Immune modifying particles for treatment of cancer

Abstract

To provide immune modifying particles for treatment of cancer.SOLUTION: The present disclosure provides methods of treating cancer and proliferative diseases by using immune-modifying particles in combination with cancer therapeutics, such as checkpoint inhibitors or biologic agents, to modulate the activity of myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), neutrophils, monocytes, and / or tumor-associated stroma associated with promoting tumor growth and metastasis.SELECTED DRAWING: Figure 3A

Description

[Technical Field]

[0001] The present disclosure generally relates to methods of treating cancer and proliferative diseases by altering myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), neutrophils, and monocytes in the tumor microenvironment using immunomodulatory particles in combination with cancer therapeutics, such as checkpoint modulators, small molecules, or biologics. [Background technology]

[0002] It is now widely recognized that distinct populations of bone marrow-derived cells are part of the tumor microenvironment. These cells include monocytes, tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), and dendritic cells (Kumar et al., Trends Immunol., 37(3):208-220 (2016); Richards et al., Cancer Microenviron., 6(2):179-91 (2013)). Significant research suggests that these cells are actively recruited to tumor and metastatic sites, where they may trigger changes in the local environment to favor an immunosuppressive microenvironment (Kumar et al., Trends Immunol., 37(3):208-220(2016); Kitamura et al., Front Immunol., 8:2004(2018); Kitamura et al., J Exp Med., 212(7):1043-59(2015)). The immunosuppressive activity of these cells has been shown to promote tumor growth, proliferation, angiogenesis, metastasis, and tumor immune evasion. Furthermore, the presence of MDSCs and TAMs in the tumor microenvironment (TME) correlates with unfavorable disease outcomes (Gabrilovich et al., Nat Rev Immunol., 12(4):253-68(2012); Ouzounova et al., Nat Comm., 8:14979(2017); Marvel et al J Clin Invest., 125(9):3356-64(2015)).

[0003] In addition to immune cells, the tumor stroma also plays a role in shaping the tumor microenvironment and influencing tumor growth and progression. The cellular and molecular elements that comprise the tumor-associated stroma, including fibroblasts, mesenchymal stromal cells, adipocytes, endothelium, and extracellular matrix (ECM), have all been shown to contribute to tumorigenesis (Valkenburg et al., 2011). Tissue-resident and bone marrow mesenchymal stem cell (MSC)-derived cancer-associated fibroblasts (CAFs) have been shown to secrete growth factors and proteins that alter antitumor immune responses and promote tumor growth and metastasis (Valkenburg et al., Nat Rev Clin Oncol., 15(6):366-381(2018); Shiga et al., Cancers, 7, 2443-2548(2015)). Similarly, ECM proteins, mesenchymal stromal cells, endothelial cells, and adipocytes have been reported to weaken antitumor immunity and promote tumor progression (Lu et al., J Cell Biol., 196(4):395-406(2012); Kumar et al., Cancer Cell 32(5):654-668.e5(2017); Quante et al., Cancer Cell., 19,257-272(2011); Park et al., Endocr Rev., 32(4):550-70(2011); Young et al., Cancer Immunol Immunother., 61(10):1609-16(2012); Hida et al., Int J Mol Sci.,19(5):1272(2018)).

[0004] Although there have been great advances in the development of novel anti-cancer drugs, the efficacy of these drugs remains limited. These targets are less promising due to the fact that they are tumor rather than immunosuppressive factors in the tumor microenvironment (TME) that inhibit antitumor immune function and promote tumor progression.

[0005] Signaling through immune checkpoint receptors (e.g., programmed cell death protein 1 (PD-1) and CTLA-4) and their ligands (e.g., PD-L1) has been shown to regulate the activity of cytotoxic T cells and play a crucial role in regulating inflammatory immune responses. Importantly, several tumor types are known to hijack the PD-1 / PD-L1 and CTLA-4 immune checkpoint signaling pathways to evade T cell-mediated antitumor immune responses. For this reason, targeting immune checkpoint signaling pathways using specific inhibitors, such as monoclonal antibodies, has emerged as an attractive front-line therapeutic option for several cancers (Alsaab et al., Front Pharmacol 8:561 (2017)). However, in addition to tumor-associated stroma, the presence of myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) in the tumor microenvironment has been shown to weaken the antitumor efficacy of immune checkpoint inhibitors such as anti-PD1 monoclonal antibodies (Weber et al., Front Immunol., 9:1310, (2018); Highfill et al., Sci Transl Med., 6(237):237ra67 (2014); Zhao et al., Cancer Immunol Res., 6(12):1459-1471 (2016); Wang et al., Nat Commun., 9(1):3503 (2018)).

[0006] Immunomodulatory particles (IMPs) are negatively charged nanoparticles with immunomodulatory properties (see, e.g., U.S. Patent Publication Nos. US20150010631, US20130323319). Summary of the Invention

[0007] The present disclosure provides methods for treating cancer and proliferative diseases, including administering immunomodulatory particles that do not contain antigens or other bioactive agents and are capable of suppressing monocytes and other phagocytes in a subject in combination with a cancer therapy. Without being bound by theory, it is hypothesized that IMPs alter immunosuppressive monocyte-derived cells in the tumor microenvironment, which in turn enhances the efficacy of other cancer therapies when administered to a subject. It is suggested herein that combination therapy using IMPs and cancer therapeutics, which can not only target tumor cells but also overcome the immunosuppressive tumor microenvironment by targeting MDSCs, TAMs, neutrophils, other monocyte-derived cells, and tumor-associated stroma, may provide enhanced therapeutic benefit compared to monotherapy with cancer therapeutics, such as immune checkpoint inhibitors.

[0008] In various embodiments, the present disclosure provides methods of treating cancer in a subject, comprising administering to the subject negatively charged particles in combination with a cancer therapeutic agent, wherein the particles do not contain attached peptides or antigenic moieties or other bioactive agents, and wherein the administration alters populations of myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), neutrophils, monocytes, dendritic cells, and / or stroma at the tumor site.

[0009] In various embodiments, the present disclosure provides methods of treating cancer in a subject, comprising administering to the subject negatively charged particles in combination with a cancer therapeutic agent, wherein the particles do not contain attached peptides or antigenic moieties or other bioactive agents, and wherein the administration alters tumor-associated stroma.

[0010] In various embodiments, the present disclosure provides a method of treating cancer in a subject, comprising administering to the subject negatively charged particles in combination with a cancer therapeutic agent, wherein the particles bind The method further provides a method for treating cancer, wherein the therapeutic agent does not contain a peptide or antigenic moiety or other bioactive agent that inhibits the growth of cancer cells, and wherein administration alters stromal connective tissue, fibroblasts, endothelium, adipose tissue, extracellular matrix, pericytes, cancer stem cells, mesenchymal stem cells and / or mesenchymal stromal cells.

[0011] In various embodiments, the present disclosure provides methods of treating cancer in a subject, comprising administering to the subject negatively charged particles in combination with a cancer therapeutic agent, wherein the particles do not contain attached peptides or antigenic moieties or other bioactive agents, and wherein the administration reduces tumor size and / or tumor growth. In various embodiments, the administration induces tumor cell death, apoptosis, and / or necrosis by direct particle uptake by tumor cells.

[0012] In various embodiments, the present disclosure provides methods of treating cancer in a subject, comprising administering to the subject negatively charged particles in combination with a cancer therapeutic agent, wherein the particles do not contain attached peptides or antigenic moieties or other bioactive agents, and wherein the administration modulates an anti-tumor immune response.

[0013] In various embodiments, the present disclosure provides methods of treating cancer in a subject, comprising administering to the subject negatively charged particles in combination with a cancer therapeutic agent, wherein the particles do not contain attached peptides or antigenic moieties or other bioactive agents, and wherein the administration alters or modulates a tumor-specific immune response.

[0014] In various embodiments, the present disclosure provides methods of treating a proliferative disease in a subject, comprising administering to the subject negatively charged particles in combination with a cancer therapeutic agent, wherein the particles do not contain attached peptides or antigenic moieties or other bioactive agents, and wherein the administration alters populations of myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), dendritic cells, neutrophils, and / or monocytes at the tumor site.

[0015] In various embodiments, the present disclosure provides a method of treating a proliferative disease in a subject, comprising administering to the subject negatively charged particles in combination with a cancer therapeutic agent, wherein the particles do not contain attached peptides or antigenic moieties or other bioactive agents, and wherein the administration alters tumor-associated stroma.

[0016] In various embodiments, the present disclosure provides methods of treating a proliferative disorder in a subject, comprising administering to the subject negatively charged particles in combination with a cancer therapeutic agent, wherein the particles do not contain attached peptides or antigenic moieties or other bioactive agents, and wherein the administration alters stromal connective tissue, fibroblasts, endothelium, adipose tissue, extracellular matrix, pericytes, mesenchymal stem cells and / or mesenchymal stromal cells.

[0017] In various embodiments, the present disclosure provides methods of treating a proliferative disease in a subject, comprising administering to the subject negatively charged particles in combination with a cancer therapeutic agent, wherein the particles do not contain attached peptides or antigenic moieties or other bioactive agents, and wherein the administration reduces tumor size and / or tumor growth.

[0018] In various embodiments, the present disclosure provides methods of treating a proliferative disease in a subject, comprising administering to the subject negatively charged particles in combination with a cancer therapeutic agent, wherein the particles do not contain attached peptides or antigenic moieties or other bioactive agents, and wherein the administration modulates an anti-tumor immune response.

[0019] In various embodiments, the present disclosure provides a method of treating a proliferative disease in a subject, comprising administering to the subject negatively charged particles in combination with a cancer therapeutic agent, wherein the particles The method provides for the administration of a compound that does not contain an attached peptide or antigenic moiety or other bioactive agent, and whose administration modulates a tumor-specific immune response.

[0020] In various embodiments, the present disclosure provides methods of treating cancer or a proliferative disorder in a subject, comprising administering to the subject negatively charged particles in combination with a cancer therapeutic agent, wherein the particles do not contain attached peptides or antigenic moieties or other bioactive agents, and wherein the administration modulates cancer stem cells.

[0021] In various embodiments, the negatively charged particles are polyglycolic acid (PGA) particles, polylactic acid (PLA) particles, polystyrene particles, poly(lactic-co-glycolic acid) (PLGA) particles, diamond particles, or iron, zinc, cadmium, gold, or silver particles, or combinations thereof.

[0022] In some embodiments, the negatively charged particles are poly(lactic-co-glycolic acid) (PLGA) particles. In various embodiments, the particles comprise about 50:50, about 80:20 to about 100:0 polylactic acid:polyglycolic acid, or about 50:50 to 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 about 99:1 to about 1:99 polylactic acid:polyglycolic acid, including all values ​​and ranges therebetween.

[0023] In various embodiments, the particles are surface functionalized. In various embodiments, surface functionalization is achieved by carboxylation. In further embodiments, surface functionalization is achieved by the addition of a targeting agent. In some embodiments, the targeting agent comprises a polypeptide, an antibody, a nucleic acid, a lipid, a small molecule, a carbohydrate, and a surfactant. In various embodiments, the surface-functionalized nanoparticles preferentially target monocytes, neutrophils, macrophages, dendritic cells, T cells, B cells, NK cells, NK T 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.

[0024] In various embodiments, the particles have a zeta potential of -100 mV to -1 mV. In various embodiments, the particles have a zeta potential of -80 mV to -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 -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.

[0025] In various embodiments, the negatively charged particles have a diameter of 0.1 to 10 μm. In various embodiments, the average particle diameter is about 0.2 μm to about 2 μm, about 0.3 μm to about 5 μm, about 0.5 μm to about 3 μm, or about 0.5 μm to about 1 μm. In some embodiments, the particle diameter is about 100 to 1500 nm, about 200 to 2000 nm, about 100 to 10,000 nm, about 300 to 1000 nm, about 400 to 800 nm, or about 200 to 700 nm. In various embodiments, 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 subranges therebetween. In some embodiments, the negatively charged particles have a diameter of 400-800 nm.

[0026] In various embodiments, 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, colon cancer, colorectal cancer, rectal 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 for the methods are disclosed in the detailed description.

[0027] In various embodiments, the cancer therapeutic agent is a chemotherapeutic agent selected from the group consisting of a growth inhibitor, a DNA replication inhibitor, a kinase inhibitor, a signal transduction cascade inhibitor, an angiogenesis inhibitor, a metabolic inhibitor, an amino acid synthesis inhibitor, a selective inhibitor of oncogenic proteins, an inhibitor of metastasis, an inhibitor of anti-apoptotic factors, an inducer of apoptosis, a nucleoside signaling inhibitor, an enzyme inhibitor, and a DNA damaging agent.

[0028] In various embodiments, the cancer therapeutic comprises one or more biopharmaceuticals selected from the group consisting of cytokines, anti-angiogenic agents, enzymes, immune checkpoint modulators, and monoclonal antibodies.

[0029] In various embodiments, the cytokine is selected from the group consisting of transforming growth factors, tumor necrosis factors, interferons, and interleukins. 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, such as 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).

[0030] In various embodiments, the monoclonal antibody is a monospecific, bispecific, trispecific, or bispecific T cell engager (BiTE) antibody.

[0031] 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 costimulatory molecule) (CD278), OX40 (CD134), 41BB, GITR (glucocorticoid-induced tumor necrosis factor receptor), CD40, and CD27.

[0032] In various embodiments, the immune checkpoint modulator targets programmed cell death protein 1 (PD-1), programmed cell death protein ligand-1 (PD-L1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), T-cell immunoglobulin 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.

[0033] In various embodiments, the monoclonal antibody useful in the method 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 method is selected from the group including receptor tyrosine kinases EGFR, VEGF, VEGFR, PDGF, PDGFR, TGF-β, TGF-β-LAP, SIRP-α, CD47, CD39, CD73, and fibroblast activation protein (FAP). ) is targeted.

[0034] 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, macrophages, dendritic cells, 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.

[0035] 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.

[0036] In various embodiments, the cell-based therapy is the adoptive transfer of autologous patient-derived cells. In various embodiments, the cell-based therapy is the adoptive transfer of allogeneic donor-derived cells.

[0037] In various embodiments, the cell-based therapy is the transfer of universal, donor-derived or induced pluripotent stem cell-derived cells that are not patient-specific and are suitable for long-term storage. Such therapies are also referred to as "off-the-shelf" therapies.

[0038] In various embodiments, the cancer therapeutic agent is a hormone therapy. In various embodiments, the cancer therapeutic agent comprises one or more antibody-drug conjugates. In various embodiments, the cancer therapeutic agent comprises one or more cancer vaccines. In various embodiments, the cancer vaccine is a protein, polypeptide, nucleic acid vaccine, and / or dendritic cell vaccine.

[0039] In various embodiments, the cancer therapeutic agent is an immunotherapy selected from the group including an oncolytic virus, an oncolytic bacterium or other bacterial constituent, Bacillus Calmette-Guerin (BCG), a microbiota modulator, 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 virally derived, bacterially derived, and / or synthetically produced. In various embodiments, the immunotherapy is a STING pathway modulator.

[0040] In various embodiments, the cancer therapeutic agent comprises a viral vector or a bacterial vector.In various embodiments, the viral vector is selected from the group comprising 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 x virus, comovirus, or cucumber mosaic virus.In various embodiments, the virus is an oncolytic virus.In various embodiments, the virus is a chimeric virus, an artificial virus, a mosaic virus, or a pseudotyped virus.

[0041] Additional cancer therapeutic agents contemplated for use in the methods are presented in the detailed description.

[0042] In various embodiments, the particles and / or cancer therapeutic agent are administered 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 administered once a year.

[0043] In various embodiments, the particles and / or cancer therapeutic agents are administered intravenously, orally, intranasally, intramuscularly, intraocularly, transdermally, or subcutaneously.

[0044] In various embodiments, the subject is a mammal, hi various embodiments, the subject is a human.

[0045] In various embodiments, the 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 in the subject, tumor metastasis, and levels of inflammatory cells in the tumor or tumor microenvironment. In various embodiments, the administration reduces tumor size or tumor burden by 10%, 20%, 30% or more. In various embodiments, the administration reduces monocytes, macrophages, granulocytes, dendritic cells, and / or neutrophils in the tumor.

[0046] In various embodiments, the particles are formulated in a composition comprising a pharmaceutically acceptable carrier, diluent, or excipient. In various embodiments, the cancer therapeutic is formulated in a composition comprising a pharmaceutically acceptable carrier, diluent, or excipient. In various embodiments, the particles and the cancer therapeutic can be formulated in the same composition or in separate compositions.

[0047] Further contemplated are compositions comprising any of the above particles or cancer treatment compositions of the present disclosure, or their use in the preparation of a medicament for treating any of the disorders described herein related to inflammation and cancer and / or proliferative diseases.

[0048] It is understood that each feature or embodiment or combination described herein is a non-limiting illustrative example of any of the aspects of the invention and is therefore intended to be combinable with any other feature or embodiment or combination described herein. For example, when a feature is described using phrases such as "one embodiment," "some embodiments," "further embodiments," "specific exemplary embodiment," and / or "another embodiment," each of these classes 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. Any such feature or combination of features is compatible with any of the aspects of the present disclosure. When example values ​​falling within a range are disclosed, any of these examples are intended as endpoints of the possible range, and any and all values ​​between those endpoints are contemplated, with any and all combinations of upper and lower endpoints being envisioned. [Brief explanation of the drawings]

[0049] [Figure 1] An example treatment schedule for administration of IMP and a checkpoint inhibitor anti-PD-1 antibody is provided. [Figure 2] 1 shows that combination therapy with IMP and anti-PD-1 exhibits improved tumor growth reduction compared to monotherapy treatment in LLC cells. [Figure 3A] This figure shows the antitumor efficacy of IMP when administered alone or in combination with an anti-PD1 checkpoint inhibitor antibody. Mice were implanted with MC38 tumor cells and treated with either saline control, IMP, anti-PD1, or IMP + anti-PD1 after palpable tumors formed. Treatment with IMP inhibited tumor growth compared to control treatment. The efficacy of IMP was similar to that of anti-PD1 treatment. Treatment with IMP + anti-PD1 demonstrated synergy, enhancing tumor growth inhibition. [Figure 3B]The antitumor efficacy of IMP when administered alone or in combination with an anti-PD1 checkpoint inhibitor antibody is shown. Mice were implanted with MC38 tumor cells and, after palpable tumors formed, were treated with either saline control, IMP, anti-PD1, or IMP + anti-PD1. Treatment with IMP extended survival compared to control treatment. The survival efficacy of IMP was superior to that of anti-PD1. Treatment with IMP + anti-PD1 demonstrated synergistic effects that extended survival compared to control and each monotherapy. DETAILED DESCRIPTION OF THE INVENTION

[0050] IMPs offer an attractive opportunity to specifically target immunosuppressive monocytes in the circulation and prevent their trafficking to tumor sites where their immunosuppressive activity promotes tumor growth, proliferation, and metastasis. Combining the disruption / modification of the immunosuppressive tumor microenvironment by IMP therapy with other anti-cancer therapeutics, such as anti-PD1 monoclonal antibodies, is expected to offer significant benefits over monotherapies that target only the tumor.

[0051] Definition: Each of the publications, patent applications, patents, and other references cited herein is incorporated by reference in its entirety to the extent not inconsistent with this disclosure.

[0052] It is hereby noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include the plural references unless the context clearly dictates otherwise.

[0053] "Particle," as used herein, 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," "immunomodulatory particle," and "bead" may be used interchangeably depending on the context. Additionally, the term "particle" may be used to encompass beads and spheres.

[0054] As used herein, "surface functionalized" refers to the introduction of chemical functional groups onto the surface of a particle. Surface-functionalized particles can be prepared by free radical copolymerization of hydrophobic monomers bearing carboxylic acid, phosphoric acid, hydroxyl, sulfonic acid, phosphonic acid, and amine or ammonium groups, as well as other functional groups. A general method for making surface-functionalized nanoparticles is described, for example, in Froimowicz et al., Curr Org. Chem 17:900-912, 2013.

[0055] As used herein, "biodegradable" refers to particles comprising polymers that can undergo degradation, for example, as a result of functional groups reacting with water in solution. The term "degradation," as used herein, means 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 long periods of time, and the time required for complete degradation can be controlled. Biocompatible, biodegradable polymers useful in the present invention include polymers or copolymers of caprolactone, carbonate, amide, amino acid, orthoester, acetal, cyanoacrylate, 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, may be included in copolymers with any of the above materials to form reactive groups for conjugation with antigenic peptides and proteins or conjugation moieties. 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 may also be used in the particles described herein. For example, acrylates, ethylene vinyl acetate, acyl-substituted cellulose acetate, non-degradable urethanes, styrene, vinyl chloride, vinyl fluoride, vinyl imidazole, chlorosulfonated olefins, ethylene oxynitride ... Non-biodegradable polymers of polyethylene terephthalate (PE), vinyl alcohol, Teflon® (DuPont, Wilmington, Del.), and nylon may be employed.

[0056] "Negatively charged particles," as used herein, refer to particles that have been modified to carry a net surface charge of less than zero.

[0057] Zeta potential is the charge that develops 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), as measured by devices known in the art for calculating zeta potential, such as a NanoBrook ZetaPlus Zeta Potential Analyzer or a Malvern Zetasizer.

[0058] "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 phagocytosis / monocyte uptake of the particles from the circulatory system, for example, by interaction with scavenger receptors such as MARCO. Carboxylation of particles can be achieved using any compound that adds carboxyl groups, including, but not limited to, poly(ethylene-maleic anhydride) (PEMA).

[0059] The terms "modulate" or "alter," as used herein, refer to the modification, adjustment, or alteration of the immune response at the tumor site or tumor microenvironment. Examples of modified or altered immune responses include, but are not limited to, reducing the number or activity of immunosuppressive cells at the tumor site or tumor microenvironment, reducing immunosuppressive immune cell infiltration at the tumor site or tumor microenvironment, increasing the number of pro-inflammatory immune cells at the tumor site or tumor microenvironment, increasing pro-inflammatory immune cell infiltration at the tumor site or tumor microenvironment, improving the activity or function of pro-inflammatory immune cells at the tumor site or tumor microenvironment, increasing the number of anti-tumor immune cells at the tumor site or tumor microenvironment, increasing anti-tumor immune cell infiltration at the tumor site or tumor microenvironment, improving the anti-tumor activity or function of immune cells at the tumor site or tumor microenvironment, and / or killing of cells associated with immunosuppression.

[0060] The term "tumor microenvironment" (TME), as used herein, 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, dendritic cells, as well as lymphocytes, stroma, fibroblasts, signaling molecules, and the extracellular matrix (ECM) (Joyce et al., Science 348:74-80, 2015).

[0061] "Subject," as used herein, refers to a human or non-human animal, including a mammal or primate, to which a particle described herein is administered. Subjects can include animals such as dogs, cats, rats, mice, rabbits, horses, pigs, sheep, cows, as well as humans and other primates.

[0062] The term "therapeutically effective amount" is used to describe an amount of a target-specific composition of the present disclosure effective to ameliorate or alleviate one or more symptoms or signs of the disease or disorder being treated.

[0063] The terms "treat," "treated," "treating," and "treatment," when used in reference to the methods of the present invention, refer to the temporary or permanent, partial or complete elimination, alleviation, suppression, or amelioration of one or more clinical symptoms, signs, or progression of an event, disease, or condition. Such treatment need not be completely effective.

[0064] Checkpoint modulators Programmed cell death protein 1 (PD-1), also known as differentiation antigen 279 (CD279), is a cell surface costimulatory receptor expressed on activated T cells, B cells, and macrophages and is a component of immune checkpoint blockade (Shinohara et al., Genomics., 23(3):704, (1994); Francisco et al., Immunol Rev., 236:219, (2010)). PD-1 restricts T cell activity upon interaction with its two ligands, PD-L1 (also known as B7-H1, CD274) and PD-L2 (B7-DC, CD273) (Postow et al., J Clin Oncol., 33:9, (2015)). Interaction of PD-1 with PD-L1 and PD-L2 reduces T cell proliferation, cytokine production, and cytotoxic activity (Freeman GJ et al., J Exp Med., 192:1027-34, (2000); Brown JA et al., J Immunol., 170:1257-66, (2003)).

[0065] Two monoclonal antibodies have been approved by the U.S. Food and Drug Administration (FDA) for PD-1 blockade immunotherapy: pembrolizumab (Keytruda®, Merck Opdivo (Sharp & Dohme Corp.) is approved for use in metastatic melanoma, and nivolumab (Opdivo®, Bristol-Myers Squibb) is approved for use in metastatic melanoma and metastatic squamous non-small cell lung cancer (NSCLC). Both of these antibodies bind to the PD-1 receptor and block its interaction with its ligands, PD-L1 and PD-L2. In various embodiments, the anti-PD-1 antibody inhibits or blocks the binding of the PD-1 receptor to one or both of its ligands, PD-L1 and PD-L2.

[0066] Additional antibodies against PD-1 are described in U.S. Patent Nos. 8,735,553, 8,617,546, 8,008,449, 8,741,295, 8,552,154, 8,354,509, 8,779,105, 7,563,869, 8,287,856, 8,927,697, 8,088,905, 7,595,048, 8,168,179, 6,808,710, 7,943,743, 8,246,955, and 8,217,149.

[0067] PD-L1 inhibitors have also been shown to be effective in inhibiting solid tumors in bladder cancer, head and neck cancer, and gastrointestinal cancer (Herbst RS et al., J Clin Oncol., 31:3000(2013); Heery CR et al., J Clin Oncol., 32:5s,3064(2014); Powles T et al., J Clin Oncol., 32:5s,5011(2014); Segal NH et al., J Clin Oncol., 32:5s,3002(2014)).

[0068] CTLA-4 (cytotoxic T lymphocyte-associated protein 4) (CD152) is a protein receptor constitutively expressed on regulatory T cells but upregulated only after activation on conventional T cells. CTLA-4 is a member of the CTLA-4 / CD28; B7-1 / B7-2 costimulatory pathway of T cell signaling and APC activation (Grosso et al., Cancer Immun. 13:5, 2013) and functions as a negative regulator of T cell activation by binding to B7-1 or B7-2 and attenuating T cell responses. CTLA-4 is hypothesized to function as an immune checkpoint. CTLA-4-specific antibodies include tremelimumab and ipilimumab (Yervoy®), which are approved for the treatment of melanoma.

[0069] Lymphocyte activation gene 3 (LAG-3) (CD223) is a cell surface receptor expressed on multiple immune cells, including activated T cells. LAG-3 binds to CD8 + It is a negative regulator of T cells, and its deficiency is associated with CD8 + associated with enhanced T cell proliferation (Workman et al. al. J Immunol 174:688-695(2005)). Importantly, LAG-3 has been shown to be co-expressed with PD-1 on tumor-infiltrating lymphocytes and exhausted T cells in both preclinical tumor models and cancer patient samples, promoting tumor immune evasion (Andrews et al., Immunol Rev.,276(1):80-96(2017); Le Mercier et al. Front Immunol.,6:418(2015); Woo et al. Cancer Res.,15;72(4):917-27(2011); Zhou et al. Oncoimmunology 7(7):e1448332(2018)). LAG-3 inhibition has shown promise as a therapeutic strategy in preclinical models (Grosso et al. J Clin Invest., 117(11):3383-92(2007) and Woo et al. al. Cancer Res., 15;72(4):917-27(2011)), inhibition of LAG-3 using monoclonal antibodies, alone or in combination with other checkpoint inhibitor antibodies, such as nivolumab® and pembrolizumab®, is currently being evaluated in clinical trials for a number of cancers.

[0070] TIM-3 (T-cell immunoglobulin mucin domain-containing-3) is a transmembrane protein found to be expressed on terminally differentiated and activated T cells, where it plays a role in inhibiting T cell responses and the expression of inflammatory cytokines such as IFN-γ. TIM-3 is coexpressed with PD-1 on tumor-infiltrating lymphocytes, and its expression correlates with T cell exhaustion and suppression of T cell responses (Linhares et al., Front Immunol., 9:1909 (2018); Das et al., Immunol Rev. 276(1):97-111 (2017)). CD8 + High levels of TIM-3 expression on T cells are associated with tumor immune evasion and correlate with poor prognosis in cancer patients (Anderson et al. Immunity 17;44(5):989-1004(2016), Das et al. Immunol Rev.276(1):97-111(2017)). Furthermore, upregulation of TIM-3 in PD-1-expressing tumor-infiltrating lymphocytes has been shown to mediate resistance to anti-PD-1 therapeutics (Koyama et al., Nat Commun.,7:10501(2016)). Consistent with these findings, monoclonal antibody-mediated co-blockade of TIM-3 in conjunction with anti-PD-1 has shown promising anti-tumor effects in several preclinical tumor models (Ngiow et al., Cancer Res., 3540-3551 (2011); Anderson et al. Immunity 17;44(5):989-1004 (2016)), and several clinical trials evaluating the efficacy of anti-TIM-3 monoclonal antibodies alone or in combination with checkpoint inhibitor antibodies are currently underway.

[0071] immunomodulatory particles In some embodiments, the present disclosure provides for the use of particles having a negative zeta potential that do not contain associated antigens, peptides, or other bioactive substances in therapeutic methods. In various embodiments, the particles are surface-functionalized particles.

[0072] The particles can be formed from a variety of materials. Preferably, the particles are made of a material suitable for biological applications. For example, the particles can be made 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 made of linear or branched, substituted or unsubstituted, saturated or unsaturated, linear or crosslinked alkanyl, haloalkyl, thioalkyl, aminoalkyl, aryl, aralkyl, The particles may be composed of polyesters of alkenyl, aralkenyl, heteroaryl, or alkoxy hydroxy acids, or polyanhydrides of linear or branched, substituted or unsubstituted, saturated or unsaturated, linear or crosslinked alkanyl, haloalkyl, thioalkyl, aminoalkyl, aryl, aralkyl, alkenyl, aralkenyl, heteroaryl, or alkoxy dicarboxylic acids. In addition, the particles may be or consist of quantum dots, such as quantum dot polystyrene particles (Joumaa et al. (2006) Langmuir 22:1810-6). Particles containing a mixture of ester bonds and anhydride bonds (e.g., copolymers of glycolic acid and sebacic acid) may also be employed. For example, the particles may comprise materials including polyglycolic acid polymers (PGA), polylactic acid polymers (PLA), polysebacic acid polymers (PSA), poly(lactic-co-glycolic acid) copolymers (PLGA or PLG, the terms are interchangeable), [rho]oly(lactic-co-sebacic acid) copolymers (PLSA), poly(glycolic acid-co-sebacic acid) copolymers (PGSA), polypropylene sulfide polymers, poly(caprolactone), chitosan, etc. Other biocompatible, biodegradable polymers useful in the present invention include polymers or copolymers of caprolactone, carbonates, amides, amino acids, orthoesters, acetals, cyanoacrylates, 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, may be included in copolymers with any of the above materials to form reactive groups for conjugation to antigenic peptides and proteins or conjugation moieties.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).Biocompatible but non-biodegradable materials can also be used in the particles described herein, such as 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.

[0073] The 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 particles contemplated herein is disclosed in U.S. Patent No. 9,616,113 and International Patent Publication No. WO / 2017 / 143346. Particle properties (e.g., particle size, particle size distribution, zeta potential, morphology, hydrophobicity / hydrophilicity, polypeptide entrapment, etc.) can be controlled by manipulating the manufacturing process of PLGA particles. Particle size is affected by several factors, including but not limited to, the concentration of polymer, e.g., PLGA, the solvent used to prepare the particles, the nature of the organic phase, the surfactants used in 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 antigen or polypeptide.

[0074] In various embodiments, the particle comprises a polymer, a copolymer, a dendrimer, a diamond nanoparticle, a polystyrene nanoparticle, or a metal. In various embodiments, the particle comprises a polyglycolic acid polymer (PGA), polylactic acid (PLA), polystyrene, a copolymer of PGA and PLA (poly(lactide-co-glycolide) (PLGA)), diamond (PLGA), a liposome, PEG, cyclodextran, or a metal, such as iron. (Fe), zinc (Zn), cadmium (Cd), gold (Au) or silver (Ag).

[0075] In various embodiments, the particles are polylactic acid:polyglycolic acid or copolymers having a molar ratio of about 50:50 to about 80:20 to about 100:0 polyglycolic acid:polylactic acid, or about 50:50 to about 80:20 to about 100:0 polyglycolic acid:polylactic acid. In some embodiments, the particles are poly(lactic-co-glycolic acid) particles. In various embodiments, the particles comprise 50:50 polylactic acid:polyglycolic acid. In various embodiments, the particles comprise about 99:1 to about 1:99 polylactic acid:polyglycolic acid, including all values ​​and ranges therebetween.

[0076] In some embodiments, the zeta potential of the particles is about -100 mV to about -1 mV. In some embodiments, the zeta potential of the 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 ranges therebetween.

[0077] In some embodiments, the particles have an average diameter of about 0.1 μm to about 10 μm. In some embodiments, the particles have an average diameter of 0.2 μm to about 2 μm. In some embodiments, the particles have a diameter of about 0.3 μm to about 5 μm. In some embodiments, the particles have a diameter of about 0.5 μm to about 3 μm. In some embodiments, the particles have a diameter of about 0.5 μm to about 1 μm. In some embodiments, the particles have a diameter of about 100-1500 nm, about 200-2000 nm, about 100-10000 nm, about 300-1000 nm, about 400-800 nm, or about 200-700 nm, including all values ​​and ranges therebetween.

[0078] For administration of the particles described herein to humans or other mammals, the particles can be formulated into 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 cause allergic or other adverse reactions when administered by routes well known in the art, such as those described below. "Pharmaceutically acceptable carriers" include any and all clinically useful solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like.

[0079] The pharmaceutical compositions of the present disclosure containing the 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 appropriate for the dosage form of the present invention, but are not limited thereto. For liquid or emulsion formulations, suitable carriers include, for example, aqueous or alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Oral vehicles include sodium chloride. Intravenous vehicles may include various additives, preservatives, or fluid, nutrient, or electrolyte replenishers. Various aqueous carriers, such as sterile phosphate-buffered saline, bacteriostatic water, water, buffered water, 0.4% saline, 0.3% glycine, and the like, are suitable, and may contain other proteins, such as albumin, lipoproteins, and globulins, that have been subjected to mild chemical modifications to enhance stability.

[0080] It is contemplated that the particles may further comprise a surfactant. The surfactant may be anionic, cationic, or nonionic. Poloxamer and poloxamine 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. In addition, biodegradable and biocompatible surfactants include, but are not limited to, vitamin E TPGS (D-α-tocopheryl polyethylene glycol 1000 succinate). In certain embodiments, two surfactants are used. For example, when the particles are produced by a double emulsion method, the two surfactants may include a hydrophobic surfactant for the first emulsion and a hydrophobic surfactant for the second emulsion.

[0081] Therapeutic formulations of particles are prepared in the form of lyophilized formulations or aqueous solutions for storage by mixing particles having the desired 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 nontoxic to recipients at the dosages and concentrations employed and include buffers such as phosphates, citrates, succinates, 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; alkylparabens, such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol. 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).

[0082] Particle preparations can be stabilized by lyophilization. Addition of a cryoprotectant such as trehalose can reduce particle aggregation during lyophilization. Any suitable lyophilization and reconstitution technique can be employed. Those skilled in the art will understand that lyophilization and reconstitution can result in varying degrees of antibody activity loss, and that use levels may need to be adjusted to compensate.

[0083] How to use Provided herein are methods of treating cancer or a proliferative disorder in a subject, comprising administering negatively charged particles in combination with a cancer therapeutic agent, wherein the particles do not contain bound peptides or antigenic moieties, and wherein the administration alters populations of myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), neutrophils, dendritic cells, and / or monocytes at the tumor site.

[0084] In various embodiments, the present disclosure provides a method for treating cancer or a proliferative disorder in a subject. The present invention provides a method of treating a cancer, comprising administering to a subject a negatively charged particle as described herein in combination with a cancer therapeutic agent, wherein the administration alters tumor-associated stroma, and / or the administration alters stromal connective tissue, fibroblasts, endothelium, adipose tissue, extracellular matrix, pericytes, mesenchymal stem cells and / or mesenchymal stromal cells, and / or the administration reduces tumor size and / or tumor growth, and / or the administration modulates an anti-tumor immune response, and / or the administration modulates a tumor-specific immune response.

[0085] In various embodiments, the present disclosure provides methods of treating cancer or a proliferative disorder in a subject, comprising administering to the subject a negatively charged particle described herein in combination with a cancer therapeutic agent, wherein the administration modulates cancer stem cells.

[0086] Methods useful for determining the effects of therapeutic agents on immune cells include, but are not limited to, microscopic analysis, histological assay, cytological assay, flow cytometry, polymerase chain reaction (PCR), quantitative polymerase chain reaction (qPCR), RNA sequencing analysis (RNA-seq), single-cell RNA sequencing analysis (scRNA-seq), next-generation sequencing analysis, whole-exome sequencing analysis, epigenetic sequencing analysis, ATAC-seq, microarray analysis, and mass cytometry or CyTOF. Biomarkers that can be used alone or in combination to evaluate immune cells include cell surface markers and secreted proteins. Exemplary biomarkers 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, CD204, CD205, CD206, CD163, CD103, CD103c, F4 / 80, PD-L1, PD-L2, arginase, iNOS, ROS, These include, but are not limited to, TNF-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.

[0087] Cancer stem cells (CSCs) are considered to be a subset of tumorigenic, self-renewing, and differentiated cells found in solid and hematopoietic tumors. Several reports have documented the importance of CSCs in the pathogenesis of various tumors, tumor recurrence after therapy, and the development of therapeutic resistance. Several cell surface markers can be used to identify CSCs in solid and hematopoietic tumors. CSC markers include, but are not limited to, CD19, CD20, CD24, CD34, CD38, CD44, CD90, CD133, aldehyde dehydrogenase 1, CEACAM-6 / CD66c, BMI-1, connexin 43 / GJA1, DLL4, 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.

[0088] 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, and BRACAnalyze. s CDx, therascreen EGFR RGQ PCR kit, cobase EGFR Mutation Test v2, PD-L1 IHC 22C3 pharmDx, Abbott Real-Time IDH1, MRDx BCR-ABL Test, VENTANA ALK(D5F3)CDx Assay, Abbott Real-Time IDH2, Praxis Expanded RAS Panel, Oncomine Dx Targeted Test, LeukoStrat CDx FLT3 Mutation Assay, FoundationFocus CDx BRCA 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.

[0089] It is contemplated herein that following treatment with the negatively charged particles described herein, optionally in combination with a cancer therapeutic agent, the level of one or more biomarkers will be increased in the range of 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. After treatment with the surface-functionalized particles described herein, the level of one or more of the biomarkers is reduced by a range of 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 amount.

[0090] Exemplary diseases, conditions, or disorders that can be treated using the methods of the present invention include cancer, e.g., esophageal cancer, pancreatic cancer, metastatic pancreatic cancer, metastatic adenocarcinoma of the pancreas, bladder cancer, gastric cancer, fibrous carcinoma, glioma, malignant glioma, diffuse pontine glioma, recurrent pediatric brain neoplasm renal cell carcinoma, clear cell metastatic renal cell carcinoma, kidney cancer, prostate cancer, metastatic castration-resistant prostate cancer, stage IV prostate cancer, metastatic melanoma, melanoma, malignant melanoma, recurrent cutaneous melanoma, melanoma brain metastasis, stage I Stage IIA 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 non-small cell lung cancer; breast cancer, recurrent and metastatic breast cancer; hepatocellular carcinoma; Hodgkin lymphoma, follicular lymphoma, non-Hodgkin lymphoma; advanced 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; adult acute myeloid leukemia with t(16;16)(p13.1;q22); CBFB-MYH11; 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-RAR A; 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 Owing's sarcoma / peripheral undifferentiated neuroectodermal tumor, recurrent neuroblastoma; recurrent osteosarcoma, colorectal cancer, MSI-positive colorectal cancer; MSI-negative colorectal cancer, nasopharyngeal nonkeratinizing carcinoma; recurrent nasopharyngeal undifferentiated carcinoma, cervical adenocarcinoma; cervical adenosquamous carcinoma; cervical squamous cell carcinoma; recurrent Stage IVA cervical cancer; Stage IVB cervical cancer, anal canal squamous cell carcinoma; metastatic anal canal carcinoma; recurrent anal canal carcinoma, recurrent head and neck cancer; carcinoma, head and neck squamous cell carcinoma, head and neck squamous cell carcinoma (HNSCC), ovarian carcinoma, colon cancer, colorectal cancer, rectal cancer, gastric cancer, advanced gastrointestinal 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, bone cancer, uterine cancer, ovarian cancer, bladder cancer, stomach cancer, oral cancer, thyroid cancer, kidney cancer, testicular cancer, leukemia, lymphoma, and mesothelioma.

[0091] It is contemplated that the methods of the present invention 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%, including all values ​​and ranges therebetween.

[0092] In various embodiments, the present disclosure provides a method for reducing the number of immunosuppressive cells from a tumor site by sequestrating them in the spleen and / or liver and inducing apoptosis in a subject, the method comprising administering to the subject negatively charged particles in combination with a cancer therapeutic agent.

[0093] In various embodiments, administration of the particles in a subject prevents pathological accumulation of monocytes, macrophages, granulocytes and / or neutrophils at the tumor site or in the tumor microenvironment.

[0094] In various embodiments, the present disclosure provides methods for reducing the number of monocytes, macrophages, granulocytes, and / or neutrophils in a tumor, hi various embodiments, the number of monocytes, macrophages, granulocytes, and / or neutrophils is reduced by about 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-fold or more in the tumor.

[0095] In various embodiments, the present disclosure provides methods of altering an anti-tumor immune response in a subject, the methods comprising administering to the subject negatively charged particles in combination with a cancer therapeutic agent, hi various embodiments, the alteration increases the level or activity of anti-tumor APCs, macrophages, dendritic cells, T cells, B cells, NK T cells, and / or NK cells.

[0096] In various embodiments, administration of the particles in a subject alters the activity and / or function of immune cells at the tumor site or in the tumor microenvironment. In various embodiments, administration of the particles increases the number of inflammatory immune cells at the tumor site or in the tumor microenvironment. In various embodiments, administration of the particles in a subject enhances the anti-tumor inflammatory function or activity of immune cells at the tumor site or in the tumor microenvironment.

[0097] In various embodiments, the present disclosure provides methods of altering tumor-associated stroma, the methods comprising administering to a subject negatively charged particles in combination with a cancer therapeutic agent.

[0098] In various embodiments, administration of the particles in a subject alters fibroblasts, cancer-associated fibroblasts, adipocytes, endothelial cells, pericytes, mesenchymal stromal cells and / or ECM at the tumor site or tumor-associated stroma.

[0099] In various embodiments, the present disclosure provides methods for reducing tumor size and / or tumor growth in a subject, the methods comprising administering to the subject negatively charged particles in combination with a cancer therapeutic agent.

[0100] In various embodiments, the present disclosure provides methods for altering cancer stem cells and / or mesenchymal stem cells, the methods comprising administering to a subject negatively charged particles in combination with a cancer therapeutic agent.

[0101] In various embodiments, administration of the particles in a subject induces tumor cell death, tumor cell apoptosis, and / or tumor cell necrosis by direct particle uptake.

[0102] Administration and Dosage Contemplated herein are methods for treating a subject suffering from cancer or a proliferative disorder, comprising administering a negatively charged particle described herein in combination with a cancer therapeutic agent.

[0103] The methods of the present disclosure may be 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 administration, 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, although other routes of administration may also be used, including, but not limited to, intradermal, subcutaneous, epicutaneous, oral, intraarticular, and intrathecal administration. In various embodiments, the composition is administered to the site of a tumor.

[0104] In various embodiments, the particles are administered at a dose of about 0.1 to about 10 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, 4.0 mg / kg, 8.0 mg / kg, or 10 mg / kg. In various embodiments, the particles are administered at a dose of about 8.0 mg, 80 mg, 320 mg, 640 mg, or 800 mg. Values ​​within and between the listed dose endpoints are also contemplated. These concentrations can be administered as a single dosage form or as multiple doses.

[0105] The cancer therapeutic, if a known cancer therapeutic, is intended to 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 formulated as described herein.

[0106] The amount of immunomodulatory agent or biopharmaceutical cancer therapeutic in a given dosage can vary depending on the size of the individual receiving the therapy and the characteristics of the disorder being treated. Exemplary treatments may require administration of 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. Standard dose-response studies, first in animal models and then in clinical trials, will identify optimal dosages for specific disease conditions and patient populations.

[0107] In various embodiments, the checkpoint modulator, e.g., a PD-1 antibody, a CTLA-4 antibody, or a PD-L1 antibody, is administered at a dose range of 0.1 to 15 mg / kg, which may be administered as a single dosage form or in multiple doses.

[0108] The conditions that can be treated with 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.

[0109] In various embodiments, the particles are administered 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.

[0110] The present disclosure further contemplates a sterile pharmaceutical composition comprising a particle described herein, a cancer therapeutic agent, and a pharmaceutically acceptable carrier.

[0111] The present disclosure further contemplates a sterile pharmaceutical composition comprising the particles described herein and a pharmaceutically acceptable carrier.

[0112] The present disclosure further contemplates a sterile pharmaceutical composition comprising a cancer therapeutic agent and a pharmaceutically acceptable carrier.

[0113] 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 accompanied by suitable instructions for use, are also contemplated.

[0114] Combination therapy It is contemplated that the particles described herein may be administered in combination with a cancer therapeutic agent to treat cancer, a proliferative disorder. In various embodiments, the cancer therapeutic agent is a chemotherapeutic agent, a biologic, a cell-based therapy, a hormone therapy, an antibody-drug conjugate, an oncolytic virus, or a cancer vaccine. Hormonal therapies include tamoxifen for breast cancer, and Zoladex and aromatase inhibitors (e.g., anastrozole, letrozole, exemestane) for breast and prostate cancer. Antibody-drug conjugates include brentuximab vedotin (anti-CD30 mAB + monomethyl auristatin E) for lymphoma, adotrastuzumab emtansine (anti-Her2 / Neu + maytansinoid) for breast cancer, and inotuzumab ozogamicin (anti-CD22 + calicheamicin) for ALL. Oncolytic viruses include Imligic (Amgen®). Cancer vaccines include sipuleucel-T for prostate cancer. Several cancer vaccines are under development, including but not limited to protein, polypeptide, nucleic acid, and dendritic cell vaccines.

[0115] In various embodiments, the cancer therapeutic agent is a chemotherapeutic agent selected from the group consisting of a growth inhibitory agent, a cytotoxic agent, a DNA replication inhibitor, a kinase inhibitor, a signal transduction cascade inhibitor, an angiogenesis inhibitor, a metabolic inhibitor, an amino acid synthesis inhibitor, a selective inhibitor of oncogenic proteins, an inhibitor of metastasis, an inhibitor of anti-apoptotic factors, an inducer of apoptosis, a nucleoside signal transduction inhibitor, an enzyme inhibitor, and a DNA damaging agent.

[0116] 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 is one that does not inhibit or prevent the function of cells and / or cause destruction of cells. Non-cytotoxic agents can include agents that can be activated to become cytotoxic.

[0117] Chemotherapeutic agents contemplated for use in the methods of the present disclosure include, but are not limited to, those listed in Table I. [Table 1-1] [Table 1-2] [Table 1-3]

[0118] It is also contemplated that the cancer therapeutic agent may include one or more biologics, such as cytokines, angiogenesis inhibitors, immune checkpoint modulators, and monoclonal antibodies. 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, and IL-15. , IL-17, IL-18, IL-21, members of the transforming growth factor beta superfamily such as 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).

[0119] Biopharmaceuticals, such as immune checkpoint modulators, target PD1, PD-L1, CTLA-4, TIMP-3, LAG-3, and / or TIGIT (a 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 Corp) 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 carcinoma), cemiplimab (Libtayo®) (approved for cutaneous squamous cell carcinoma).

[0120] In various embodiments, the monoclonal antibody useful in the method 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 method targets receptor tyrosine kinases EGFR, VEGF, VEGFR, PDGF, PDGFR, TGF-β, TGF-β-LAP, SIRP-α, CD47, CD39, CD73, and fibroblast activation protein (FAP).

[0121] Biopharmaceuticals include monoclonal antibodies that are monospecific, bispecific, trispecific, or bispecific T cell engagers (BiTEs). Monoclonal antibodies useful for treating cancer include bevacizumab (Avastin®, Genetech), an antibody against VEGF-A; erlotinib (Tarceva®, Genentech and OSI Pharmaceuticals), a tyrosine kinase inhibitor that acts on EGFR; dasatinib (Sprycel®, Bristol-Myers Squibb Company), an oral Bcr-Abl tyrosine kinase inhibitor; IL-21; pegylated IFN-α2b; axitinib (Inlyta®, Pfizer, Inc.), a tyrosine kinase inhibitor; and trametinib (Mekinist®, GlaxoSmithKline), an MEK inhibitor (Philips and Atkins, Int Immunol., 27(1):39-46 (2015), which is incorporated herein by reference). Bispecific antibodies useful for treating cancer, including blinatumomab and catumaxomab, are described in Krishnamurthy et al. (Pharmacol Ther. 2018 May;185:122-134) and Yu et al. (J. Hematol Oncol 2017,10:155).

[0122] The method also enables the cancer therapeutic agent to include one or more cell-based therapies, such as adoptive cell transfer, tumor-infiltrating leukocyte therapy, chimeric antigen receptor T-cell therapy, NK cell therapy, and stem cell therapy.

[0123] The cancer therapeutic agent may be one or more immunotherapies, e.g., oncolytic viruses, oncolytic bacteria or other bacterial constructs, microbiota modulating agents, bacillus Calmette-Guerin, TLR agonists, microorganisms, or the like. Contemplated therapeutic agents include plasmodesmata modulators, STING pathway modulators, and cancer vaccines. The methods also enable the cancer therapeutic to include a viral or bacterial vector. 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 viral, bacterial, and / or synthetic.

[0124] 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.

[0125] 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, as long as there is an overlap in the period during which the agents exert their therapeutic effect. Simultaneous or sequential administration is contemplated, as is administration on different days or weeks.

[0126] It is contemplated that the particles and the cancer therapeutic agent may be given simultaneously in the same formulation. It is further contemplated that the agents may be given in separate formulations and given concurrently, where concurrently means that the agents are given within 30 minutes of each other.

[0127] In another embodiment, the cancer therapeutic agent is administered prior to the administration of the particle composition.Preceding administration refers to administering the cancer therapeutic agent within a range from one week before particle treatment to 30 minutes before particle administration.It is also contemplated that the cancer therapeutic agent is administered after the administration of the particle composition.Later administration refers to administration from 30 minutes after particle treatment to one week after administration.

[0128] kit In a further aspect, the present disclosure includes kits containing one or more compounds or compositions packaged to facilitate their use in practicing the methods of the present disclosure. In one embodiment, such kits include a compound or composition described herein (e.g., a composition comprising particles alone or in combination with a cancer therapeutic agent) in a container, such as a sealed bottle or container, with a label affixed to the container or within the packaging that describes the use of the compound or composition in practicing the method. Preferably, the compound or composition is packaged in a unit dosage form. The kit may further include a device suitable for administering the particles, cancer therapeutic agent, or composition by a particular route of administration or for conducting a screening assay. Preferably, the kit includes a label that describes the use of the inhibitor composition.

[0129] Further aspects and details of the present disclosure will become apparent from the following examples, which are intended to be illustrative rather than limiting. [Example]

[0130] Example 1 Targeted tumor therapy using IMP therapy in combination with immune checkpoint inhibitors and anti-PD-1 monoclonal antibodies To determine the antitumor efficacy of IMP therapy in combination with PD-1 monoclonal antibody (mAb), Kumar et al. (Cancer Cell 32, 654- Orthotopic or syngeneic tumor models will be established using 6-8 week old C57BL / 6 mice as described in [668, (2017)]. For example, mice will be implanted with LLC (Lewis lung carcinoma), MC38, or EL4 (9,10-dimethyl-1,2-benzanthracene-induced mouse thymoma) cells via subcutaneous injection and randomly divided into four treatment groups as follows:

[0131] Group 1: Control treatment (n=7)

[0132] Group 2: IMP only (n=10)

[0133] Group 3: Anti-PD1 mAb only (n=10)

[0134] Group 4: IMP+anti-PD1 mAb (n=7).

[0135] 24 hours after subcutaneous injection of tumor cells, mice are treated with IMP (1 mg intravenously) and / or anti-PD1 mAb (100 μg intraperitoneally) according to the following treatment schedule, an example of which is shown in Figure 1:

[0136] Day 0: Subcutaneous injection of LLC or EL4 cells. [Table A]

[0137] Mice receiving control treatment receive intravenous and / or intraperitoneal saline instead of IMP and anti-PD1 mAb. Tumor growth in each treatment group is assessed by measuring tumor area using standard calipers on days 0, 14, 19, 22, 25, and 29.

[0138] Mice receiving saline treatment lost an area of ​​500–600 mm by the end of the treatment period.2 In contrast, combination therapy with both IMP and anti-PD1 mAb is expected to result in a reduction in tumor growth and its complete elimination. In the combination treatment group, tumors remained at 5-10 mm after 40 days. 2 It is possible that the area will only grow to this extent.

[0139] Mice treated with IMP alone or anti-PD1 mAb alone are expected to show a moderate effect, with tumor growth significantly slowed compared to saline-treated mice. These groups will grow to 100-150 mm2 in area toward the end of treatment. 2 It is hypothesized that tumors in the range of 200-250 mm would be expected in these treatment groups. 2 Although tumors may grow to an area of ​​100 mm, they are not expected to reach the size of tumors seen in saline-treated mice.

[0140] In the first experiment, mice were inoculated with LLC tumor cells by subcutaneous injection on day 0. LLC tumor cells (ATCC®) were cultured in 10% FBS (Atlanta Biologicals), 5 nM glutamine, 25 mM HEPES, and 1% antibiotic (In The cells were maintained in monolayer culture in DMEM (Corning CellGro®, 10-013-CV) supplemented with 0.5 × 10 6 A suspension of LLC cells was prepared and injected into the right lower flank of 7- to 10-week-old C57BL / 6 mice. On day 1, mice were randomly assigned to treatment groups and treated according to the following schedule: Tumor growth in each treatment group was assessed by measuring tumor area using a standard caliper on days 0, 14, 19, 22, 25, and 29. PLGA particles were used as implants. [Table B]

[0141] The results are shown in Figure 2. As expected, saline-treated (control) mice developed large tumors by day 29. Monotherapy with anti-PD1 alone or IMP alone demonstrated comparable antitumor efficacy, with moderate tumor growth inhibition compared to saline treatment. Combination therapy with IMP and anti-PD1 mAb resulted in a synergistic effect, enhancing tumor growth inhibition compared to saline treatment or monotherapy with IMP or anti-PD-1 mAb.

[0142] Example 2 Therapeutic efficacy of IMP administered alone or in combination with anti-PD1 To determine the effect of IMP combination treatment on a syngeneic tumor model, MC38 (colon adenocarcinoma) tumor cells were implanted subcutaneously into the flanks of 6-8 week-old C57BL / 6 mice. MC38 mouse tumor cells were cultured in monolayers in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 2 mM L-glutamine and maintained at 37°C in a tissue culture incubator with 5% CO2. Palpable tumors (approximately 100 mm2) were observed. 3 After the formation of the grafts (size), animals were randomly assigned to one of four treatment groups as follows:

[0143] Group 1: Control treatment (n=10)

[0144] Group 2: IMP (n=10)

[0145] Group 3: anti-PD1 (n=10)

[0146] Group 4: IMP+anti-PD1 (n=10).

[0147] 1 × 10 cells resuspended in 0.1 mL of serum-free DMEM 6 A single cell suspension of 95% viable cells was prepared and injected into the right lower flank of the animals. Treatment was performed when the tumor size was approximately 100 mm 3 IMP (PLGA particles) (1 mg) was administered by intravenous (iv) injection and anti-PD-1 (100 μg) was administered by intraperitoneal (ip) injection according to the following treatment schedule: [Table C]

[0148] Tumor growth was assessed by measuring tumor size in two dimensions using calipers according to the formula V = 0.5 × a × b, where a and b are the long and short diameters of the tumor, respectively. 2 The tumor volume was calculated using the following formula. The tumor size was measured in mm 3 It is expressed as:

[0149] As shown in Figure 3A, treatment with IMP led to potent inhibition of tumor growth compared to control treatment. The efficacy of IMP was comparable to that of anti-PD1 treatment. Combined treatment with IMP and anti-PD1 demonstrated synergy, leading to enhanced tumor growth inhibition compared to either monotherapy. Treatment with IMP led to prolonged survival of MC38 tumor-bearing mice, reflecting its effect on tumor growth. The survival efficacy of IMP was superior to that of anti-PD1, and combination therapy with IMP and anti-PD1 demonstrated a synergistic effect resulting in increased survival compared to either monotherapy (Figure 3B).

[0150] Numerous modifications and variations in the present disclosure set forth in the illustrative examples above are anticipated to occur to those skilled in the art, and therefore, only such limitations should be placed on the present disclosure as appear in the appended claims.

Claims

1. 1. A method of treating cancer or a proliferative disease in a subject, comprising: administering to said subject negatively charged particles in combination with a cancer therapeutic agent. wherein the particles comprise Attaching peptides or antigenic moieties or other bioactive agents The method, wherein the method does not contain

2. 10. The method of claim 1, wherein the administration alters myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), neutrophils and / or monocytes at the tumor site.

3. 3. The method of claim 1 or claim 2, wherein said administration reduces tumor size and / or tumor growth in said subject.

4. The method of any one of claims 1 to 3, wherein said administering modulates an anti-tumor immune response.

5. The method according to any one of claims 1 to 4, wherein the administration alters cancer stem cells and / or mesenchymal stem cells.

6. The method of any one of claims 1 to 5, wherein said administration alters tumor-associated stroma.

7. The method of any one of claims 1 to 6, wherein said administration alters fibroblasts, adipocytes, endothelial cells, mesenchymal stromal cells and / or ECM in said tumor-associated stroma.

8. 10. The method of any one of the preceding claims, wherein the negatively charged particles are polyglycolic acid polymers (PGA), polylactic acid (PLA), polystyrene particles, or poly(lactic-co-glycolic acid) (PLGA) particles, diamond particles, or iron, zinc, cadmium, gold, or silver particles.

9. 10. The method of any one of the preceding claims, wherein the negatively charged particles are poly(lactic-co-glycolic acid) (PLGA) particles.

10. The particles are polylactic acid:polyglycolic acid from about 50:50, about 80:20 to about 100:0; or 10. The method of claim 8 or claim 9, comprising from about 50:50 to about 80:20 to about 100:0 polyglycolic acid:polylactic acid.

11. 10. The method of any one of the preceding claims, wherein the particles comprise 50:50 polylactic acid:polyglycolic acid.

12. 10. The method of any one of the preceding claims, wherein the particles are carboxylated.

13. 10. A method according to any one of the preceding claims, wherein the particles have a zeta potential of between -100mV and -1mV.

14. 10. A method according to any one of the preceding claims, wherein the particles have a zeta potential of between -80mV and -30mV.

15. 10. Any one of the preceding claims, wherein the negatively charged particles have a diameter of 0.1 μm to 10 μm. The method described in paragraph .

16. 10. The method of any one of the preceding claims, wherein the negatively charged particles have a diameter of between 400 nm and 800 nm.

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

18. 10. The method of any one of the preceding claims, wherein 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, inhibitors of metastasis, inhibitors of anti-apoptotic factors, apoptosis inducers, enzyme inhibitors, nucleoside signal transduction inhibitors and DNA damaging agents.

19. 18. The method of any one of claims 1 to 17, wherein the cancer therapeutic agent comprises one or more biopharmaceuticals selected from the group consisting of cytokines, enzymes, angiogenesis inhibitors, immune checkpoint modulators, and monoclonal antibodies.

20. 20. The method of claim 19, wherein the cytokine is selected from the group consisting of transforming growth factors, tumor necrosis factors, interferons, and interleukins.

21. 20. The method of claim 19, 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 mucin domain-containing 3 (TIM-3), lymphocyte-activation gene-3 (LAG-3), and / or TIGIT (T-cell immunoreceptor with Ig and ITIM domains).

22. 22. The method of claim 21, wherein the immune checkpoint modulator is an antibody selected from the group consisting of ipilimumab, tremelimumab, pembrolizumab, nivolumab, atezolizumab, avelumab, cemiplimab, and durvalumab.

23. 20. The method of claim 19, wherein the monoclonal antibody is a monospecific, bispecific, trispecific, or bispecific T cell engager (BiTE).

24. 20. The method of claim 19, wherein the monoclonal antibodies comprise alemtuzumab, bevacizumab, brentuximab, cetuximab, denosumab, ibritumomab, trastuzumab, panitumumab, pertuzumab, and rituximab.

25. 20. The method of claim 19, wherein the monoclonal antibody targets a receptor tyrosine kinase, EGFR, VEGF, VEGFR, PDGF, PDGFR, TGF-β, TGF-β-LAP, SIRP-α, CD47, CD39, CD73 and / or fibroblast activation protein (FAP).

26. 18. The method of any one of claims 1 to 17, wherein the cancer therapeutic agent comprises one or more cell-based therapies selected from the group consisting of adoptive cell transfer, intratumoral infiltrating leukocyte therapy, chimeric antigen receptor T-cell therapy (CAR-T), NK cell therapy, and stem cell therapy.

27. The method according to any one of claims 1 to 17, wherein the cancer treatment drug is a hormone therapy.

28. The method of any one of claims 1 to 17, wherein the cancer therapeutic comprises one or more antibody-drug conjugates.

29. The method of any one of claims 1 to 17, wherein the cancer therapeutic agent comprises one or more cancer vaccines.

30. 18. The method of any one of claims 1 to 17, wherein the cancer therapeutic agent is an immunotherapy comprising an oncolytic virus, an oncolytic bacterium or other bacterial construct, Bacillus Calmette-Guerin (BCG), a microbiota modulator, a STING pathway modulator, and / or a toll-like receptor (TLR) agonist.

31. 10. The method of any one of the preceding claims, wherein the particles and / or the cancer therapeutic agent are administered 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.

32. 10. The method of any one of the preceding claims, wherein the particles are administered intravenously, orally, intranasally, intramuscularly, intraocularly, transdermally or subcutaneously.

33. 10. The method of any one of the preceding claims, wherein the subject is a human.

34. 10. The method of any one of the preceding claims, wherein said administering ameliorates one or more symptoms of said cancer, a proliferative disorder.

35. 35. The method of claim 34, wherein the one or more symptoms are selected from the group consisting of tumor size or tumor burden in the subject, tumor metastasis, and levels of inflammatory cells in the tumor.

36. 36. The method of claim 35, wherein said administering reduces said tumor size or tumor burden by 10%, 20%, 30% or more.

37. 35. The method of claim 34, wherein said administration reduces the number of monocytes, macrophages, granulocytes and / or neutrophils in said tumor.

38. 10. The method of any one of the preceding claims, wherein the particles are formulated into a composition comprising a pharmaceutically acceptable carrier, diluent or excipient.

39. 10. The method of any one of the preceding claims, wherein the cancer therapeutic agent is formulated in a composition comprising a pharmaceutically acceptable carrier, diluent or excipient.

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