day 3 after tumor injection, animals were treated with saline or CNP-301 for six consecutive days. 24 hours after the last dose, macrophages (CD11b) in the lung were significantly increased. + / F4 / 80 + ), monocytes (CD11b + Ly6C + ), MDSCs (CD11b + / Ly6C lo / - / Ly6G + ), and dendritic cells (CD11c + The levels of pulmonary MDSCs were assessed by flow cytometry. As shown in Figure 13B, treatment with CNP-301 caused a significant decrease in the level of pulmonary MDSCs compared with saline (p<0.0001). CNP-301 did not alter the levels of monocytes, macrophages, and dendritic cells in the lungs.

[0298]

[0307] Overall, these data demonstrate that surface-functionalized particles, such as CNP-301, reduce the number of MDSCs in the blood and at metastatic sites.

[0299] Example 9 Assay of cellular uptake of surface-functionalized particles in tumor-bearing mice

[0308] Fluorescently labeled CNP-301 particles were used to study the cellular uptake of surface-functionalized particles, such as CNP-301, in tumor-bearing mice in a syngeneic LLC tumor model. CNP-301 particles encapsulating fluorescently labeled (Alexa-Fluor 647) ovalbumin were used. Simply put, 5 x 10 5 Tumor cells were injected subcutaneously into the shaved flank of mice. Palpable tumors (approximately 50 mm) were observed. 2 ) mice were randomized into one of the following treatment groups: Saline (control) CNP-301

[0300]

[0309] Mice were administered saline or CNP-301 (1 mg / mouse) by intravenous injection. Two hours after the single intravenous injection, the mice were sacrificed and CNP-301-positive cells were analyzed in the spleen and LLC tumors by flow cytometry. As shown in Figures 14A and 14B, TAMs (CD11b + F4 / 80 + ), M-MDSC (CD11b + Ly6C + Ly6G - ), and PMN-MDSC (CD11b + Ly6C - Ly6G + The majority of cells were positive for CNP-301 2 hours after injection. Furthermore, approximately 75% of fibroblasts (CD45 - CD140a + ) were also positive for CNP-301. Similarly, CNP-301-positive TAMs (CD11b + F4 / 80 + ), M-MDSC (CD11b + Ly6C + Ly6G - ), and PMN-MDSC (CD11b + Ly6C - Ly6G + ) was also found in the spleen (Figures 14C and 14D).

[0301]

[0310] Overall, these data demonstrate that CNP-301 is taken up by bone marrow-derived cells and fibroblasts after iv infusion.

[0302] Example 10 Effect of surface-functionalized particles on gene expression in bone marrow-derived cells and fibroblasts of LLC tumor-bearing mice

[0311] The effect of surface-functionalized particles, e.g., CNP-301, on gene expression in bone marrow-derived cells and fibroblasts was investigated in LLC tumor-bearing mice. 5 Tumor cells were injected subcutaneously into the shaved flank of mice. Palpable tumors (approximately 50 mm) were observed. 2 ) mice were randomized into one of the following treatment groups: Saline (control) CNP-301

[0303]

[0312] Mice were administered saline or CNP-301 (1 mg / mouse) by intravenous injection. Mice received the indicated treatments twice a week for 2 weeks. Treatment period At the end of the treatment period, the mice were sacrificed and tumors were harvested. Fluorescence-activated cell sorting (FACS) was performed to identify TAMs (CD11b + F4 / 80 + ) and fibroblasts (CD45 - CD140a +) were isolated from tumors. The effects of CNP-301 treatment on gene expression in TAMs and fibroblasts were determined by quantitative polymerase chain reaction (qPCR). Compared with saline, CNP-301 treatment resulted in a clear trend toward a shift in TAM phenotype from an anti-inflammatory / tumor-promoting M2 to a pro-inflammatory / anti-tumor M1 phenotype at the gene expression level. After CNP-301 treatment, a trend toward increased expression of the pro-inflammatory Ifnγ and Nos2 genes associated with M1 TAMs and decreased expression of the Cd206 and Ym-1 genes associated with tumor-promoting M2 TAMs was observed. Furthermore, the expression of Mmp9, encoding an ECM-remodeling protease involved in tumor progression and metastasis, was also reduced in TAMs after CNP-301 treatment (Figure 15A).

[0304]

[0313] As shown in Figure 15B, treatment with CNP-301 caused a statistically significant reduction in the expression of the Fap, Cxcl1, αSma, and Vim genes in fibroblasts isolated from LLC tumors. These genes are known to be associated with pro-tumorigenic cancer-associated fibroblasts, suggesting that CNP-301 treatment reduces the expression of cancer-associated fibroblasts. It is shown that the tumorigenicity-promoting function of blast cells was inhibited.

[0305]

[0314] Overall, these data demonstrate that CNP-301 treatment results in phenotypic changes in TAMs and fibroblasts in LLC tumors that are associated with a shift in the tumor microenvironment from a pro-tumor to an anti-tumor state.

[0306]

[0315] It is anticipated that numerous modifications and variations in the present disclosure set forth in the above illustrative examples will occur to those skilled in the art. Consequently, only limitations appearing in the claims should be placed on the present disclosure.

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Claims

1. 1. A method of treating cancer in a subject comprising administering a surface-functionalized particle, alone or in combination with a cancer therapeutic agent, wherein the subject has one or more immune-evasive tumors.

2. 1. A method for treating an immune-evasive tumor in a subject, comprising: (i) diagnosing the subject as having an immune-evasive tumor; and (ii) administering to the subject surface-functionalized particles, alone or in combination with a cancer therapeutic agent.

3. 3. The method of claim 2, wherein the diagnosis comprises assaying biomarkers / characteristics associated with immune evasive tumors, microsatellite stability / instability, tumor mutational burden, resistance to therapy, tumor heterogeneity, or a combination thereof.

4. 4. The method of claim 2 or 3, further comprising: (iii) determining whether the subject's tumor becomes immunoresponsive; and then (iv) administering surface-functionalized particles in combination with immunotherapy.

5. 1. A method for treating a subject having cancer who has previously undergone immunotherapy or whose cancer is refractory to immunotherapy, comprising administering to the subject surface-functionalized particles alone or in combination with a cancer therapeutic agent.

6. 6. The method of any one of claims 1 to 5, wherein the particles comprise polyglycolic acid (PGA) polymer, polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), polystyrene, chitosan, polysaccharides, one or more lipids, diamond, or iron, zinc, cadmium, gold, or silver.

7. The method of any one of claims 1 to 6, wherein the surface-functionalized particles are poly(lactic-co-glycolic acid) (PLGA) particles.

8. 8. The method of claim 7, wherein the particles comprise polylactic acid:polyglycolic acid in a ratio of about 1:99 to about 99:

1.

9. 9. The method of claim 8, wherein the particles comprise about 50:50 or about 80:20 to about 100:0 polylactic acid:polyglycolic acid, or about 50:50 or about 80:20 to about 100:0 polyglycolic acid:polylactic acid.

10. 10. The method of any one of claims 1 to 9, wherein the particles comprise 50:50 polylactic acid:polyglycolic acid.

11. The method according to any one of claims 1 to 10, wherein the particles are surface functionalized by the addition of one or more carboxyl groups.

12. The method of any one of claims 1 to 11, wherein the particles are negatively charged particles.

13. The method of claim 12 , wherein the particles do not include a therapeutic agent.

14. 14. The method of claim 12 or 13, wherein the particles do not contain attached peptide or antigenic moieties or other bioactive agents.

15. 15. The method according to claim 1, wherein the particles have a zeta potential between −100 mV and −1 mV. The method according to any one of claims 1 to 5.

16. 16. The method of any one of claims 1 to 15, wherein the particles have a zeta potential between -80mV and -30mV.

17. The method according to any one of claims 1 to 16, wherein the diameter of the surface-functionalized particles is between 0.1 μm and 10 μm.

18. The method according to any one of claims 1 to 17, wherein the diameter of the surface-functionalized particles is between 400 nm and 800 nm.

19. The method of any one of claims 1 to 18, wherein the surface-functionalized particles and / or cancer therapeutic agent are administered in a composition.

20. 20. The method of claim 19, wherein the composition comprises a pharmaceutically acceptable excipient, diluent, or carrier.

21. The method of any one of claims 1 to 20, wherein the subject has one or more immunological cold tumors.

22. 22. The method of any one of claims 1 to 21, wherein the subject has one or more tumors with a low tumor mutational burden.

23. 23. The method of any one of claims 1 to 22, wherein the subject has one or more microsatellite-stable tumors.

24. 24. The method of any one of claims 1 to 23, wherein the subject has one or more tumors with low microsatellite instability.

25. 25. The method of any one of claims 1 to 24, wherein the subject has one or more tumors with a low tumor immune infiltrate.

26. The method of any one of claims 1 to 25, wherein said administering alters the tumor immune infiltrate.

27. 27. The method of claim 25 or 26, wherein the tumor immune infiltrate comprises antigen-presenting cells, myeloid cells, and lymphoid cells.

28. 28. The method of claim 27, wherein the antigen-presenting cells comprise macrophages and / or dendritic cells.

29. 28. The method of claim 27, wherein the myeloid cells include monocytes, neutrophils, myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages (TAMs).

30. 30. The method of claim 29, wherein the tumor-associated macrophages comprise M1 macrophages, M2 macrophages, and / or MARCO+ macrophages.

31. 28. The method of claim 27, wherein the lymphoid cells include T cells, B cells, NKT cells, and NK cells.

32. The method of any one of claims 1 to 31, wherein said administering modulates an anti-tumor immune response.

33. The method of any one of claims 1 to 32, wherein said administering alters the tumor microenvironment.

34. 34. The method of claim 33, wherein the tumor microenvironment comprises tumor cells, cancer stem cells, immune cells, and stroma.

35. 35. The method of claim 34, wherein the stroma comprises fibroblasts, adipocytes, endothelium, vasculature, mesenchymal stromal cells, and / or extracellular matrix.

36. 36. The method of any one of claims 1 to 35, wherein said administering converts an immunologically cold tumor into an immunologically hot tumor.

37. 37. The method of any one of claims 1 to 36, wherein said administering reduces tumor size and / or inhibits tumor growth.

38. 38. The method of any one of claims 1 to 37, wherein the subject has a cancer selected from the group consisting of brain cancer, skin cancer, eye cancer, breast cancer, prostate cancer, pancreatic cancer, lung cancer, esophageal cancer, head and neck cancer, cervical cancer, liver cancer, colorectal cancer, bone cancer, uterine cancer, ovarian cancer, bladder cancer, endometrial cancer, gastric cancer, stomach cancer, oral cancer, thyroid cancer, kidney cancer, testicular cancer, leukemia, lymphoma, and mesothelioma.

39. 39. The method of any one of claims 1 to 38, wherein the cancer therapeutic agent administered in combination with the particles is a chemotherapeutic agent selected from the group consisting of growth inhibitors, DNA replication inhibitors, kinase inhibitors, receptor tyrosine kinase inhibitors, signal transduction cascade inhibitors, angiogenesis inhibitors, metabolism inhibitors, amino acid synthesis inhibitors, selective inhibitors of oncogenic proteins, metastasis inhibitors, inhibitors of anti-apoptotic factors, apoptosis inducers, enzyme inhibitors, nucleoside signal transduction inhibitors, antibody-drug conjugates, and DNA damaging agents.

40. 39. The method of any one of claims 1-38, wherein the cancer therapeutic agent administered in combination with the particles comprises one or more biologic agents selected from the group consisting of cytokines, angiogenesis inhibitors, receptor tyrosine kinase inhibitors, immune checkpoint modulator enzymes, and monoclonal antibodies.

41. 41. The method of claim 40, wherein the cytokine is selected from the group consisting of transforming growth factor, tumor necrosis factor, interferon, and interleukin.

42. 41. The method of claim 40, wherein the immune checkpoint modulator targets programmed cell death protein 1 (PD1), programmed cell death protein ligand-1 (PD-L1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), T-cell immunoglobulin and mucin domain-containing-3 (TIM-3), lymphocyte-activation gene-3 (LAG-3), and / or TIGIT (T-cell immunoreceptor with Ig and ITIM domains).

43. 43. The method of claim 42, wherein the immune checkpoint modulator is an antibody selected from the group consisting of ipilimumab, tremelimumab, pembrolizumab, nivolumab, atezolizumab, avelumab, cemiplimab, and durvalumab.

44. 41. The method of claim 40, wherein the monoclonal antibody comprises a monospecific, bispecific, or trispecific antibody.

45. 41. The method of claim 40, wherein the monoclonal antibody comprises a bispecific T cell engager (BiTE).

46. 41. The method of claim 40, wherein the monoclonal antibody is selected from the group comprising alemtuzumab, bevacizumab, brentuximab, cetuximab, denosumab, ibritumomab, trastuzumab, panitumumab, pertuzumab, and rituximab.

47. 39. The method of any one of claims 1 to 38, wherein the cancer therapeutic administered in combination with the particles comprises one or more cell-based therapies selected from the group consisting of adoptive cell transfer, tumor-infiltrating leukocyte therapy, chimeric antigen receptor T-cell therapy (CAR-T), NK cell therapy, and stem cell therapy.

48. The method of any one of claims 1 to 38, wherein the cancer therapeutic agent administered in combination with the particles is a hormone therapy.

49. 39. The method of any one of claims 1 to 38, wherein the cancer therapeutic administered in combination with the particles comprises one or more cancer vaccines.

50. 39. The method of any one of claims 1-38, wherein the cancer therapeutic agent administered in combination with the particles is one or more immunotherapies including an oncolytic virus, an oncolytic bacterium or other bacterial composition, Bacillus Calmette-Guerin (BCG), a microbiome modulating agent, a STING pathway modulating agent, and / or a Toll-like receptor (TLR) modulating agent.

51. 1. A method of treating cancer in a subject, comprising: a. determining the immunological status of the tumor and / or the tumor mutational burden and / or the microsatellite instability status of the tumor; b. diagnosing the tumor as immunologically protected and / or immunologically cold and / or with a low tumor immune infiltrate and / or with a low tumor mutational burden and / or microsatellite stable and / or refractory; c. Administering the surface-functionalized particles alone or in combination with a cancer therapeutic agent; A method comprising:

52. 52. The method of any one of claims 1-51, wherein the particles and / or the cancer therapeutic agent are administered once a day, twice a day, three times a day, seven times a week, six times a week, five times a week, four times a week, three times a week, twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once every two months, once every three months, once every six months, or once a year.

53. 53. The method of any one of claims 1 to 52, wherein the particles are administered intravenously, orally, intranasally, intramuscularly, intraocularly, transdermally, or subcutaneously.

54. 54. The method of any one of claims 1 to 53, wherein the subject is a human.

55. 55. The method of any one of claims 1-54, wherein said administering ameliorates one or more symptoms of said cancer.

56. 56. The method of any one of claims 1 to 55, wherein the surface-functionalized particles are negatively charged particles that do not contain attached peptide or antigenic moieties or other bioactive agents.

57. The particles have a zeta potential between -80 and -30 mV and a diameter between 200 and 2000 nm.

57. The method of claim 56, wherein the PLGA particles have a diameter of 1.0 mm.

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