Immunomodulatory nanoparticle conjugatesand uses thereof

Abstract

Disclosed herein are immunomodulatory nanoparticle conjugates useful for therapeutics and / or diagnostics. In certain embodiments, each immunomodulatory nanoparticle conjugate comprises a nanoparticle that has a plurality of cellular receptor- and / or antigen- targeting ligands (e.g., wherein the plurality of cellular receptor- and / or antigen- targeting ligands targets domains that are expressed on the surface of tumor cells, e.g., wherein the plurality of cellular receptor- and / or antigen- targeting ligands targets MUC16ecto domain expressed on the surface of tumor cells, e.g., wherein the plurality of cellular receptor- and / or antigen- targeting ligands comprises anti-MUC16, e.g., wherein the plurality of cellular receptor- and / or antigen- targeting ligands comprises a plurality of immunomodulatory / stimulatory ligands (of the same species or of differing species) (e.g., a plurality of oligodeoxynucleotides (ODNs) (e.g., an ODN containing an unmethylated cytosine-phosphate-guanine (CpG) motif (or CpG ODNs)) conjugated to the nanoparticle) conjugated to the nanoparticle.

Description

IMMUNOMODULATORY NANOPARTICLE CONJUGATES AND USES THEREOFCross Reference to Related Applications

[0001] This application claims priority to and benefit of U.S. Provisional Patent Application No. 63 / 542,564 filed October 5, 2023, the contents of which are incorporated by reference herein in its entirety.Field of the Invention

[0002] This invention relates generally to combinatorial methods and compositions for the treatment of a disease or condition [e.g., cancer, e.g., ovarian cancer, e.g., relapsed and refractory ovarian cancer, e.g., breast cancer, e.g., colon cancer, e.g., lung cancer, e.g., pancreas cancer, endometrial cancer, e.g., benign conditions such as ovarian cysts, endometriosis, pancreatitis, inflammatory disease (e.g., pelvic inflammatory disease), early pregnancy, hepatitis, cirrhosis, or pericarditis] in subjects. More specifically, in certain embodiments, the invention relates to enhanced particle-driven therapies for combinatorial treatment of a disease or condition by targeting mucin- 16 (MUC16) in combination with engineered cellular therapy (e.g., CAR T cell therapy).Summary of the Invention

[0003] The present disclosure describes immunomodulatory nanoparticles and their conjugates, the latter enhancing cellular uptake, as agents with intrinsic therapeutic capabilities that, among other things, (1) modulate the tumor microenvironment (TME) toward a pro-inflammatory phenotype, (2) increase immune cell activation and cytotoxicity in the TME, and / or (3) target cancer cells directly for cell death through the mechanism of fcrroptosis and / or other cell death processes. Unlike otherparticle-based tools, the development of a single highly specialized multi-therapeutic ultrasmall particle-based agent that can be delivered systemically and can specifically engage ferroptosis, while also activating separable, ferroptosis-independent pro-inflammatory and cytotoxic responses in the TME is unexpected. These therapeutic activities can, in turn, potentially reduce immunosuppressive factors that limit the normal functioning of effector cells, underscoring an advantage described by the present disclosure: leveraging these death-inducing and pro-inflammatory mechanisms for treating relapsed and refractory solid tumors and their metastases, and which also have relevance for treating human disease. Activation of Type I / II interferons and other immunostimulatory pathways using immunomodulatory nanoparticle conjugates described herein, therefore, presents a unique approach to transform a suppressive TME into one that can enhance CAR T cell cytotoxicity, improve efficacy, and expand the window for treating these aggressive tumors.

[0004] In one aspect, the invention is directed to a method of treating a disease or condition [e.g., cancer, e.g., ovarian cancer, e.g., relapsed and refractory ovarian cancer, e.g., breast cancer, e.g., colon cancer, e.g., lung cancer, e.g., pancreas cancer, endometrial cancer, e.g., benign conditions such as ovarian cysts, endometriosis, pancreatitis, inflammatory disease (e.g., pelvic inflammatory disease), early pregnancy, hepatitis, cirrhosis, or pericarditis], the method comprising: administering to a subject a first pharmaceutical composition comprising: an immunomodulatory nanoparticle conjugate, wherein the immunomodulatory nanoparticle conjugate comprises: (1) a nanoparticle; and (2) a plurality of cellular receptor- and / or antigen- targeting ligands (e.g., comprising one or more species of targeting ligands) [e.g., wherein the plurality of cellular receptor- and / or antigen- targeting ligands targets domains that arc expressed on the surface of tumor cells, e.g., wherein the plurality of cellular receptor- and / or antigen- targeting ligands targets a MUC16ectodomain expressed on the surface of tumor cells,e.g., wherein the plurality of cellular receptor- and / or antigen- targeting ligands comprises an anti- MUC16 antibody fragment (e.g., anti-MUC16 scFv) (e.g., conjugated to the nanoparticle), e.g., wherein the plurality of cellular receptor- and / or antigen- targeting ligands comprises a plurality of immunomodulatory / stimulatory ligands (of the same species or of differing species) (e.g., a plurality of oligodeoxynucleotides (ODNs) (e.g., an ODN containing an unmethylated cytosine-phosphate-guanine (CpG) motif (or CpG ODNs)) (e.g., conjugated to the nanoparticle)], wherein the nanoparticle has a diameter (e.g., average diameter) no greater than 20 nanometers (e.g., as measured by dynamic light scattering (DLS) in aqueous solution, e.g., as measurement by fluorescence correlation spectroscopy, e.g., saline solution) (e.g., wherein the average nanoparticle diameter is from 1 to 20 nm, e.g., from 1 to 15 nm, e.g., from 1 to 10 nm, e.g., from 1 to 8 nm, e.g., from 4 to 10 nm , e.g., from 4 to 8 nm) (e.g., wherein the immunomodulatory nanoparticle conjugate has an average diameter no greater than 50 nm, e.g., no greater than 40 nm, e.g., no greater than 30 nm, e.g., no greater than 20 nm, e.g., no greater than 15 nm, e.g., no greater than 10 nm) (e.g., wherein the nanoparticle comprises silica), wherein the subject also receives a second pharmaceutical composition comprising engineered cells (e.g., engineered immune cells, e.g., CAR T cells, e.g., wherein the engineered cells are or comprise MUC16-targeting engineered cells, e.g., wherein the engineered cells secrete IL-12, e.g., wherein the engineered cells are or comprise MUC16-targeting, IL- 12 secreting CAR T cells) (e.g., wherein the first pharmaceutical composition is being used to enhance activity of the second pharmaceutical composition) (e.g., wherein the first pharmaceutical composition activates anti-tumor immune responses in the tumor microenvironment (TME), e.g., without off-target effects) (e.g., wherein the first pharmaceutical composition is administered before, after, or at the same time as the second pharmaceutical composition).

[0005] In another aspect, the invention is directed to a method of treating a disease or condition [e.g., cancer, e.g., ovarian cancer, e.g., relapsed and refractory ovarian cancer, e.g., breast cancer, e.g., colon cancer, e.g., lung cancer, e.g., pancreas cancer, endometrial cancer, e.g., benign conditions such as ovarian cysts, endometriosis, pancreatitis, inflammatory disease (e.g., pelvic inflammatory disease), early pregnancy, hepatitis, cirrhosis, or pericarditis], the method comprising: administering to a subject (1) a first pharmaceutical composition comprising (a) an immunomodulatory nanoparticle conjugate, wherein the immunomodulatory nanoparticle conjugate comprises: (i) a nanoparticle; and (ii) a plurality of cellular receptor- and / or antigen- targeting ligands (e.g., comprising one or more species of targeting ligands) (e.g., wherein the plurality of cellular receptor- and / or antigen- targeting ligands targets domains that are expressed on the surface of tumor cells (e.g., wherein the plurality of cellular receptor- and / or antigen- targeting ligands targets MUC16ect0domain expressed on the surface of tumor cells (e.g., wherein the plurality of cellular receptor- and / or antigen- targeting ligands comprises an anti-MUC16 antibody fragment) conjugated to the nanoparticle), wherein the nanoparticle has a diameter (e.g., average diameter) no greater than 20 nanometers (e.g., as measured by dynamic light scattering (DLS) in aqueous solution, e.g., as measurement by fluorescence correlation spectroscopy, e.g., saline solution) (e.g., wherein the average nanoparticle diameter is from 1 to 20 nm, e.g., from 1 to 15 nm, e.g., from 1 to 10 nm, e.g., from 1 to 8 nm, e.g., from 4 to 10 nm , e.g., from 4 to 8 nm) (e.g., wherein the immunomodulatory nanoparticle conjugate has an average diameter no greater than 50 nm, e.g., no greater than 40 nm, e.g., no greater than 30 nm, e.g., no greater than 20 nm, e.g., no greater than 15 nm, e.g., no greater than 10 nm) (e.g., wherein the nanoparticle comprises silica); and (2) a second pharmaceutical composition comprising engineered immune cells (e.g., CAR T cells, e.g., wherein the engineered cells are or comprise MUC16-targeting engineered cells, e.g., wherein theengineered cells expies s / secrete IL-12, e.g., wherein the engineered cells are or comprise MUC16- targeting, IL- 12 secreting CAR T cells) (e.g., wherein the first pharmaceutical composition is being used to enhance activity of the second pharmaceutical composition) (e.g., wherein the first pharmaceutical composition activates anti-tumor immune responses in the tumor microenvironment (TME), e.g., without off-target effects) (e.g., wherein the first pharmaceutical composition is administered before, after, or at the same time as the second pharmaceutical composition).

[0006] In another aspect, the invention is directed to a method of treating a disease or condition [e.g., cancer, e.g., ovarian cancer, e.g., relapsed and refractory ovarian cancer, e.g., breast cancer, e.g., colon cancer, e.g., lung cancer, e.g., pancreas cancer, endometrial cancer, e.g., benign conditions such as ovarian cysts, endometriosis, pancreatitis, inflammatory disease (e.g., pelvic inflammatory disease), early pregnancy, hepatitis, cirrhosis, or pericarditis], the method comprising: administering to a subject a first pharmaceutical composition comprising engineered immune cells (e.g., CAR T cells, e.g., wherein the engineered cells are or comprise MUC16-targeting engineered cells, e.g., wherein the engineered cells expres s / secrete IL-12, e.g., wherein the engineered cells are or comprise MUC16- targeting, IL- 12 secreting CAR T cells), wherein the subject also receives a second pharmaceutical composition comprising an immunomodulatory nanoparticle conjugate, wherein the immunomodulatory nanoparticle conjugate comprises: (1) a nanoparticle; and (2) a plurality of cellular receptor- and / or antigen- targeting ligands (e.g., comprising one or more species of targeting ligands) [e.g., wherein the plurality of cellular receptor- and / or antigen- targeting ligands targets domains that are expressed on the surface of tumor cells, e.g., wherein the plurality of cellular receptor- and / or antigen- targeting ligands targets MUC16ectodomain expressed on the surface of tumor cells, e.g., wherein the plurality of cellular receptor- and / or antigen- targeting ligands comprises an anti-MUC16 antibody fragment) (e.g., conjugated to the nanoparticle), e.g., wherein the plurality of cellular receptor- and / or antigen- targeting ligands comprises a plurality of immunomodulatory / stimulatory ligands (of the same species or of differing species) (e.g., a plurality of oligodeoxynucleotides (ODNs) (e.g., an ODN containing an unmethylated cytosine-phosphate-guanine (CpG) motif (or CpG ODNs)) (e.g., conjugated to the nanoparticle)], wherein the nanoparticle has a diameter (e.g., average diameter) no greater than 20 nanometers (e.g., as measured by dynamic light scattering (DLS) in aqueous solution, e.g., as measurement by fluorescence correlation spectroscopy, e.g., saline solution) (e.g., wherein the average nanoparticle diameter is from 1 to 20 nm, e.g., from 1 to 15 nm, e.g., from 1 to 10 nm, e.g., from 1 to 8 nm, e.g., from 4 to 10 nm , e.g., from 4 to 8 nm) (e.g., wherein the immunomodulatory nanoparticle conjugate has an average diameter no greater than 50 nm, e.g., no greater than 40 nm, e.g., no greater than 30 nm, e.g., no greater than 20 nm, e.g., no greater than 15 nm, e.g., no greater than 10 nm) (e.g., wherein the nanoparticle comprises silica) (e.g., wherein the second pharmaceutical composition is being used to enhance activity of the first pharmaceutical composition) (e.g., wherein the second pharmaceutical composition activates anti-tumor immune responses in the tumor microenvironment (TME), e.g., without off-target effects) (e.g., wherein the first pharmaceutical composition is administered before, after, or at the same time as the second pharmaceutical composition).

[0007] In certain embodiments, the plurality of cellular receptor- and / or antigen- targeting ligands comprises one or more species of targeting ligands.

[0008] In certain embodiments, the plurality of cellular receptor- and / or antigen- targeting ligands targets domains that are expressed on the surface of tumor cells.

[0009] In certain embodiments, the plurality of cellular receptor- and / or antigen- targeting ligands comprises a plurality of immunomodulatory / stimulatory ligands (of the same species or of differing species).

[0010] In certain embodiments, the plurality of immunomodulatory / stimulatory ligands comprises or is a plurality of oligodeoxynucleotides (ODNs).

[0011] In certain embodiments, the plurality of ODNs comprises or is a plurality of ODNs containing an unmethylated cytosine-phosphate-guanine (CpG) motif (or CpG ODNs)) (e.g., conjugated to the nanoparticle).

[0012] In certain embodiments, the plurality of ODNs (e.g., CpG ODNs) bind to Toll-like receptor 9 (TLR9) (e.g., thereby promoting expression of co-stimulatory molecules, secretion of inflammatory cytokines, and / or development of CD8+ T cell responses).

[0013] In certain embodiments, the plurality of cellular receptor- and / or antigen- targeting ligands is a member selected from the set consisting of a recombinant antibody fragment (Fabs), a single chain variable fragment (scFv), and a single domain antibody (sdAb) fragment (e.g., anti- MUC16 antibody fragment).

[0014] In certain embodiments, the plurality of cellular receptor- and / or antigen- targeting ligands is a single chain variable fragment (scFv) (e.g., anti-MUC16 scFv).

[0015] In certain embodiments, the plurality of cellular receptor- and / or antigen- targeting ligands comprises an anti-MUC16 antibody fragment (e.g., anti-MUC16 scFv) (e.g., conjugated to the nanoparticle).

[0016] In certain embodiments, the antibody fragment or scFv comprises a first linker sequence(e.g., between variable light sequence and variable heavy chain sequence) (e.g., wherein the first linkersequence comprises one or more glycine residues; e.g., wherein the first linker sequence comprises one or more serine residues).

[0017] In certain embodiments, the first linker sequence comprises a sequence having at least 80% identity to, e.g., at least 85% identity to, e.g., at least 90% identity to, e.g., at least 95% identity to, e.g., at least 98% identity to, e.g., at least 100% identity to GGGGSGGGGSGGGGS (SEQ ID NO: 3).

[0018] In certain embodiments, the scFv comprises a second linker sequence attached (e.g., covalently or non-covalently) to the C-terminus end of the scFv. In certain embodiments, the second linker sequence comprises a cysteine residue. In certain embodiments, the second linker sequence comprises a sequence having at least 85% identity to, e.g., at least 100% identity to GGSCGSG (SEQ ID NO: 3).

[0019] In certain embodiments, the plurality of cellular receptor- and / or antigen- targeting ligands comprises species having a sequence of at least 50 (e.g., at least 100; e.g., at least 150; e.g., at least 200; e.g., at least 250) amino acid sequences with at least 80% identity to a portion of SEQ ID NO: 1 (e.g., wherein the portion is a variable region) (e.g., at least 85% identity, e.g., at least 90% identity, e.g., at least 91% identity, e.g., at least 92% identity, e.g., at least 93% identity, e.g., at least 94% identity, e.g., at least 95% identity, e.g., at least 96% identity, e.g., at least 97% identity, e.g., at least 98% identity, e.g., at least 99% identity, e.g., 100% sequence identity to the portion of SEQ ID NO: 1).

[0020] In certain embodiments, the plurality of cellular receptor- and / or antigen- targeting ligands comprises a species having a sequence of at least 50 (e.g., at least 100; e.g., at least 150; e.g., at least 200; e.g., at least 250) amino acid sequences with at least 80% identity to a portion of SEQ ID NO:2 (e.g., wherein the portion is a variable region) (e.g., at least 85% identity, e.g., at least 90%identity, e.g., at least 91% identity, e.g., at least 92% identity, e.g., at least 93% identity, e.g., at least 94% identity, e.g., at least 95% identity, e.g., at least 96% identity, e.g., at least 97% identity, e.g., at least 98% identity, e.g., at least 99% identity, e.g., 100% sequence identity to the portion of SEQ ID NO: 2).

[0021] In certain embodiments, the plurality of cellular receptor- and / or antigen- targeting ligands is covalently or non-covalently bonded to the nanoparticle (e.g., via a linker or covalently or non-covalently bonded directly to the nanoparticle, or associated with the nanoparticle or a moiety surrounding the nanoparticle, e.g., via van der Waals forces).

[0022] In certain embodiments, the nanoparticle is coated with an organic polymer (e.g., polyethylene glycol (PEG)) (e.g., wherein the composition comprises a chelator).

[0023] In certain embodiments, the plurality of cellular receptor- and / or antigen- targeting ligands comprises from 1 to 75 cellular receptor- and / or antigen- targeting ligands (e.g., of a single species) (e.g., 1 to 50 ligands, e.g., 2 to 50, e.g., 5 to 30 ligands, e.g., about 6 to 8 ligands, e.g., about 1 to 5 ligands, e.g., about 1 to 2 ligands) conjugated to the nanoparticle.

[0024] In certain embodiments, the immunomodulatory nanoparticle conjugate comprises a radiotherapeutic agent (e.g., a radiolabel, e.g., a radioisotope, e.g., 225-Actinium) (e.g., formulated at a therapeutically effective dose, e.g., at least 0.27 microcuries / kg, e.g., at least 1.0 microcuries / kg, e.g., at least 2 microcuries / kg, e.g., at least 2.7 microcuries / kg, e.g., relative to a microenvironment control).

[0025] In certain embodiments, the nanoparticle comprises silica.

[0026] In certain embodiments, the nanoparticle comprises a silica core.

[0027] In certain embodiments, the nanoparticle comprises a dye-encapsulated core and a silica shell surrounding the core.

[0028] In certain embodiments, the nanoparticle comprises a fluorescent compound within the core.

[0029] In certain embodiments, the average nanoparticle diameter is from 1 to 20 nm, e.g., from 1 to 15 nm, e.g., from 1 to 10 nm, e.g., from 1 to 8 nm, e.g., from 4 to 10 nm, e.g., from 4 to 8 nm.

[0030] In certain embodiments, the immunomodulatory nanoparticle conjugate has an average diameter no greater than 50 nm, e.g., no greater than 40 nm, e.g., no greater than 30 nm, e.g., no greater than 20 nm, e.g., no greater than 15 nm, e.g., no greater than 10 nm).

[0031] In certain embodiments, the nanoparticle comprises a silica composition such that ferroptosis is not induced (e.g., ferroptosis is switched “off’).

[0032] In certain embodiments, the nanoparticles have a silica composition such that ferroptosis may be induced (e.g., ferroptosis is not switched “off’).

[0033] In certain embodiments, the immunomodulatory nanoparticle conjugate further comprises a therapeutic agent (e.g., wherein the therapeutic agent is attached to the nanoparticle, or to the plurality of cellular receptor- and / or antigen- targeting ligands, or to both the nanoparticle and the plurality of cellular receptor- and / or antigen- targeting ligands, e.g., wherein the attachment is covalent or non-covalent).

[0034] In certain embodiments, the immunomodulatory nanoparticle conjugate further comprises a radiotherapeutic agent (e.g., formulated at a therapeutically effective dose, e.g., at least 0.27 microcuries / kg, e.g., at least 1.0 microcuries / kg, e.g., at least 2 microcuries / kg, e.g., at least 2.7 microcuries / kg, e.g., relative to a microenvironment control).

[0035] In certain embodiments, the nanoparticle further comprises a targeting ligand.

[0036] In certain embodiments, the first pharmaceutical composition and / or second pharmaceutical composition further comprises a carrier.

[0037] In certain embodiments, the engineered immune cells comprises CAR T cells.

[0038] In certain embodiments, the engineered cells are MUC 16-targeting engineered cells.

[0039] In certain embodiments, the engineered cells secrete IL-12.

[0040] In certain embodiments, the engineered cells are or comprise MUC 16-targeting, IL- 12 secreting CAR T cells.

[0041] In certain embodiments, the first pharmaceutical composition is being used to enhance activity of the second pharmaceutical composition.

[0042] In certain embodiments, the first pharmaceutical composition activates anti-tumor immune responses in the tumor microenvironment (TME), e.g., without off-target effects.

[0043] In certain embodiments, the first pharmaceutical composition is administered before, after, or at the same time as the second pharmaceutical composition.

[0044] In certain embodiments, the method increases CD8+ / Tregs ratio at least about 5-fold (e.g., at least about 10-fold, e.g., at least about 20-fold, e.g., at least about 30-fold, e.g., at least about 40-fold, e.g., at least about 50-fold, e.g., at least about 60-fold, e.g., at least about 70-fold, e.g., at least about 80-fold, e.g., at least about 90-fold, e.g., at least about 100-fold) (e.g., said increase in the CD8+ / Tregs ratio due at least in part to administration of the first pharmaceutical composition).

[0045] In certain embodiments, the method increases co-stimulatory molecules (e.g., CD40, e.g., CD86) on Ml macrophages and / or (overall) dendritic cell (DC) population (e.g., said increase(s) due at least in part to administration of the first pharmaceutical composition).

[0046] In certain embodiments, the method results in a M1 / M2 ratio greater than 0.5 (e.g., greater than 1.0, e.g., greater than 1.25, e.g., greater than 1.5) (e.g., over a period of four days) (e.g., due at least in part to administration of the first pharmaceutical composition).

[0047] In certain embodiments, the method decreases myeloid-derived suppressor cells (MDSCs) by a factor of at least 2, e.g., at least 3 (e.g., over a period of 10 days) (e.g., said decrease due at least in part to administration of the first pharmaceutical composition).

[0048] In another aspect, the invention is directed to a immunomodulatory nanoparticle conjugate comprising: a nanoparticle; and a plurality of cellular receptor- and / or antigen- targeting ligands (e.g., anti-MUC16 antibody fragment, oligodeoxynucleotides (ODNs), e.g., CpG-ODNs) conjugated to the nanoparticle, wherein the nanoparticle has a diameter (e.g., average diameter) no greater than 20 nanometers (e.g., as measured by dynamic light scattering (DLS) in aqueous solution, e.g., as measurement by fluorescence correlation spectroscopy, e.g., saline solution) (e.g., wherein the average nanoparticle diameter is from 1 to 20 nm, e.g., from 1 to 15 nm, e.g., from 1 to 10 nm, e.g., from 1 to 8 nm, e.g., from 4 to 10 nm , e.g., from 4 to 8 nm) (e.g., wherein the immunomodulatory nanoparticle conjugate has an average diameter no greater than 50 nm, e.g., no greater than 40 nm, e.g., no greater than 30 nm, e.g., no greater than 20 nm, e.g., no greater than 15 nm, e.g., no greater than 10 nm) (e.g., wherein the nanoparticle comprises silica),

[0049] for use with an engineered cellular therapy (e.g., engineered immune cellular therapy, e.g., comprising engineered cells such as CAR T cells, e.g., wherein the engineered cells are or comprise MUC16-targeting engineered cells, e.g., wherein the engineered cells secrete IL-12, e.g., wherein the engineered cells are or comprise MUC16-targeting, IL-12 secreting CAR T cells) (e.g., wherein the immunomodulatory nanoparticle conjugate is being used to enhance activity of theengineered cellular therapy) (e.g., wherein the immunomodulatory nanoparticle conjugate activates anti-tumor immune responses in the tumor microenvironment (TME), e.g., without off-target effects) (e.g., wherein the immunomodulatory nanoparticle conjugate is administered before, after, or at the same time as the engineered cellular therapy).

[0050] In another aspect, the invention is directed a composition (e.g., pharmaceutical composition, e.g., a radiotherapeutic composition) comprising an immunomodulatory nanoparticle conjugate and engineered cells (e.g., engineered immune cells, e.g., CAR T cells, e.g., wherein the engineered cells are or comprise MUC16-targeting engineered cells, e.g., wherein the engineered cells secrete IL-12, e.g., wherein the engineered cells are or comprise MUC16-targeting, IL-12 secreting CAR T cells) for use in engineered cellular therapy (e.g., CAR T cell therapy) (e.g., wherein the immunomodulatory nanoparticle conjugate is being used to enhance activity of the engineered cells) (e.g., wherein the immunomodulatory nanoparticle conjugate activates anti-tumor immune responses in the tumor microenvironment (TME), e.g., without off-target effects).

[0051] In certain embodiments, the composition is formulated at a therapeutically effective dose (e.g., at least 0.27 microcuries / kg, e.g., at least 1.0 microcuries / kg, e.g., at least 2 microcuries / kg, e.g., at least 2.7 microcuries / kg, e.g., relative to a microenvironment control).

[0052] In another aspect, the invention is directed to a treatment comprising: (a) a therapeutically effective amount of an immunomodulatory nanoparticle conjugate for use in combination with engineered cells (e.g., engineered immune cells, e.g., CAR T cells, e.g., wherein the engineered cells are or comprise MUC16-targeting engineered cells, e.g., wherein the engineered cells secrete IL-12, e.g., wherein the engineered cells are or comprise MUC16-targeting, IL-12 secretingCAR T cells); or (b) a therapeutically effective amount of engineered cells (e.g., engineeredimmune cells, e.g., CAR T cells, e.g., wherein the engineered cells are MUC16-targeting engineered cells, e.g., wherein the engineered cells secrete IL- 12, e.g., wherein the engineered cells are or comprise MUC16-targeting, IL-12 secreting CAR T cells) for use in combination with an immunomodulatory nanoparticle conjugate, for use in a method of treating a disease or condition [e.g., cancer, e.g., ovarian cancer, e.g., relapsed and refractory ovarian cancer, e.g., breast cancer, e.g., colon cancer, e.g., lung cancer, e.g., pancreas cancer, endometrial cancer, e.g., benign conditions such as ovarian cysts, endometriosis, pancreatitis, inflammatory disease (e.g., pelvic inflammatory disease), early pregnancy, hepatitis, cirrhosis, or pericarditis] or preventing disease or condition occurrence or recurrence in a subject (e.g., wherein the immunomodulatory nanoparticle conjugate is being used to enhance activity of the engineered cells) (e.g., wherein the immunomodulatory nanoparticle conjugate activates anti-tumor immune responses in the tumor microenvironment (TME), e.g., without off-target effects) (e.g., wherein the immunomodulatory nanoparticle conjugate is administered before, after, or at the same time as the engineered cells).

[0053] In another aspect, the invention is directed to a radiotherapeutic composition (e.g., a pharmaceutical radiotherapeutic composition) comprising an immunomodulatory nanoparticle conjugate and engineered cells (e.g., engineered immune cells, e.g., CAR T cells, e.g., wherein the engineered cells are or comprise MUC16-targeting engineered cells, e.g., wherein the engineered cells secrete IL-12, e.g., wherein the engineered cells are or comprise MUC16-targeting, IL-12 secreting CAR T cells) for use in engineered cellular therapy (e.g., CAR T cell therapy), wherein the immunomodulatory nanoparticle conjugate activates anti-tumor immune responses in the tumor microenvironment (TME) and wherein the nanoparticle conjugate comprises a radiotherapeutic agent(e.g., a radiotherapy).

[0054] In certain embodiments, the composition is formulated at a therapeutically effective dose. In certain embodiments, the therapeutically effective dose is at least 0.27 microcuries / kg, e.g., at least 1.0 microcuries / kg, e.g., at least 2 microcuries / kg, e.g., at least 2.7 microcuries / kg, e.g., relative to a microenvironment control.

[0055] In another aspect, the invention is directed to an immunomodulatory nanoparticle conjugate comprising: a nanoparticle; and a plurality of immunomodulatory / stimulatory ligands (of the same species or of differing species) conjugated to the nanoparticle, wherein the nanoparticle has a diameter (e.g., average diameter) no greater than 20 nanometers (e.g., as measured by dynamic light scattering (DLS), e.g., as measurement by fluorescence correlation spectroscopy, in aqueous solution, e.g., saline solution) (e.g., wherein the average nanoparticle diameter is from 1 to 20 nm, e.g., from 1 to 15 nm, e.g., from 1 to 10 nm, e.g., from 1 to 8 nm, e.g., from 4 to 10 nm , e.g., from 4 to 8 nm) (e.g., wherein the immunomodulatory nanoparticle conjugate has an average diameter no greater than 50 nm, e.g., no greater than 40 nm, e.g., no greater than 30 nm, e.g., no greater than 20 nm, e.g., no greater than 15 nm, e.g., no greater than 10 nm).

[0056] In certain embodiments, the plurality of immunomodulatory ligands comprises or is a plurality of pattern recognition receptors (PRR) agonist moieties. In certain embodiments, the plurality of PRR agonist moieties is or comprises toll-like receptors (TLRs) agonist moieties, RIG-1 like receptors (RLRs) agonist moieties, NOD-like receptors (NLRs) agonist moieties, C-type lectin receptors (CLRs) agonist moieties, or cytosolic DNA sensors (e.g., a Stimulator of Interferon Genes (STING)).

[0057] In certain embodiments, the plurality of immunomodulatory ligands comprises or is a plurality of oligodeoxynucleotides (ODNs) (e.g., an ODN containing an unmethylated cytosine-phosphate-guanine (CpG) motif (or CpG ODNs)). In certain embodiments, the plurality of CpG ODNs bind to Toll-like receptor 9 (TLR9) (e.g., thereby promoting expression of co- stimulatory molecules, secretion of inflammatory cytokines, and / or development of CD8+ T cell responses).

[0058] In certain embodiments, the plurality of ODNs comprises or is a plurality of ODNs containing an unmethylated cytosine-phosphate-guanine (CpG) motif (or CpG ODNs)), and wherein the CpG motif comprises or is a sequence according to 5’-TCCATGACGTTCCTGACGTT-3’ (SEQ ID NO: 4).

[0059] In certain embodiments, the plurality of immunomodulatory / stimulatory ligands is covalently or non-covalently bonded to the nanoparticle (e.g., via a linker or covalently or non- covalently bonded directly to the nanoparticle, or associated with the nanoparticle or a moiety surrounding the nanoparticle, e.g., via van der Waals forces).

[0060] In certain embodiments, the nanoparticle is coated with an organic polymer (e.g., polyethylene glycol (PEG)) (e.g., wherein the composition comprises a chelator).

[0061] In certain embodiments, the plurality of immunomodulatory / stimulatory ligands comprises from 1 to 75 immunomodulatory / stimulatory ligands (e.g., of a single species) (e.g., 1 to 50, e.g., 2 to 50, e.g., e.g., 5 to 30 ligands, e.g., about 6 to 8 ligands, e.g., about 1 to 5 ligands, e.g., about 1 to 2 ligands) conjugated to the nanoparticle.

[0062] In certain embodiments, the immunomodulatory nanoparticle conjugate comprises a radiolabel.

[0063] In certain embodiments, nanoparticle comprises silica.

[0064] In certain embodiments, the nanoparticle comprises a silica core.

[0065] In certain embodiments, the nanoparticle comprises a dye-encapsulating core and a silica shell surrounding the core.

[0066] In certain embodiments, the nanoparticle comprises a fluorescent compound within the core.

[0067] In certain embodiments, the immunomodulatory nanoparticle conjugate further comprises a therapeutic agent (e.g., wherein the therapeutic agent is attached to the nanoparticle, or to the plurality of immunomodulatory / stimulatory ligands, or to both the nanoparticle and the plurality of immunomodulatory / stimulatory ligands, e.g., wherein the attachment is covalent or non-covalent).

[0068] In certain embodiments, the plurality of immunomodulatory / stimulatory ligands conjugated to the nanoparticle enhance immunomodulatory properties of the nanoparticle (e.g., properties present in the nanoparticle even in the absence of the immunomodulatory ligands).

[0069] In certain embodiments, the immunomodulatory ligands are immunostimulatory (e.g., the ligands enhance ongoing immune responses).

[0070] In another aspect, the invention is directed to a method of treating a disease or condition, the method comprising administering (e.g., systemically) to a subject a pharmaceutical composition comprising a immunomodulatory nanoparticle conjugate as described herein (e.g., to target a particular type of tissue, e.g., cancer, e.g., ovarian cancer, e.g., relapsed and refractory ovarian cancer, e.g., breast cancer, e.g., colon cancer, e.g., lung cancer, e.g., pancreas cancer, endometrial cancer, e.g., benign conditions such as ovarian cysts, endometriosis, pancreatitis, inflammatory disease (e.g., pelvic inflammatory disease), early pregnancy, hepatitis, cirrhosis, or pericarditis.

[0071] In certain embodiments, the immunomodulatory nanoparticle conjugate modulates the tumor microenvironment.

[0072] In certain embodiments, the nanoparticle does or does not modulate the tumor microenvironment.

[0073] In certain embodiments, the plurality of immunomodulatory / stimulatory ligands does or does not modulate the tumor microenvironment.

[0074] In certain embodiments, the plurality of immunomodulatory / stimulatory ligands activate one or more pattern recognition receptors (PRR) (e.g., thereby activating different pro-inflammatory transcription factors (e.g. nuclear factor kappa B or NF-KB) (e.g., thereby promoting expression of costimulatory molecules, secretion of inflammatory cytokines, and development of CD8+ T cell responses), augment proinflammatory responses, and / or innate and adaptive immune responses beyond that of the particle itself.

[0075] In certain embodiments, the PRR is or comprises toll-like receptors (TLRs), RIG-1 like receptors (RLRs), NOD-like receptors (NLRs), C-type lectin receptors (CLRs), and cytosolic DNA sensors, such as the Stimulator of Interferon Genes (STING).

[0076] In certain embodiments, the method comprises administering a therapeutic radioisotope (e.g., wherein the therapeutic radioisotope is attached to a second nanoparticle having a diameter (e.g., average diameter) no greater than 20 nanometers (e.g., as measured by dynamic light scattering (DLS) in aqueous solution, e.g., as measurement by fluorescence correlation spectroscopy, e.g., saline solution) (e.g., wherein the radioisotope is attached to the nanoparticle or the second nanoparticle via a second chelator)) (e.g., wherein the second nanoparticle has a diameter from 1 to 20 nm, e.g., from 1 to 15 nm, e.g., from 1 to 10 nm, e.g., from 1 to 8 nm, e.g., from 4 to 10 nm , e.g., from 4 to 8 nm).

[0077] In certain embodiments, the pharmaceutical composition further comprises a carrier.

[0078] Elements of embodiments involving one aspect of the invention (e.g., methods) can be applied in embodiments involving other aspects of the invention (e.g., systems), and vice versa.Brief Description of the Drawing

[0079] The foregoing and other objects, aspects, features, and advantages of the present disclosure will become more apparent and better understood by referring to the following description taken in conjunction with the accompanying drawing, in which:

[0080] FIG. 1 is an illustrative schematic of MUC16 showing a retained MUC16ectodomain and a 4H11 mAb binding site of a retained portion of MUC16 (arrow).

[0081] FIG. 2A shows results from adult female wild type C57BL6 mice, which was inoculated with IxlO7ID8-Muc16ectocells intraperitoneally (i.p.) and treated for 35 days and later with 2xl06indicated CAR T cells i.p., *p < 0.02.

[0082] FIG. 2B shows results from adult female wild type C57BL6 mice, which was inoculated with IxlO7ID8-Muc16ectocells intraperitoneally (i.p.) and treated for 42 days, and later with 2xl06indicated CAR T cells i.p., *p < 0.02.

[0083] FIG. 3 shows concentration and time-dependent gene expression modulation by C dots in ID8 cells.

[0084] FIG. 4A shows concentration-dependent uptake of PEG-Cy5-C’ dots in ovarian cancer ID8 cells over a 24h period by flow cytometry (FC).

[0085] FIG. 4B shows a control image of no C’ dots in ovarian cancer ID8 cells by confocal microscopy. Hoechst (blue). Insert, scale bar = 10pm.

[0086] FIG. 4C shows internalization of PEG-Cy5-C’ dots in ovarian cancer ID8 cells by confocal microscopy. Hoechst (blue), C dot (red). Insert, scale bar = 10pm.

[0087] FIG. 4D shows HPLC absorbance graphs of anti-hMUC16 scFv-PEG-C’ dots (upper graph) and unmodified C’ dots (lower graph).

[0088] FIG. 4E shows a histogram of ovarian cancer ID8 cells’ uptake of MUC16-PEG-Cy5-C’ dots relative to PEG-Cy5-C’ dots and saline vehicle using FC.

[0089] FIG. 4F shows a bar graph corresponding to the histogram of FIG. 4E of ovarian cancer ID8 cells’ uptake of MUC16-PEG-Cy5-C’ dots relative to PEG-Cy5-C’ dots and saline vehicle using FC.

[0090] FIG. 5A shows a HPLC graph of anti-MUC16 scFv-PEG-C’ dots (upper panel) and PEG-Cy5-C’ dots (lower panel).

[0091] FIG. 5B shows concentration-dependent uptake at 24h of PEG-Cy5- and anti-MUC16- scFv-PEG-Cy5-C’ dots in MUC16+ versus MUC16- cells by flow cytometry (FC). All studies were performed in triplicate; mean+ / -s.e.m. computed by unpaired Student t-tests (*P<0.05; **P<0.01; ***P<0.005).

[0092] FIG. 5C shows particle internalization of non-targeted C’ dots by confocal microscopy (insert, scale bar=10pm). Hoechst stain (blue), C dot (red).

[0093] FIG. 5D shows particle internalization of MUC16-targeted C’ dots by confocal microscopy (insert, scale bar=10pm). Hoechst stain (blue), C dot (red).

[0094] FIG. 5E shows a cytokine release profiling of particle-exposed MP cells vs saline vehicle. All studies were performed in triplicate; mean+ / -s.e.m. computed by unpaired Student t-tests(*P<0.05; **P<0.01; ***P<0.005).

[0095] FIG. 6A shows C’ dot modulation of the transcriptome based on genotypic status through the use of bulk RNA-seq analyses of murine ID8(L), MP, and MPB1 cells exposed to C’ dots (15pM, 24h), normalized to saline vehicle. KEGG pathway analysis shows representative upregulated and downregulated pathways per established protocols. Each quadrant reflects n=3 biological replicates.

[0096] FIG. 6B shows C’ dot modulation of the transcriptome based on genotypic status through the use of bulk RNA-seq analyses of murine ID8(L), MP, and MPB1 cells exposed to C’ dots (15pM, 24h), normalized to saline vehicle. Reactome pathway analysis shows representative upregulated and downregulated pathways per established protocols. Each quadrant reflects n=3 biological replicates.

[0097] FIG. 7A is a heatmap showing time-dependent modulation of gene expression profiles by 15pM C’ dot- versus saline-exposed ID8(L), ID8(H), MP, and MPB1 cells over 72h using RT-qPCR for selected classes of genes.

[0098] FIG. 7B shows cytokine / chemokine profiles derived from culture media of particle- vs saline-exposed cells (from FIG. 7A) treated after 24h.

[0099] FIG. 7C shows cell cycle analyses of 1D8(L), MP, and MPB1 cells at 24h using phosphorylated histone 3 (PPH3 / PI) staining and FC for assessing cell cycle phase. All studies were performed in triplicate and mean+ / -s.e.m. computed by unpaired Student t-tests (*=p<.05, **=p<.01, ***=p<_005).

[0100] FIG. 8A shows viability of luc+ ID8(L), ID8(H), MP, MPB1 cells 24h after incubation with (i) media alone (“Control”), (ii) C’ dots alone (15 pM, 24h; “C’ dots”) alone or (iii) followed byco-culture with anti-MUC16-CAR T cells (1 : 1 E:T) cell ratio) for 24h; “C’ dots + CAR T cells”), or (iv) anti-MUC16-CAR T cells alone (E:T 1 : 1; “CAR T Cells”).

[0101] FIG. 8B shows comparative cell-specific viability for combination treatments from FIG. 8A.

[0102] FIG. 8C shows viability of luc+ ID8(L) 24h after incubation following the same treatments as in FIG. 8A, except using MUC16-targeting C’ dots.

[0103] FIG. 8D shows a bar graphs of cytokine IFN-y concentrations derived from co-culture supernatants. Data reflects analyses of four biological replicates / group, mean+s.e.m. (1-way ANOVA per cell line per cytokine (***P<0.005; ****P<0.0001; boxes).

[0104] FIG. 8E shows a bar graphs of cytokine TNF-a concentrations derived from co-culture supernatants. Data reflects analyses of four biological replicates / group, mean+s.e.m. (1-way ANOVA per cell line per cytokine (***P<0.005; ****P<0.0001; boxes).

[0105] FIG. 8F shows a bar graphs of IL-2 concentrations derived from co-culture supernatants. Data reflects analyses of four biological replicates / group, mean+s.e.m. (1-way ANOVA per cell line per cytokine (***P<0.005; ****P<0.0001; boxes).

[0106] FIG. 9A shows optical BL1 imaging (y-axis on color scale shows luminescence) in a luc+ ID8 model 7 days post-i.v. injection of PEG-Cy5-C’ dots or saline vehicle (control) with correlative ex vivo evaluation (y-axis on color scale shows radiant efficiency.

[0107] FIG. 9B shows fluorescence imaging (y-axis on color scale shows radiant efficiency) in a luc+ ID8 model 7 days post-i.v. injection of PEG-Cy5-C’ dots or saline vehicle (control) with correlative ex vivo evaluation (y-axis on color scale shows radiant efficiency).

[0108] FIG. 9C shows BLI of ex vivo organs harvested from control animals (y-axis shows luminescence).

[0109] FIG. 9D shows BLI of ex vivo organs harvested from animals injected with C’ dots (y- axis shows luminescence).

[0110] FIG. 9E shows fluorescence imaging of ex vivo organs harvested from control mice (y- axis on color scale shows radiant efficiency).

[0111] FIG. 9F shows fluorescence imaging of ex vivo organs harvested from PEG-Cy5-C’ dot- injected mice (y-axis on color scale shows radiant efficiency).

[0112] FIG. 9G shows a maximum intensity project (MIP) PET image in a tumor-bearing (T) mouse following iv injection of89Zr-PEG-C’ dots (y-axis color scale shows radiant efficiency).

[0113] FIG. 9H shows ex vivo fluorescence imaging of vehicle- and particle-exposed ascites (y-axis shows radiant efficiency).

[0114] FIG. 10A shows an exemplary experimental schema.

[0115] FIG. 10B shows gene expression profiles for tumor measured as fold changes (n=4) with respect to ID8(L) mice treated with vehicle (CT, control) from cohorts of ID8(L) mice (n=3 mice / cohort) treated with (i) triply injected i.v. C’ dots on Days 0, 3, and 6 (36 nmoles; 60pM), (ii) single-dose i.p. -injected CAR T cells, or (iii) a combination of triply injected C’ dots and CAR T cells, the latter given 4 days after the final C’ dot dose.

[0116] FIG. 10C shows gene expression profiles for spleen measured as fold changes (n=4) with respect to ID8(L) mice treated with vehicle (CT, control) from cohorts of ID8(L) mice (n=3 mice / cohort) treated with (i) triply injected i.v. C’ dots on Days 0, 3, and 6 (36 nmoles; 60pM), (ii)single-dose i.p. -injected CAR T cells, or (iii) a combination of triply injected C’ dots and CAR T cells, the latter given 4 days after the final C’ dot dose.

[0117] FIG. 10D shows cytokine profiles of ascites specimens.

[0118] FIG. 10E shows persistence of CAR T cells in blood.

[0119] FIG. HA shows single cell dissociates of ID8(L) tumor specimens stained for surface markers at 4 or 10 days after CAR T cell or vehicle administration for CD8+ T cells.

[0120] FIG. 11B shows single cell dissociates of ID8(L) tumor specimens stained for surface markers at 4 or 10 days after CAR T cell or vehicle administration for CAR T cells.

[0121] FIG. 11C shows single cell dissociates of ID8(L) tumor specimens stained for surface markers at 4 or 10 days after CAR T cell or vehicle administration for Tregs.

[0122] FIG. HD shows single cell dissociates of ID8(L) tumor specimens stained for surface markers at 4 or 10 days after CAR T cell or vehicle administration for CXCR3+CAR T cells.

[0123] FIG. HE shows the CD8+ / Treg ratio of single cell dissociates of ID8(L) tumor specimens stained for surface markers at 4 or 10 days after CAR T cell or vehicle administration.

[0124] FIG. 12A shows %CD 1 lb+F4 / 80+CD40+for CT, C’ dots, CAR T, and Combo treatments at 4 and 10 days after administration of treatment.

[0125] FIG. 12B shows %iNOS+of CD 1 lb+F4 / 80+for CT, C’ dots, CAR T, and Combo treatments at 4 and 10 days after administration of treatment.

[0126] FIG. 12C shows M1 / M2 for CT, C’ dots, CAR T, and Combo treatments at 4 and 10 days after administration of treatment.

[0127] FIG. 12D shows %Ly6C+Ly6G+of CD 1 lb+for CT, C’ dots, CAR T, and Combo treatments at 4 and 10 days after administration of treatment.

[0128] FIG. 12E shows %Argl+of CDllb+F4 / 80+for CT, C’ dots, CAR T, and Combo treatments at 4 and 10 days after administration of treatment.

[0129] FIG. 12F shows %CDllc+MHCII+for CT, C’ dots, CAR T, and Combo treatments at 4 days after administration of treatment.

[0130] FIG. 13A shows a UMAP of droplet-based scRNA-seq profiles from 10 samples (C’ dot treated n=5, and saline controls n=5), colored by cluster number.

[0131] FIG. 13B shows a volcano plot of differentially expressed genes between C’ dot and vehicle-treated samples in tumor cells.

[0132] FIG. 13C shows a volcano plot of differentially expressed genes between C’ dot and vehicle-treated samples in macrophages.

[0133] FIG. 13D shows a volcano plot of differentially expressed genes between C’ dot and vehicle-treated samples in DCs.

[0134] FIG. 13E shows a volcano plot of differentially expressed genes between C’ dot and vehicle-treated samples in T cells.

[0135] FIG. 13F shows gene set enrichment analysis of genes significantly up- (red-blue) or down-regulated (green) in tumor cells; y-axis shows the enrichment score, x-axis shows rank in ordered data set for enrichment profile (green), hits (black), or ranking metric scores (light grey).

[0136] FIG. 13G shows gene set enrichment analysis of genes significantly up- (red-blue) or down-regulated (green) in tumor cells; y-axis shows the enrichment score, x-axis shows rank in ordered data set for enrichment profile (green), hits (black), or ranking metric scores (light grey).

[0137] FIG. 13H shows gene set enrichment analysis of genes significantly up- (red-blue) or down-regulated (green) in tumor cells; y-axis shows the enrichment score, x-axis shows rank in ordered data set for enrichment profile (green), hits (black), or ranking metric scores (light grey).

[0138] FIG. 14 shows elimination of antigen escape variants using MUC16-targeting C’ dots and MUC16+ CAR T cells.

[0139] FIG. 15A shows a C’ dot rendering (left panel), surface conjugated with one CpG-ODN to compare sizes and encapsulating Cy5 dye, according to an illustrative embodiment.

[0140] FIG. 15B shows characterization of CpG-PEG-Cy5-C' dots using a gel permeation chromatography (GPC) elugram.

[0141] FIG. 15C shows characterization of CpG-PEG-Cy5-C' dots using a fluorescence correlation spectroscopy (FCS) correlation curve with fit.

[0142] FIG. 16A shows BMDCs cultured with media alone, lOOnM PEG-C’ dots, or lOOnM CpG-C’ dots for 48 h and analyzed by flow cytometry for % dendritic cells (%DCs). *p<0.05, ***p<0.005, ****p<0.001; by one-way ANOVA.

[0143] FIG. 16B shows BMDCs cultured with media alone, lOOnM PEG-C’ dots, or lOOnM CpG-C’ dots for 48 h and analyzed by flow cytometry for dendritic cells (DCs) positive for MHC11. *p<0.05, ***p<0.005, ****p<0.001; by one-way ANOVA.

[0144] FIG. 16C shows BMDCs cultured with media alone, lOOnM PEG-C’ dots, or lOOnM CpG-C’ dots for 48 h and analyzed by flow cytometry for overall MHCII. *p<0.05, ***p<0.005, ****p<0.001; by one-way ANOVA.

[0145] FIG. 16D shows BMDCs cultured with media alone, lOOnM PEG-C’ dots, or lOOnMCpG-C’ dots for 48 h and analyzed by flow cytometry for TLR9 expression. *p<0.05, ***p<0.005, ****p<0.001; by one-way ANOVA.

[0146] FIG. 16E shows RT-qPCR of BMDCs treated as in FIG. 15 A.

[0147] FIG. 16F shows a bead-based assay of media from FIG. 15A for cytokine and chemokine production.

[0148] FIG. 16G shows BMDCs exposed to CpG-C’ dots (red) and lOkD dextran (green) showing colocalization (yellow) by super-resolution microscopy. Scale bai- 10pm.

[0149] FIG. 16H shows a phagocytosis assay of NP-exposed BMDCs co-cultured with B 16- GM cells for 48h. *p<0.05, ***p<0.005, ****p<0.001; by one-way ANOVA.

[0150] FIG. 17 shows B16-GM growth inhibition in mice following n=3 doses of i.v.-injectedCpG-C’ dots (6 nmoles (lOpM), red), as against PEG-C’ dots (gray, dotted line), and saline vehicle (blue), initiated when tumors reached ~50 mm3(day 0), and repeated on days 3 and 6 thereafter. Data represents analyses of n=3 mice per group, mean+s.e.m. **p<0.01, ****p<0.0001 using a nonparametric 2-way ANOVA.

[0151] FIG. 18A shows the population of M2 macrophages as % CD45+ cells in due to CpG-C’ dot treatment in B16-GM tumor mice triply injected with CpG-C’ dots or C’ dots (6 nmoles; lOpM) or saline vehicle (control) every 3 days when tumor volumes reached 50mm3. *=p<.05, **p<0.01, ***=p<_005, ****p<0.001 (ordinary one-way ANOVA).

[0152] FIG. 18B shows the population of Ml macrophages as % CD45+ cells in due to CpG-C’ dot treatment in B16-GM tumor mice triply injected with CpG-C’ dots or C’ dots (6 nmoles; lOpM) orsaline vehicle (control) every 3 days when tumor volumes reached 50mm3. *=p<.05, **p<0.01, ***=p<_005, ****p<0.001 (ordinary one-way ANO VA).

[0153] FIG. 18C shows the ratio of M1 / M2 macrophages in due to CpG-C’ dot treatment in B 16-GM tumor mice triply injected with CpG-C’ dots or C’ dots (6 nmoles; lOpM) or saline vehicle (control) every 3 days when tumor volumes reached 50mm3. *=p<.05, **p<0.01, ***=p<.005, ****p<0.001 (ordinary one-way ANOVA).

[0154] FIG. 18D shows the population of Treg cells as % CD45+ cells due to CpG-C’ dot treatment in B16-GM tumor mice triply injected with CpG-C’ dots or C’ dots (6 nmoles; lOpM) or saline vehicle (control) every 3 days when tumor volumes reached 50mm3. *=p<.05, **p<0.01, ***=p<_005, ****p<0.001 (ordinary one-way ANOVA).

[0155] FIG. 18E shows the ratio of CD8 / Treg cells due to CpG-C’ dot treatment in B16-GM tumor mice triply injected with CpG-C’ dots or C’ dots (6 nmoles; lOpM) or saline vehicle (control) every 3 days when tumor volumes reached 50mm3. *=p<.05, **p<0.01, ***=p<.005, ****p<0.001 (ordinary one-way ANOVA).

[0156] FIGS. 19A-19B shows that treatment with aMSH-PEG-C’ dots synergizes with amino acid starvation to kill 1D8 cells through ferroptosis. (FIG. 19A) Graph (left) shows percent cell death of ID8 cells cultured for 24 or 48 h in full or amino acid (AA)-free medium, in the presence or absence of 15pM aMSH-C’ dots. Data show percentages out of 500 cells from five independent microscopic fields of view; error bars show s.d. Images (right) show representative fields of view from time-lapse imaging of control and aMSH C’ dot-treated cells, cultured in AA-free medium. (FIG. 19B) Growth inhibition in ID8 flank tumor mice after n=3 doses of i.v.-injected aMSH-PEG-C’ dots (36 nmoles), red) versus aMSH-PEG-C’ doH- liproxstatin- 1 (green), and vehicle (blue) were initiated when tumors reached ~50mm3 (day 0), then days 3 and 6. Data reflects mean +s.e.m (n=4 mice / group). *P<0.05, **P<0.01 (2- way ANOVA).

[0157] Features and advantages of the present disclosure will become more apparent from the detailed description of certain embodiments that is set forth below, particularly when taken in conjunction with the figures.Definitions

[0158] In order for the present disclosure to be more readily understood, certain terms are first defined below. Additional definition for the following terms and other terms are set forth throughout the specification.

[0159] Administration: As used herein, the term “administration” typically refers to the administration of a composition comprising a nanoparticle to a subject or system. In general, any route of administration may be utilized including, for example, parenteral (e.g., intravenous), oral, topical, subcutaneous, peritoneal, intraarterial, inhalation, vaginal, rectal, nasal, introduction into the cerebrospinal fluid, or instillation into body compartments. In certain embodiments, administration is oral. Additionally or alternatively, in certain embodiments, administration is parenteral. In certain embodiments, administration is intravenous. In certain embodiments, administration is intraperitoneal.

[0160] Agent'. The term “agent”, as used herein, may refer to a compound, molecule, or entity of any chemical and / or biological class including, for example, a small molecule, polypeptide, nucleic acid, saccharide, lipid, metal, or a combination or complex thereof. In certain embodiments, the term “agent” may refer to a compound, molecule, or entity that comprises a polymer. In certain embodiments, the term may refer to a compound or entity that comprises one or more polymeric moieties. In certain embodiments, the term may refer to a compound, molecule, or entity that lacks oris substantially free of any polymer or polymeric moiety. In some embodiments, the term may refer to a nanoparticle.

[0161] Antibody: As used herein, the term “antibody” refers to a polypeptide that includes canonical immunoglobulin sequence elements sufficient to confer specific binding to a particular target antigen. As is known in the art, intact antibodies as produced in nature are approximately 150 kD tetrameric agents comprised of two identical heavy chain polypeptides (about 50 kD each) and two identical light chain polypeptides (about 25 kD each) that associate with each other into what is commonly referred to as a “Y-shaped” structure. Each heavy chain is comprised of at least four domains (each about 110 amino acids long)- an amino-terminal variable (VH) domain (located at the tips of the Y structure), followed by three constant domains: CHI, CH2, and the carboxy-terminal CH3 (located at the base of the Y’s stem). A short region, known as the “switch”, connects the heavy chain variable and constant regions. The “hinge” connects CH2 and CH3 domains to the rest of the antibody. Two disulfide bonds in this hinge region connect the two heavy chain polypeptides to one another in an intact antibody. Each light chain is comprised of two domains - an amino-terminal variable (VL) domain, followed by a carboxy-terminal constant (CL) domain, separated from one another by another “switch”. Intact antibody tetramers are comprised of two heavy chain-light chain dimers in which the heavy and light chains are linked to one another by a single disulfide bond; two other disulfide bonds connect the heavy chain hinge regions to one another, so that the dimers are connected to one another and the tetramer is formed. Naturally-produced antibodies are also glycosylated, typically on the CH2 domain. Each domain in a natural antibody has a structure characterized by an “immunoglobulin fold” formed from two beta sheets (e.g., 3-, 4-, or 5-stranded sheets) packed against each other in a compressed antiparallel beta barrel. Each variable domain contains three hypervariable loops known as“complement determining regions” (CDR1, CDR2, and CDR3) and four somewhat invariant “framework” regions (FR1, FR2, FR3, and FR4). When natural antibodies fold, the FR regions form the beta sheets that provide the structural framework for the domains, and the CDR loop regions from both the heavy and light chains are brought together in three-dimensional space so that they create a single hypervariable antigen binding site located at the tip of the Y structure. The Fc region of naturally-occurring antibodies binds to elements of the complement system, and also to receptors on effector cells, including for example effector cells that mediate cytotoxicity. As is known in the art, affinity and / or other binding attributes of Fc regions for Fc receptors can be modulated through glycosylation or other modification. In some embodiments, antibodies produced and / or utilized in accordance with the present invention include glycosylated Fc domains, including Fc domains with modified or engineered such glycosylation. For purposes of the present disclosure, in certain embodiments, any polypeptide or complex of polypeptides that includes sufficient immunoglobulin domain sequences as found in natural antibodies can be referred to and / or used as an “antibody”, whether such polypeptide is naturally produced (e.g., generated by an organism reacting to an antigen), or produced by recombinant engineering, chemical synthesis, or other artificial system or methodology. In some embodiments, an antibody is polyclonal; in some embodiments, an antibody is monoclonal. In some embodiments, an antibody has constant region sequences that are characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, antibody sequence elements are humanized, primatized, chimeric, etc., as is known in the art. Moreover, the term “antibody” as used herein, can refer in appropriate embodiments (unless otherwise stated or clear from context) to any of the art- known or developed constructs or formats for utilizing antibody structural and functional features in alternative presentation. For example, an antibody utilized in accordance with certain embodiments ofthe present invention is in a format selected from, but not limited to, intact IgA, IgG, IgE or IgM antibodies; bi- or multi- specific antibodies (e.g., Zybodies®, etc.); antibody fragments such as Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fd’ fragments, Fd fragments, and isolated CDRs or sets thereof; single chain Fvs; polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof); cameloid antibodies; masked antibodies (e.g., Probodies®); Small Modular ImmunoPharmaceuticals (“SMIPs™ ); single chain or Tandem diabodies (TandAb®); VHHs; Anticalins®; Nanobodies® minibodies; ankyrin repeat proteins or DARPINs®; Avimers®; DARTs; TCR-like antibodies;, Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®, Centyrins®; and KALBITOR®s.

[0162] Antibody agent'. As used herein, the term “antibody agent” refers to an agent that specifically binds to a particular antigen. In some embodiments, the term encompasses any polypeptide or polypeptide complex that includes immunoglobulin structural elements sufficient to confer specific binding. Exemplary antibody agents include, but are not limited to monoclonal antibodies or polyclonal antibodies. In some embodiments, an antibody agent may include one or more constant region sequences that are characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, an antibody agent may include one or more sequence elements are humanized, primatized, chimeric, etc., as is known in the art. In many embodiments, the term “antibody agent” is used to refer to one or more of the art-known or developed constructs or formats for utilizing antibody structural and functional features in alternative presentation. For example, embodiments, an antibody agent utilized in accordance with certain embodiments of the present invention is in a format selected from, but not limited to, intact IgA, IgG, IgE or IgM antibodies; bi- or multi- specific antibodies (e.g.,Zybodies®, etc.); antibody fragments such as Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fd’fragments, Fd fragments, and isolated CDRs or sets thereof; single chain Fvs; polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof); cameloid antibodies; masked antibodies (e.g., Probodies®); Small Modular ImmunoPharmaceuticals (“SMIPs™ ); single chain or Tandem diabodies (TandAb®); VHHs; Anticalins®; Nanobodies® minibodies; ankyrin repeat proteins or DARPINs®; Avimers®; DARTs; TCR-like antibodies;, Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®, Centyrins®; and KALBITOR®s. In some embodiments, an antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally. In some embodiments, an antibody may contain a covalent modification (e.g., attachment of a glycan, a payload or other pendant group). In many embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence includes one or more structural elements recognized by those skilled in the art as a complementarity determining region (CDR); in some embodiments an antibody agent is or comprises a polypeptide whose amino acid sequence includes at least one CDR (e.g., at least one heavy chain CDR and / or at least one light chain CDR) that is substantially identical to one found in a reference antibody. In some embodiments an included CDR is substantially identical to a reference CDR in that it is either identical in sequence or contains between 1-5 amino acid substitutions as compared with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that it shows at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that it shows at least 96%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that at least one amino acid within the included CDR is deleted, added,or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical with that of the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that 1-5 amino acids within the included CDR are deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that at least one amino acid within the included CDR is substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical with that of the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that 1-5 amino acids within the included CDR arc deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR. In some embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence includes structural elements recognized by those skilled in the art as an immunoglobulin variable domain. In some embodiments, an antibody agent is a polypeptide protein having a binding domain which is homologous or largely homologous to an immunoglobulin-binding domain.

[0163] Antigen'. The term “antigen”, as used herein, refers to an agent that elicits an immune response; and / or (ii) an agent that binds to a T cell receptor e.g., when presented by an MHC molecule) or to an antibody. In some embodiments, an antigen elicits a humoral response (e.g., including production of antigen-specific antibodies); in some embodiments, an elicits a cellular response (e.g., involving T-cells whose receptors specifically interact with the antigen). In some embodiments, an antigen binds to an antibody and may or may not induce a particular physiological response in an organism.

[0164] Antigen presenting cell: The phrase “antigen presenting cell” or “APC,” as used herein, has its art understood meaning referring to cells which process and present antigens to T-cells. Exemplary antigen cells include dendritic cells, macrophages and certain activated epithelial cells.

[0165] Biocompatible: The term “biocompatible”, as used herein, refers to materials that do not cause significant harm to living tissue when placed in contact with such tissue, e.g., in vivo. In certain embodiments, materials are “biocompatible” if they are not toxic to cells. In certain embodiments, materials are “biocompatible” if their addition to cells in vitro results in less than or equal to 20% cell death. In certain embodiments, materials are biodegradable.

[0166] Cancer. As used herein, the term “cancer” refers to a malignant neoplasm or tumor (Stedman’s Medical Dictionary, 25th ed.; Hensly ed.; Williams & Wilkins: Philadelphia, 1990). Exemplary cancers include, but are not limited to, acoustic neuroma; adenocarcinoma; adrenal gland cancer; anal cancer; angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma); appendix cancer; benign monoclonal gammopathy; biliary cancer (e.g., cholangiocarcinoma); bladder cancer; breast cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast, mammary cancer, medullary carcinoma of the breast); brain cancer (e.g., meningioma, glioblastomas, glioma (e.g., astrocytoma, oligodendroglioma), medulloblastoma); bronchus cancer; carcinoid tumor; cervical cancer (e.g., cervical adenocarcinoma); choriocarcinoma; chordoma; craniopharyngioma; connective tissue cancer; epithelial carcinoma; ependymoma; endotheliosarcoma (e.g., Kaposi’s sarcoma, multiple idiopathic hemorrhagic sarcoma); endometrial cancer (e.g., uterine cancer, uterine sarcoma); esophageal cancer (e.g., adenocarcinoma of the esophagus, Barrett’s adenocarcinoma); Ewing’s sarcoma; eye cancer (e.g., intraocular melanoma, retinoblastoma); familiar hypereosinophilia; gall bladder cancer; gastric cancer (e.g., stomachadenocarcinoma); gastrointestinal stromal tumor (GIST); germ cell cancer; head and neck cancer (e.g., head and neck squamous cell carcinoma, oral cancer (e.g., oral squamous cell carcinoma), throat cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer)); hematopoietic cancers (e.g., leukemia such as acute lymphocytic leukemia (ALL) (e.g., B cell ALL, T cell ALL), acute myelocytic leukemia (AML) (e.g., B cell AML, T cell AML), chronic myelocytic leukemia (CML) (e.g., B cell CML, T cell CML), and chronic lymphocytic leukemia (CLL) (e.g., B cell CLL, T cell CLL)); lymphoma such as Hodgkin lymphoma (HL) (e.g., B cell HL, T cell HL) and non Hodgkin lymphoma (NHL) (e.g., B cell NHL such as diffuse large cell lymphoma (DLCL) (e.g., diffuse large B cell lymphoma), follicular lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), mantle cell lymphoma (MCL), marginal zone B cell lymphomas (e.g., mucosa associated lymphoid tissue (MALT) lymphomas, nodal marginal zone B cell lymphoma, splenic marginal zone B cell lymphoma), primary mediastinal B cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma (e.g., Waldenstrom’s macroglobulinemia), hairy cell leukemia (HCL), immunoblastic large cell lymphoma, precursor B lymphoblastic lymphoma and primary central nervous system (CNS) lymphoma; and T cell NHL such as precursor T lymphoblastic lymphoma / leukemia, peripheral T cell lymphoma (PTCL) (e.g., cutaneous T cell lymphoma (CTCL) (e.g., mycosis fungoides, Sezary syndrome), angioimmunoblastic T cell lymphoma, extranodal natural killer T cell lymphoma, enteropathy type T cell lymphoma, subcutaneous panniculitis like T cell lymphoma, and anaplastic large cell lymphoma); a mixture of one or more leukemia / lymphoma as described above; and multiple myeloma (MM)), heavy chain disease (e.g., alpha chain disease, gamma chain disease, mu chain disease); hemangioblastoma; hypopharynx cancer; inflammatory myofibroblastic tumors; immunocytic amyloidosis; kidney cancer (e.g., nephroblastoma a.k.a. Wilms’ tumor, renal cellcarcinoma); liver cancer (e.g., hepatocellular cancer (HCC), malignant hepatoma); lung cancer (e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non small cell lung cancer (NSCLC), adenocarcinoma of the lung); leiomyosarcoma (LMS); mastocytosis (e.g., systemic mastocytosis); muscle cancer; myelodysplastic syndrome (MDS); mesothelioma; myeloproliferative disorder (MPD) (e.g., polycythemia vera (PV), essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM) a.k.a. myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myelocytic leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES); neuroblastoma; neurofibroma (e.g., neurofibromatosis (NF) type 1 or type 2, schwannomatosis); neuroendocrine cancer (e.g., gastroenteropancreatic neuroendocrine tumor (GEP NET), carcinoid tumor); osteosarcoma (e.g., bone cancer); ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma); papillary adenocarcinoma; pancreatic cancer (e.g., pancreatic adenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), Islet cell tumors); penile cancer (e.g., Paget’s disease of the penis and scrotum); pinealoma; primitive neuroectodermal tumor (PNT); plasma cell neoplasia; paraneoplastic syndromes; intraepithelial neoplasms; prostate cancer (e.g., prostate adenocarcinoma); rectal cancer; rhabdomyosarcoma; salivary gland cancer; skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cell carcinoma (BCC)); small bowel cancer (e.g., appendix cancer); soft tissue sarcoma (e.g., malignant fibrous histiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma); sebaceous gland carcinoma; small intestine cancer; sweat gland carcinoma; synovioma; testicular cancer (e.g., seminoma, testicular embryonal carcinoma); thyroid cancer (e.g., papillary carcinoma of the thyroid, papillary thyroid carcinoma (PTC), medullary thyroid cancer); urethral cancer; vaginal cancer; and vulvar cancer (e.g., Paget’s disease of the vulva).

[0167] Cellular receptor- and / or antigen- targeting ligand: In general, the term “cellular receptor- and / or antigen- targeting ligand” is used herein to refer to any entity that binds to a target of interest as described herein (e.g., a target that is expressed on the surface of cells, e.g., expressed on the surface of tumor cells). In many embodiments, a cellular receptor- and / or antigen- targeting ligand of interest is one that binds specifically with its target in that it discriminates its target from other potential binding partners in a particular interaction context. In general, a cellular receptor- and / or antigentargeting ligand may be or comprise an entity of any chemical class (e.g., polymer, non-polymer, small molecule, polypeptide, carbohydrate, lipid, nucleic acid, etc.). In some embodiments, a cellular receptor- and / or antigen- targeting ligand is a single chemical entity. In some embodiments, a cellular binding ligand is a complex of two or more discrete chemical entities associated with one another under relevant conditions by non-covalent interactions. For example, those skilled in the art will appreciate that in some embodiments, a cellular receptor- and / or antigen- targeting ligand may comprise a “generic” binding moiety (e.g., a class-specific antibody) and a “specific” binding moiety (e.g., an antibody or aptamers with a particular molecular target) that is linked to the partner of the generic biding moiety. In some embodiments, cellular receptor- and / or antigen- targeting ligand are or comprise polypeptides (including, e.g., antibodies or antibody fragments). In some embodiment, a cellular receptor- and / or antigen- targeting ligand comprises a recombinant antibody fragment (fAbs), a single chain variable fragment (scFv), and a single domain antibody (sdAb) fragment. In some embodiment, a cellular receptor- and / or antigen- targeting ligand comprises a recombinant antibody fragment (fAbs). In some embodiments, a cellular receptor- and / or antigen- targeting ligand comprises a single chain variable fragment (scFv). In some embodiments, a cellular receptor- and / or antigentargeting ligand comprises a single domain antibody (sdAb) fragment.

[0168] Chemotherapeutic Agent: As used herein, the term “chemotherapeutic agent” or “oncolytic therapeutic agent”(e.g., anti-cancer drug, e.g., anti-cancer therapy, e.g., immune cell therapy) has its art-understood meaning referring to one or more pro-apoptotic, cytostatic and / or cytotoxic agents, and / or hormonal agents, for example, specifically including agents utilized and / or recommended for use in treating one or more diseases, disorders or conditions associated with undesirable cell proliferation. In many embodiments, chemotherapeutic agents and / or oncolytic therapeutic agents are useful in the treatment of cancer. In some embodiments, a chemotherapeutic agent and / or oncolytic therapeutic agents may be or comprise one or more hormonal agents (e.g., androgen inhibitors), one or more alkylating agents, one or more anthracy clines, one or more cytoskeletal disruptors (e.g., microtubule targeting agents such as taxanes, maytansine and analogs thereof, of), one or more epothilones, one or more histone deacetylase inhibitors HDACs), one or more topoisomerase inhibitors (e.g., inhibitors of topoisomerase I and / or topoisomerase II), one or more kinase inhibitors, one or more nucleotide analogs or nucleotide precursor analogs, one or more peptide antibiotics, one or more platinumbased agents, one or more retinoids, one or more vinca alkaloids, and / or one or more analogs of one or more of the following (i.e., that share a relevant anti-proliferative activity). In some particular embodiments, a chemotherapeutic agent may be or comprise one or more of Actinomycin, all-trans retinoic acid, an Auiristatin, Azacitidine, Azathioprine, Bleomycin, Bortezomib, Carboplatin, Capecitabine, Cisplatin, Chlorambucil, Cyclophosphamide, curcumin, Cytarabine, Daunorubicin, Docetaxel, Doxifluridine, Doxorubicin, Epirubicin, Epothilone, Etoposide, Fluorouracil, Gemcitabine, Hydroxyurea, Idarubicin, Imatinib, Irinotecan, Maytansine and / or analogs thereof (e.g., DM1) Mechlorethamine, Mercaptopurine, Methotrexate, Mitoxantrone, a Maytansinoid,Oxaliplatin, Paclitaxel, Pemetrexed, Teniposide, Tioguanine, Topotecan, Valrubicin, Vinblastine,Vincristine, Vindesine, Vinorelbine, and combinations thereof. In some embodiments, a chemotherapeutic agent may be utilized in the context of an antibody-drug conjugate. In some embodiments, a chemotherapeutic agent is one found in an antibody-drug conjugate selected from the group consisting of: hLLl -doxorubicin hRS7-SN-38, hMN-14-SN-38, hLL2-SN-38, hA20-SN-38, hPAM4-SN-38, hLLl-SN-38, hRS7- Pro-2-P-Dox, hMN-14-Pro-2-P-Dox, hLL2-Pro-2-P-Dox, hA20- Pro-2-P-Dox, hPAM4-Pro-2-PDox, hLLl-Pro-2-P-Dox, P4 / D 1 O-doxorubicin, gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, glembatumomab vedotin, SAR3419, SAR566658, BIIB015, BT062, SGN-75, SGN-CD19A, AMG-172, AMG-595, BAY-94-9343, ASG-5ME, ASG-22ME, ASG-16M8F, MDX-1203, MLN-0264, anti-PSM A ADC, RG- 7450, RG-7458, RG-7593, RG-7596, RG-7598, RG-7599, RG-7600, RG-7636, ABT-414, IMGN-853, IMGN-529, vorsetuzumab mafodotin, and lorvotuzumab mertansine. In some embodiments, a chemotherapeutic agent may be or comprise one or more of famesyl-thiosalicylic acid (FTS), 4- (4- Chloro-2-methylphenoxy)-N-hydroxybutanamide (CMH), estradiol (E2), tetramethoxystilbene (TMS), 5-tocatrienol, salinomycin, or curcumin. In certain embodiments, chemotherapeutic agents and / or oncolytic therapeutic agents for anti-cancer treatment comprise (e.g., are) biological agents such as tumor-infiltrating lymphocytes, CAR T-cells, antibodies, antigens, therapeutic vaccines (e.g., made from a patient’s own tumor cells or other substances such as antigens that are produced by certain tumors), immune-modulating agents (e.g., cytokines, e.g., immunomodulatory drugs or biological response modifiers), checkpoint inhibitors) or other immunologic / pharmacologic agents (e.g., PI3KD- selective inhibitor targeting myeloid cells or IPI-549). In certain embodiments, immunologic agents include immunoglobins, immunostimulants (e.g., bacterial vaccines, colony stimulating factors, interferons, interleukins, therapeutic vaccines, vaccine combinations, viral vaccines) and / orimmunosuppressive agents (e.g., calcineurin inhibitors, interleukin inhibitors, TNF alpha inhibitors). In certain embodiments, hormonal agents include agents for anti-androgen therapy (e.g., Ketoconazole, ABiraterone, TAK-700, T0K-001, Bicalutamide, Nilutamide, Flutamide, Enzalutamide, ARN-509).

[0169] Identity, identical'. As used herein, the terms “identity” and “identical” refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Methods for the calculation of a percent identity as between two provided sequences are known in the art. Calculation of the percent identity of two nucleic acid or polypeptide (e.g., amino acid) sequences, for example, can be performed by aligning the two sequences (or the complement of one or both sequences) for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). The nucleotides or amino acids at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences and, optionally, taking into account the number of gaps and the length of each gap, which may need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a computational algorithm, such as BLAST (basic local alignment search tool).

[0170] Marker: A “marker”, as used herein, refers to an entity or moiety whose presence or level is a characteristic of a particular state or event. In some embodiments, presence or level of a particular marker may be characteristic of presence or stage of a disease, disorder, or condition. Togive but one example, in some embodiments, the term refers to a gene expression product that is characteristic of a particular immune cell type, immune cell subclass, activation of immune cells, and / or polarization of immune cells. Alternatively or additionally, in some embodiments, a presence or level of a particular marker correlates with activity (or activity level) of a particular signaling pathway, for example that may be characteristic of a particular class of immune cells. The statistical significance of the presence or absence of a marker may vary depending upon the particular marker. In some embodiments, detection of a marker is highly specific in that it reflects a high probability that the cell is of a particular immune cell type and / or subclass. In certain embodiments, a marker is a cytokine. In certain embodiments, a marker is a chemokine. In certain embodiments, a marker is a receptor. In certain embodiments, a marker is a genetic marker (e.g., mRNA, RNA) indicative of activation of a gene.

[0171] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers. In certain embodiments, active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In certain embodiments, pharmaceutical compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, ora controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces.

[0172] Radiolabel: As used herein, “radiolabel” refers to a moiety comprising a radioactive isotope of at least one element. Exemplary suitable radiolabels include but are not limited to those described herein. In certain embodiments, a radiolabel is one used in positron emission tomography (PET). In certain embodiments, a radiolabel is one used in single-photon emission computed tomography (SPECT). In certain embodiments, radioisotopes comprise "mTc,inIn,MCu,67Ga,186Re,188Re,153Sm,177Lu,67Cu,123I,1241,125I,nC,43N,150 ,18F,161Ho,149Pm,90Y,213Bi,103Pd,159Gd,140La,198Au,199Au,169Yb,175Yb,165Dy,166Dy,105Rh,ulAg,225Ac,192Ir, and89Zr.

[0173] Subject'. As used herein, the term “subject” includes humans and mammals (e.g., mice, rats, pigs, cats, dogs, and horses). In many embodiments, subjects are mammals, particularly primates, especially humans. In certain embodiments, subjects are livestock such as cattle, sheep, goats, cows, swine, and the like; poultry such as chickens, ducks, geese, turkeys, and the like; and domesticated animals particularly pets such as dogs and cats. In certain embodiments (e.g., particularly in research contexts) subject mammals will be, for example, rodents (e.g., mice, rats, hamsters), rabbits, primates, or swine such as inbred pigs and the like.

[0174] Therapeutically ejfective amount: as used herein, is meant an amount that produces the desired effect for which it is administered. In certain embodiments, the term refers to an amount that is sufficient, when administered to a population suffering from or susceptible to a disease, disorder, and / or condition in accordance with a therapeutic dosing regimen, to treat the disease, disorder, and / or condition. In certain embodiments, a therapeutically effective amount is one that reduces the incidenceand / or severity of, and / or delays onset of, one or more symptoms of the disease, disorder, and / or condition. Those of ordinary skill in the art will appreciate that the term "therapeutically effective amount" does not in fact require successful treatment be achieved in a particular individual. Rather, a therapeutically effective amount may be that amount that provides a particular desired pharmacological response in a significant number of subjects when administered to patients in need of such treatment. In certain embodiments, reference to a therapeutically effective amount may be a reference to an amount as measured in one or more specific tissues (e.g., a tissue affected by the disease, disorder or condition) or fluids (e.g., blood, saliva, serum, sweat, tears, urine, etc.). Those of ordinary skill in the art will appreciate that, in certain embodiments, a therapeutically effective amount of a particular agent or therapy may be formulated and / or administered in a single dose. In certain embodiments, a therapeutically effective agent may be formulated and / or administered in a plurality of doses, for example, as part of a dosing regimen.

[0175] Therapeutic agent: As used herein, the phrase “therapeutic agent” in general refers to any agent that has a therapeutic effect and / or elicits a desired biological and / or pharmacological effect when administered to a subject.

[0176] Treatment'. As used herein, the term “treatment” (also “treat” or “treating”) refers to administration of a therapy that partially or completely alleviates, ameliorates, relives, inhibits, delays onset of, reduces severity of, and / or reduces incidence of one or more symptoms, features, and / or causes of a particular disease, disorder, and / or condition. In some embodiments, such treatment may be of a subject who does not exhibit signs of the relevant disease, disorder and / or condition and / or of a subject who exhibits only early signs of the disease, disorder, and / or condition. Alternatively or additionally, such treatment may be of a subject who exhibits one or more established signs of therelevant disease, disorder and / or condition. In some embodiments, treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and / or condition. In some embodiments, treatment may be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant disease, disorder, and / or condition.

[0177] Tumor. As used herein, the term “tumor” refers to an abnormal growth of cells or tissue. In some embodiments, a tumor may comprise cells that are precancerous (e.g., benign), malignant, pre- metastatic, metastatic, and / or non-metastatic. In some embodiments as discussed herein, a tumor is associated with, or is a manifestation of, a cancer. In some embodiments as discussed herein, a tumor may be a solid tumor.

[0178] Drawings are presented herein for illustration purposes, not for limitation.Detailed Description

[0179] It is contemplated that methods, compositions, and processes of the claimed invention encompass variations and adaptations developed using information from the embodiments described herein. Adaptation and / or modification of the methods, compositions, and processes described herein may be performed, as contemplated by this description.

[0180] Throughout the description, where methods, compositions, and processes are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present invention that consist essentially of, or consist of, the recitedcomponents, and that there are processes and methods according to the present invention that consist essentially of, or consist of, the recited steps.

[0181] It should be understood that the order of steps or order for performing certain action is immaterial so long as the invention remains operable. Moreover, two or more steps or actions may be conducted simultaneously.

[0182] The mention herein of any publication, for example, in the Background section, is not an admission that the publication serves as prior art with respect to any of the claims presented herein. The Background section is presented for purposes of clarity and is not meant as a description of prior art with respect to any claim. Documents arc incorporated herein by reference as noted. Where there is any discrepancy in the meaning of a particular term, the meaning provided in the Definition section above is controlling. Headers are provided for the convenience of the reader - the presence and / or placement of a header is not intended to limit the scope of the subject matter described herein.

[0183] The present disclosure describes immunomodulatory nanoparticles and their conjugates, the latter enhancing cellular uptake, as agents with intrinsic therapeutic capabilities that, among other things, (1) modulate the tumor microenvironment (TME) toward a pro-inflammatory phenotype, (2) increase immune cell activation and cytotoxicity in the TME, and / or (3) target cancer cells directly for cell death through the mechanism of ferroptosis and / or other cell death processes. Unlike other particle-based tools, the development of a single highly specialized multi-therapeutic ultrasmall particle-based agent that can be delivered systemically and can specifically engage ferroptosis, while also activating separable, ferroptosis-independent pro-inflammatory and cytotoxic responses in the TME is unexpected. These therapeutic activities can, in turn, potentially reduce immunosuppressive factors that limit the normal functioning of effector cells, underscoring an advantage described by thepresent disclosure: leveraging these death-inducing and pro-inflammatory mechanisms for treating relapsed and refractory solid tumors and their metastases, and which also have relevance for treating human disease. Activation of Type I / II interferons and other immunostimulatory pathways using immunomodulatory nanoparticle conjugates described herein, therefore, presents a unique approach to transform a suppressive TME into one that can enhance CAR T cell cytotoxicity, improve efficacy, and expand the window for treating these aggressive tumors.In some embodiments, the present disclosure describes a custom-designed immunomodulatory nanoparticle conjugate (e.g., an scFv-conjugated nanoparticle) for examining biological responses in ovarian cancer models. Such immunomodulatory nanoparticle conjugate comprises a plurality of cellular receptor- and / or antigen- targeting ligands attached thereto. In some embodiments, the immunomodulatory nanoparticle conjugates target a retained extracellular domain of MUC16, MUC16ecto, found on the surface of a number of cancer cell types. MUC16 is a glycoprotein overexpressed on a majority of ovarian cancer cells and other malignant solid tumors. The extracellular portion of MUC16 (or CA-125) is cleaved and released, making this portion of the target unsuitable for targeting by CAR T cells (FIG. 1).

[0184] As described by the present disclosure, the ectodomain, MUC16ecto, which is retained on the tumor cell surface, is well-suited for targeting with antibodies, targeted T cells, or targeted particle-based probes. For example, a second-generation CAR, 4Hll-28z, specific to the MUC16 ectodomain, and containing the cytoplasmic signaling domain of the CD28 co-stimulatory receptor, can be used in accordance with embodiments described by the present disclosure. The 4H11 mAb, from which 4Hll-28z is derived, binds to a majority of ovarian tumor clinical samples. The present disclosure describes that human 4Hll-28z CAR T cells can successfully eradicate MUC16ecto+ tumorcells in SCID-Beige mice. For example, murine T cells expressing the murine 4Hll-28z CAR were shown to successfully eradicate murine ID8 ovarian tumor cells in a syngeneic orthotopic model. Accordingly, CAR T cell targeting of MUC16ecto for treatment of relapsed and refractory ovarian cancers can be achieved.

[0185] The present disclosure also describes that modified CAR T cells can be used in accordance with the immunomodulatory nanoparticle conjugates and methods described herein. Modification of CAR T cells to secrete the pro-inflammatory cytokine, IL- 12, leads to significant antitumor efficacy in orthotopic ID8 models. FIGs. 2A and 2B show enhanced anti-tumor efficacy of MUC16cctotargeted CAR T cells in human (h)MUC16ccto+ ID8 orthotopic tumors in C57BL6 mice, where CAR T cells had further been modified to secrete IL- 12. IL- 12- secreting CAR T cells prolong survival in an ID8 ovarian orthotopic tumor model. In FIGs. 2A and 2B, results are shown from adult female wild type C57BL6 mice, which were inoculated with IxlO7ID8-Mucl6ect0cells intraperitoneally (i.p.) and treated 35 days (FIG. 2A) or 42 days (FIG. 2B) later with 2xl06indicated CAR T cells i.p., *p < 0.02. The number of mice per group: n=5 (NT CAR T cells), n=5 (non-T CAR T-IL12), n=6 (T CAR T cells), n=8 (T CAR T-IL12 cells). Results were pooled from 2 independent studies. Statistical analysis used a log-rank (Mantel-Cox) test.

[0186] In addition to observing improved survival (FIGs. 2A and 2B) in this advanced syngeneic high-grade cancer model, IL- 12 armored CAR T cells demonstrated prolonged in vivo persistence and enhanced expansion. Moreover, treatment of these orthotopic ID8 tumors with IL- 12- secreting (armored) CAR T cells resulted in the depletion of immune-suppressive TAMs, as well as resistance to PD-L1 mediated T cell suppression. This orthotopic MUC16ccto+ ID8 model is, therefore, one of several models that may be well-suited for use in the tumor microenvironment.

[0187] Moreover, in some embodiments, the present disclosure describes development of a MUC16-targeting immunomodulatory nanoparticle conjugate that targets the MUC16ectodomain - highly expressed on ovarian cancer cells and other solid tumor cells - by attaching anti-hMUC16 single chain (scFv) fragments to the particle surface (see, e.g., Example 2). Such immunomodulatory nanoparticle conjugates enhance biological cellular and molecular activities within the TME described herein. This paradigm shift marks a departure from traditional cancer nanomedicine drug delivery approaches, which rely on incorporating cytotoxic agents or, more recently, cytokines or adjuvants, to ultrasmall nanoparticles for enhancing efficacy or improving immunotherapeutic responses, respectively. The present disclosure also describes use of advanced materials characterization tools, including high-performance liquid chromatography (HPLC), to quantitatively and precisely characterize the surface chemical properties of the nanoparticles described herein.

[0188] In certain embodiments, the described immunomodulatory nanoparticle conjugates comprise ultrasmall nanoparticles (e.g., “C or C’ dots”), which are fluorescent, organo-silica core shell particles that have diameters controllable down to the sub- 10 nm range with a range of modular functionalities. C or C’ dots are described by U.S. Patent No. 8298677 B2 “Fluorescent silica-based nanoparticles”, U.S. Publication No. 2013 / 0039848 Al “Fluorescent silica-based nanoparticles”, and U.S. Publication No. US 2014 / 0248210 Al “Multimodal silica-based nanoparticles”, the contents of which are incorporated herein by reference in their entireties. Incorporated into the silica matrix of the core are near-infrared dye molecules, such as Cy5.5, which provides its distinct optical properties. Surrounding the core is a layer or shell of silica. The silica surface is covalently modified with silylpolyethylene glycol (PEG) groups to enhance stability in aqueous and biologically relevant conditions.These particles have been evaluated in vivo and exhibit excellent clearance properties owing largely totheir size and inert surface. Among the additional functionalities incorporated into C or C’ dots are chemical sensing, non-optical (PET) image contrast and in vitro! in vivo targeting capabilities, which enable their use in visualizing lymph nodes for surgical applications, and melanoma detection in cancer. C or C’ dots provide a unique platform for drug delivery due to their physical properties as well as demonstrated human in vivo characteristics. These immunomodulatory nanoparticle conjugates are ultrasmall (e.g., sub-8 nm) and benefit from targeted delivery (e.g., TLR-9) and EPR effects in tumor microenvironments, while retaining favorable bulk renal clearance and pharmacokinetic properties. To this end, in certain embodiments, drug constructs are covalently attached to C dots (or other nanoparticles). C dot-based immunomodulatory nanoparticle conjugates for drug delivery provide good biostability, minimize premature drug release, and exhibit controlled release of the bioactive compound. In certain embodiments, peptide-based linkers are used for the described immunomodulatory nanoparticle conjugates and other applications described herein. These linkers, in the context of antibodies and polymers, are stable both in vitro and in vivo, with highly predictable release kinetics that rely on enzyme catalyzed hydrolysis by lysosomal proteases. For example, cathepsin B, a highly expressed protease in lysosomes, can be utilized to facilitate drug release from macromolecules. By incorporating a short, protease sensitive peptide between the macromolecular backbone and the drug molecule, controlled release of the drug can be obtained in the presence of the enzyme. Interestingly, the described nanoparticles exhibit intrinsic therapeutic capabilities that (1) modulate the tumor microenvironment (TME) toward a pro-inflammatory phenotype, (2) increase immune cell activation and cytotoxicity in the TME, and (3) target cancer cells directly for cell death through mechanisms, such as ferroptosis.

[0189] In certain embodiments, the immunomodulatory nanoparticle conjugates comprise an ultrasmall (e.g., sub-50 nm diameter, e.g., sub-20 nm diameter, e.g., sub- 15 nm diameter, e.g., sub- 10 nm diameter, e.g., sub-8 nm diameter) silica nanoparticle containing a deep red / near-infrared dye (e.g., Cy5; absorption peak: 650 nm) that is covalently encapsulated within the silica-matrix. In this embodiment, due to the encapsulation of the dye and the specific design on the immunomodulatory nanoparticle conjugates, the brightness is dramatically improved (e.g., at least 2-times, e.g., at least 10- times, e.g., at least 50-times, e.g., at least 100-times, e.g., at least 600-times) as compared to the free dye.

[0190] In certain embodiments, cellular binding ligands can be attached to the nanoparticle as described herein. In certain embodiments, the immunomodulatory nanoparticle conjugates comprise from 1 to 100 discrete cellular receptor- and / or antigen- targeting ligands (e.g., of the same type or different types), wherein the cellular receptor- and / or antigen- targeting ligands bind to receptors on tumor cells (e.g., wherein the immunomodulatory nanoparticle conjugates have an average diameter no greater than 15 nm, e.g., no greater than 10 nm, e.g., from about 5 nm to about 7 nm, e.g., about 6 nm). In certain embodiments, the immunomodulatory nanoparticle conjugates comprise a plurality of cellular receptor- and / or antigen- targeting ligands (e.g., anti-MUC16 antibody fragment, oligodeoxynucleotides (ODNs), e.g., CpG ODNs). In certain embodiments, the immunomodulatory nanoparticle conjugates comprise from 1 to 100 discrete cellular receptor- and / or antigen- targeting ligands, e.g., from 1 to 30 discrete cellular receptor- and / or antigen- targeting ligands, e.g., from 1 to 20 discrete cellular receptor- and / or antigen- targeting ligands, e.g., from 1 to 10 discrete cellular receptor- and / or antigen- targeting ligands.

[0191] In certain embodiments, the immunomodulatory nanoparticle conjugates comprise (e.g., has attached) one or more cellular binding ligands, e.g., for targeting cancer tissue / cells of interest. In certain embodiments, the immunomodulatory nanoparticle conjugate comprises one or more cellular binding ligands (e.g., attached thereto), such as, but not limited to, small molecules (e.g., folates, dyes, etc.), aptamers (e.g., A10, AS 1411), polysaccharides, small biomolecules (e.g., folic acid, galactose, bisphosphonate, biotin), oligonucleotides, and / or proteins (e.g., (poly)peptides (e.g., anti-MUC16 antibody fragment, aMSH, RGD, octreotide, AP peptide, epidermal growth factor, chlorotoxin, transferrin, etc.), antibodies, antibody fragments, proteins, etc.). In certain embodiments, the immunomodulatory nanoparticle conjugate comprises one or more immune adjuvants (e.g., pattern recognition receptors, e.g., toll-like receptor agonists, e.g., antibody fragments) (and, optionally, a targeting agent). In certain embodiments, the immunomodulatory nanoparticle conjugate comprises one or more contrast / imaging agents (e.g., fluorescent dyes, (chelated) radioisotopes (SPECT, PET), MR-active agents, CT-agents), and / or therapeutic agents (e.g., small molecule drugs, therapeutic (poly)peptides, therapeutic antibodies, (chelated) radioisotopes, etc.).

[0192] The immunomodulatory nanoparticle conjugates may comprise one or more polymers, e.g., one or more polymers that have been approved for use in humans by the U.S. Food and Drug Administration (FDA) under 21 C.F.R. § 177.2600, including, but not limited to, polyesters (e.g., polylactic acid, poly(lactic-co-glycolic acid), polycaprolactone, polyvalerolactone, poly(l,3-dioxan-2- one)); polyanhydrides (e.g., poly(sebacic anhydride)); polyethers (e.g., polyethylene glycol); polyurethanes; polymethacrylates; polyacrylates; polycyanoacrylates; copolymers of PEG and poly (ethylene oxide) (PEO). In certain embodiments, the diameter of the immunomodulatory nanoparticle conjugates is not substantially increased by the one or more polymers.

[0193] The immunomodulatory nanoparticle conjugates may comprise one or more degradable polymers, for example, certain polyesters, polyanhydrides, polyorthoesters, polyphosphazenes, polyphosphoesters, certain polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, polyfamino acids), polyacetals, polyethers, biodegradable polycyanoacrylates, biodegradable polyurethanes and polysaccharides. For example, specific biodegradable polymers that may be used include but are not limited to polylysine, poly(lactic acid) (PLA), poly(glycolic acid) (PGA), poly(caprolactone) (PCL), poly(lactide-co-glycolide) (PLG), poly(lactide-co-caprolactone) (PLC), and poly(glycolide-co-caprolactone) (PGC). Another exemplary degradable polymer is poly (beta-amino esters), which may be suitable for use in accordance with the present application.

[0194] In certain embodiments, an immunomodulatory nanoparticle conjugate can have or be modified to have one or more functional groups. Such functional groups (within or on the surface of a nanoparticle) can be used for association with any agents (e.g., detectable entities, targeting entities, therapeutic entities, or PEG). In addition to changing the surface charge by introducing or modifying surface functionality, the introduction of different functional groups allows the conjugation of linkers (e.g., (cleavable or (bio-)degradable) polymers such as, but not limited to, polyethylene glycol, polypropylene glycol, PLGA, etc.), targeting / homing agents, and / or combinations thereof.

[0195] Moreover, a label for imaging and / or radiotherapy can be attached to the nanoparticle as described herein. In certain embodiments, the immunomodulatory nanoparticle conjugate comprises a therapeutic agent, e.g., a drug moiety (e.g., a chemotherapy drug) and / or a therapeutic radioisotope. As used herein, "therapeutic agent" refers to any agent that has a therapeutic effect and / or elicits a desired biological and / or pharmacological effect, when administered to a subject.

[0196] In some embodiments, the immunomodulatory nanoparticle conjugates described herein demonstrate enhanced penetration of tumor tissue and diffusion within the tumor interstitium, e.g., for treatment of cancer, as described in International Patent Application No. PCT / US 17 / 30056 (“Compositions and Methods for Targeted Particle Penetration, Distribution, and Response in Malignant Brain Tumors,” filed April 28, 2016) by Bradbury et al., the contents of which is hereby incorporated by reference in its entirety. Further described are methods of targeting tumor-associated macrophages, microglia, and / or other cells in a tumor microenvironment using such immunomodulatory nanoparticle conjugates.

[0197] In some embodiments, the immunomodulatory nanoparticle conjugates described herein can be used to induce ferroptosis, as described in International Patent Application No.PCT / US 16 / 34351 (“Methods of Treatment Using Ultrasmall Nanoparticles to Induce Cell Death of Cancer Cells via Ferroptosis,” filed on May 26, 2016) by Bradbury et al., the contents of which is hereby incorporated by reference in its entirety. In some embodiments, the immunomodulatory nanoparticle conjugates described herein can be used to induce ferroptosis, as described in International Patent Application No. PCT / US 18 / 63751 (“Methods of Cancer Treatment via Regulated Ferroptosis,” filed on December 4, 2018) by Bradbury et al., the contents of which is hereby incorporated by reference in its entirety. In some embodiments, the immunomodulatory nanoparticle conjugates described herein can be used to activate tumor cells and / or innate and adaptive immune responses within the tumor microenvironment, as well as induce immune-related necrosis among other cell death processes, as described in International Patent Application No. PCT / US 19 / 66944 (“Inducing Favorable Effects on Tumor Microenvironment via Administration of Nanoparticle Compositions,” filed onDecember 17, 2019, by Bradbury et al., the contents of which is hereby incorporated by reference in itsentirety. In some embodiments, the immunomodulatory nanoparticle conjugates described herein can be used to activate the tumor microenvironment, as described in International Application No. PCT / US22 / 34224 (“Nanoparticle-mediated Enhancement of Immunotherapy to Promote Ferroptosis- induced Cytotoxicity and Antitumor Immune Responses,” filed on June 21 , 2022) by Bradbury et al., the contents of which is hereby incorporated by reference in its entirety.

[0198] Moreover, diagnostic, therapeutic, and theranostic (diagnostic and therapeutic) platforms featuring such immunomodulatory nanoparticle conjugates are described for treating targets in both the tumor and surrounding microenvironment, as well as modulate remote targets (e.g., serum, spleen) towards a more anti-tumor phenotype, thereby enhancing efficacy of cancer treatment, e.g., immunotherapies and reducing off- target effects. Use of the immunomodulatory nanoparticle conjugates described herein with other conventional therapies, including chemotherapy, radiotherapy, immunotherapy, CAR T cell therapy, experimental inhibitors, and the like, is also envisaged.

[0199] Multi-targeted kinase inhibitors and combinations of single-targeted kinase inhibitors have been developed to overcome therapeutic resistance. Importantly, multimodality combinations of targeted agents, including particle-based compositions designed to carry small molecule inhibitors (SMls), chemotherapeutic s, radiotherapeutic labels, and / or immunotherapies can enhance treatment efficacy and / or improve treatment planning of malignant tumors, including malignant brain tumors. Coupled with molecular imaging labels, these vehicles permit monitoring of drug delivery, accumulation, and retention, which may, in turn, lead to optimal therapeutic indices.

[0200] Moreover, use of radiolabels and / or fluorescent markers attached to (or incorporated in or on, or otherwise associated with) the nanoparticles provide quantitative assessment of immunomodulatory nanoparticle conjugates uptake at the target site and within the body, as well aspermit monitoring of treatment response. In various embodiments, modular linkers are described for incorporating targeting ligands to develop a drug delivery system with controlled pharmacological properties. The described platforms determine the influence of targeting on immunomodulatory nanoparticle conjugates penetration and accumulation, thereby establishing an adaptable platform for improved delivery of a range of tractable SMIs, for example, to primary and metastatic brain tumors.

[0201] In certain embodiments, PET (Positron Emission Tomography) tracers are used as imaging agents. In certain embodiments, PET tracers comprise89Zr,MCu, [18F] fluorodeoxyglucose. In certain embodiments, the immunomodulatory nanoparticle conjugate includes these and / or other therapeutics radiolabels that may induce additional cytotoxic responses within the microenvironment.

[0202] In certain embodiments, the immunomodulatory nanoparticle conjugate comprises one or more fluorophores. Fluorophores comprise fluorochromes, fluorochrome quencher molecules, any organic or inorganic dyes, metal chelates, or any fluorescent enzyme substrates, including protease activatable enzyme substrates. In certain embodiments, fluorophores comprise long chain carbophilic cyanines. In other embodiments, fluorophores comprise Dil, DiR, DiD, and the like. Fluorochromes comprise far red, and near infrared fluorochromes (NIRF). Fluorochromes include but are not limited to a carbocyanine and indocyanine fluorochromes. In certain embodiments, imaging agents comprise commercially available fluorochromes including, but not limited to methylene blue, Cy5.5, Cy5 and Cy7 (GE Healthcare); AlexaFlour660, AlexaFlour680, AlexaFluor750, and AlexaFluor790 (Invitrogen); VivoTag680, VivoTag-S680, and VivoTag-S750 (VisEn Medical); Dy677, Dy682, Dy752 and Dy780 (Dyomics); DyLight547, DyLight647 (Pierce); HiLyte Fluor 647, HiLyte Fluor 680, and HiLyte Fluor 750 (AnaSpec); IRDye 800CW, IRDye 800RS, and IRDye 700DX (Li-Cor); methylene blue; and ADS780WS, ADS830WS, and ADS832WS (American Dye Source) and Kodak X-SIGHT650, Kodak X-SIGHT 691, Kodak X-SIGHT 751 (Carestream Health). In certain embodiments, a multi-wavelength camera as described by Bradbury et al. US Publication No. US 2015 / 0182118 Al, “Systems, Methods, and Apparatus for Multichannel Imaging of Fluorescent Sources in Real Time”, the disclosure of which is hereby incorporated by reference in its entirety. In certain embodiments, the imaging system used to image the lesion provides both static and functional assessments of the area of treatment (and its surroundings).

[0203] Example therapeutics and / or drugs that can be used include RTK inhibitors, such as dasatinib and gefitinib, can target either platelet-derived growth factor receptor (PDGFR) or EGFRmt-i- expressed by primary tumor cells of human or murine origin (e.g., genetically engineered mouse models of high-grade glioma, neurospheres from human patient brain tumor explants) and / or tumor cell lines of non-neural origin. Dasatinib and gefitinib analogs can be synthesized to enable covalent attachment to several linkers without perturbing the underlying chemical structure defining the active binding site. In certain embodiments, a checkpoint inhibitor or an inhibitor specifically targeting immune cell populations (e.g., IPI-549, CSF1-R) can be used as therapeutics and / or drugs for treatment of disease such as cancer.

[0204] The surface chemistry, uniformity of coating (where there is a coating), surface charge, composition, concentration, frequency of administration, shape, and / or size of the nanoparticle conjugates can be adjusted to produce a desired therapeutic effect.

[0205] In certain embodiments, the nanoprobes comprises a chelator, for example, 1,4, 8,1 1- tetraazabicyclo[6.6.2]hexadecane-4,l 1- diyl)diacetic acid (CB-TE2A); desferoxamine (DFO); diethylenetriaminepentaacetic acid (DTPA); 1,4,7, 10-tetraazacyclotetradecane- 1,4,7, 10- tetraacetic acid (DOTA); thylenedi aminetetraacetic acid (EDTA); ethylene glycolbis(2-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA); 1,4,8, 1 l-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA); ethylenebis-(2-4 hydroxy-phenylglycine) (EHPG); 5-C1-EHPG; 5Br-EHPG; 5- Me-EHPG; 5t-Bu-EHPG; 5-sec-Bu-EHPG; benzodiethylenetriamine pentaacetic acid (benzo-DTPA); dibenzo-DTPA; phenyl-DTPA, diphenyl-DTPA; benzyl-DTPA; dibenzyl DTPA; bis-2 (hydroxybenzyl)-ethylene- diaminediacetic acid (HBED) and derivatives thereof; Ac-DOTA; benzo-DOTA; dibenzo-DOTA; 1,4,7- triazacyclononane N,N',N"- triacetic acid (NOTA); benzo-NOTA; benzo-TETA, benzo-DOTMA, where DOTMA is 1,4,7, 10-tetraazacyclotetradecane-l,4,7,10-tetra(methyl tetraacetic acid), benzo- TETMA, where TETMA is 1,4,8, 1 l-tetraazacyclotetradecane-1,4,8,1 l-(methyl tetraacetic acid); derivatives of 1,3-propylenediaminetetraacetic acid (PDTA); tri ethylenetetraaminehexaacetic acid (TTHA); derivatives of l,5,10-N,N',N"-tris(2,3- dihydroxybenzoyl)-tricatecholate (LICAM); and 1,3,5- N,N',N"-tris(2,3- dihydroxybenzoyl)aminomethylbenzene (MECAM), or other metal chelators.

[0206] In certain embodiments, the immunomodulatory nanoparticle conjugate comprises an azide moiety. In certain embodiments, an azide moiety is attached to an antibody fragment for conjugation to a nanoparticle described herein. In certain embodiments, azide moieties are attached a plurality of cellular receptor- and / or antigen- targeting ligands (e.g., anti-MUC16 antibody fragment, oligodeoxynucleotides (ODNs), e.g., CpG ODNs), for instance, for conjugation to a nanoparticle described herein.

[0207] In certain embodiments, the nanoconjugate comprises more than one chelator.

[0208] In certain embodiments the radioisotope-chelator pair is89Zr-DFO. In certain embodiments the radioisotope-chelator pair is177Lu-DOTA. In certain embodiments, the radioisotopechelator pair is225Ac-DOTA.

[0209] In some embodiments, the immunomodulatory nanoparticle conjugates may be associated with PET labels and / or optical probes. Immunomodulatory nanoparticle conjugates may be observed in vivo (e.g., via PET) to evaluate drug accumulation in a target site. For example, immunomodulatory nanoparticle conjugates with PET labels (e.g., without drug substances) may be administered first. Then, by analyzing the in vivo PET images of the immunomodulatory nanoparticle conjugates, drug (e.g., conjugated with immunomodulatory nanoparticle conjugates) concentration and accumulation rate in the tumor may be estimated. The dose may be determined based on the obtained estimation to provide personalized medicine (e.g., tumor size rather than the patient’s body weight). In some embodiments, a radiolabeled drug may be traced in vivo. A highly concentrated chemotherapy drug is potentially dangerous if it is not targeted. In some embodiments, immunomodulatory nanoparticle conjugates with optical probes (e.g., fluorophore) may be used for intraoperative imaging (e.g., where surface of tissue / tumor is exposed) and / or biopsies of tumors.

[0210] While many ongoing clinical trials in melanoma patients have shown improved immune responses with PRR agonists, such as unmethylated cytosine-phosphate-guanine oligodeoxynucleotides (CpG-ODNs), no single approved product has yet emerged. Moreover, only modest efficacy has been found using STING agonists. These clinical trial findings, among others, highlight a critical need for the development of newer, more potent, agents. Without wishing to be bound to any particular theory, the present disclosure suggests that the described nanoparticles themselves and / or immunomodulatory nanoparticle conjugates can serve as a PRR agonist for improving therapeutic efficacy via its broadranging modulation of multiple signaling pathways: STING-, TLR-, RIG-I, and interferon (IFN).

[0211] In some embodiments, immunomodulatory nanoparticle conjugates are comprised of intrinsically therapeutic ultrasmall silica nanoparticles to which a plurality ofimmunomodulatory / stimulatory ligands (e.g., a plurality of pattern recognition receptors (PRR) agonist moieties (e.g., a plurality of oligodeoxy nucleotides (ODNs)) containing an unmethylated cytosine- phosphate-guanine (CpG) motif (or CpG ODNs))) can be conjugated thereto. Unlike other particlebased platforms, the described base nanoparticles themselves exhibit significant intrinsic proinflammatory activities (e.g., activities that modulate macrophage phenotype, e.g., via augmentation of interferons that stimulate the immune system) within the tumor microenvironment. These responses can be further enhanced by surface attachment of immunomodulatory / stimulatory ligands (e.g., proinflammatory ligands), such as CpG, and used in conjunction with immune checkpoint blockade (ICB) therapeutics and other immunotherapies.

[0212] In some embodiments, systemic administration of the described nanoparticle with or without immunomodulatory / stimulatory ligands attached thereto in certain models can functionally serve as a potent exogenous agonist of multiple pattern recognition receptors or PRRs, such as STING-, TLR-, and IFN-related pathways, and offer the potential to activate tumor cells, as well as innate and adaptive immune cell populations, within the tumor microenvironment (TME). Further, the described base nanoparticle itself shows enhanced immunologic responses (e.g., affecting upregulation of the R1G / MDA5-MAVS axis) in ovarian models treated systemically. As described herein, such effects can be enhanced by attachment of a plurality of immunomodulatory / stimulatory ligands to the base nanoparticle. Other variables that can influence immunologic responses described herein include size of the nanoparticle, material composition of the nanoparticles, and surface chemistry of the nanoparticle, as described herein.Nanomedicine strategies for targeting suppressive myeloid cells and limiting resistance.

[0213] Despite their promise as therapeutics, small molecule immunomodulators, such as those described herein, and agents producing inflammatory responses within the tumor microenvironment (TME) (e.g., peptides, drugs) usually demonstrate suboptimal PK and are prone to degradation.Moreover, such agents often exhibit non-specific interactions with proteases, nucleases and immune cells, which can diminish their immunomodulatory activities and lead to adverse reactions compromising their safety. To address these limitations, a wide variety of biocompatible nanoparticles (NPs) have emerged for enhancing the target-specific delivery of exogenous molecules such as cytokines, antigens, or Toll-like receptor (TLR) agonists, either through encapsulation or surface conjugation. The payload is thus afforded protection from degradation and off-target effects, with improved biological stability - all crucial for maximizing immunomodulation. Importantly, several recent studies have shown that the physicochemical properties of engineered materials (e.g., size, shape, hydrophobicity, surface chemistry) are critical determinants of immune responsiveness. Specifically, smaller core particle sizes (e.g., 15 nm) were found to improve the immunostimulatory properties of CpG-conjugated gold NPs, over larger size particles (greater than 30 nm) and exhibited higher specificity for targeting immune receptors. Relatively smaller sizes also resulted in more rapid lymphatic drainage, better antigen presentation, and more favorable distribution profiles, typically not observed with larger-sized (e.g., 30-100 nm in diameters) NPs, as the latter may exhibit considerable off-target toxicity if not administered locally and / or engage complement activation or damage endosomes, thereby inducing oxidative stress and cell death after uptake. In summary, these data suggest that the use of smaller particle sizes is desirable for achieving more potent anti-tumor immune modulation.

[0214] Towards this end, among other things, the present disclosure provides a newer- generation, renally-clearable FDA Investigational New Drug (IND)-approved hybrid organic-inorganic fluorescent core-shell silica nanoparticle with sizes smaller than 15 nm (e.g., 8 nm) and surface chemistry not yet explored for modulation of the TME. Prior work has used a first-generation base particle for targeted detection of primary tumors including melanoma and metastases as well as image- guided nodal mapping in melanoma patients driven by its favorable safety and PK profiles. In contrast to prior work, the present disclosure provides that the described nanoparticles themselves exhibit significant immunomodulatory and cytotoxic particle-intrinsic activities localized only to the tumor microenvironment. In addition to being the first particle probe to induce cancer cell death at high rates by engaging ferroptosis, an iron-dependent cell death mechanism, enhancement of anti-tumor immune cytotoxic responses can occur independently of ferroptosis via alternative (e.g., immune-related) cell death processes. Interestingly, engagement of the tumor microenvironment through ICB was recently shown to involve ferroptosis playing a cooperative role to achieve maximum efficacy, an exciting finding that suggests that ferroptosis and the immune cell activating properties of the described nanoparticles may be predicted to synergize.Synthesis of Nanoparticles

[0215] In certain embodiments, nanoparticles comprising C’ dots are synthesized as discussed herein.

[0216] PEGylated, Cy5-dye encapsulating and aMSH-ligand bearing targeted fluorescent coreshell silica nanoparticles (e.g., aMSH-PEG-Cy5-C’ dots) together with their non-targeted (PEG-Cy5-C’ dots) controls, is synthesized in an aqueous solution.

[0217] Cy5-maleimido derivatives is first coupled to a mercapto-silane to form a dye-silane conjugate. The dye-silane conjugate is subsequently co-condensed with TMOS in aqueous solutions at basic pH to form the Cy5 dye-encapsulating silica core. In certain embodiments, silica particle growth is quenched at appropriate time intervals to control silica core size by adding either monofunctional PEG-silane (6-9 EO units per chain), resulting in untargeted PEG-Cy5-C’ dots. In certain embodiments, first hetero-bifunctional PEG, functionalized on one end with a silane and on the other with aMSH peptide, immediately followed by monofunctional PEG-silane, is used to quench the reaction. In certain embodiments, the nanoparticle comprises aMSH ligands. In certain embodiments, ligand density is varied between 5 and 15 ligands per particle by adding increasing amounts of heterobifunctional PEG to the growing silica cores. In certain embodiments, subsequent purification from unreacted precursors and / or particle aggregates is performed using gel permeation chromatography (GPC).

[0218] In certain embodiments, nanoparticles as discussed herein are characterized using a particle characterization technique (e.g., fluorescence correlation spectroscopy (FCS), dynamic ligh scattering (DLS), zeta-potential, UV-VIS absorption, emission spectroscopy, transmission electron microscopy).

[0219] For example, fluorescence correlation spectroscopy (FCS) determines particle hydrodynamic size and concentration. Dynamic light scattering (DLS) and / or zeta-potential measurements determine hydrodynamic size and / or surface charge. UV-VIS absorption and emission spectroscopy determine a number of dyes and / or aMSH ligands per particle (e.g., in conjunction with FCS). Transmission electron microscopy (TEM) determines silica core size.Controlling Hydrophobic Particle Surface Patchiness

[0220] In certain embodiments, hydrophobic “patchiness” of nanoparticles is controlled using the methods and techniques described herein. The surface patchiness of the nanoparticles is used to control, among other things, tumor microenvironment response to nanoparticles.

[0221] In certain embodiments, two Cy5-maleimido dye derivatives with different net charges are used: negatively charged sulfo-Cy5(-)-maleimide dye (GE) or positively charged Cy5(+)-maleimide dye (Lumiprobe). As a result of Coulombic interactions with negatively charged ~2 nm sized silica clusters, initially formed in the sol-gel synthesis of silica, negatively charged sulfo-Cy5 dye preferentially ends up on the silica core surface, while positively charged Cy5 can be fully encapsulated.

[0222] In certain embodiments, control over the surface patchiness can be exerted by controlling the number of Cy5 dyes on the surface of the silica core of a nanoparticle by using different concentrations of ammonia as sol-gel catalyst. In certain embodiments, there are between zero and four Cy5 dyes on the silica core surface. In certain embodiments, patchiness has an effect on ferroptosis induction. In certain embodiments, patchiness has an effect on immune cell priming and / or activation. Hydrophobic patchiness from Cy5 dyes ending up on the C’ dot surface can be verified by HPLC. For example, a HPLC using 150 mm Waters Xbridge BEH C4 protein separation columns with 300 A pore size and 3.5 pm particle size, and a water / acetonitrile mixture as mobile phase may be used.Controlling Silica Core and PEG / Ligand Shell Size

[0223] In certain embodiments, the synthesis of the silica core of the nanoparticle is controlled as described herein. The water-based synthesis of C’ dots enables control of the silica core size at thelevel of a single atomic SiCh layer. As described herein, the exceptional degree of particle size control allows generation of nanoparticles (e.g., C’ dots) with overall particle size maintained below the cut-off for renal clearance (e.g., below 15nm) to reduce unwanted off-target accumulations (e.g., in the liver), while varying sizes of core and / or shell. Silica core size is reduced by increasing reaction temperature and / or by decreasing the time of core growth before PEG-silane is added. In certain embodiments, the length of the PEG-silane chains (Gelest) is increased to maintain an overall hydrodynamic size of the nanoparticle. Changing relative sizes of silica core and / or PEG shell of otherwise same hydrodynamic size of nanoparticles (e.g., C’ dots) allows decoupling contributions of silica core and PEG shell to ferroptosis and / or immune cell priming.Controlling Silica Core Composition

[0224] In certain embodiments, addition of compounds (e.g., aluminum-containing compounds) to the nanoparticle core can boost activation of cell populations within the tumor microenvironment.

[0225] In certain embodiments, the silica core composition of nanoparticles is modulated as described herein. In certain embodiments, modulation of the composition of the silica cores affects affinity of iron to C’ dots, which will be chelated by silanol (-SiOH) surface groups in micropores of the sol-gel derived silica core. Silica core composition can be varied, e.g. by the addition of aluminum sec-butoxide, mercapto-silane, and / or iodo-silane moieties into the aqueous sol-gel reaction mixture.

[0226] In certain embodiments, Cy5 dye-silane conjugate can be co-condensed with tetramethoxy- silane (TMOS) and two other precursors (e.g., aluminum-tri-sec-butoxide (Al(OBus)3) / 3-(trihydroxysilyl)propyl methylphosphonate (TPMP) for Al / P incorporation) at appropriate pH to form a Cy5-encapsulating core.

[0227] In certain embodiments, affinity of iron to a silica core is modulated through phosphonate- silane conjugates co-condensed with TMOS in the silica core synthesis. Phosphonates are known for their high affinity to metal ions like iron. Beyond about 15 mole% of phosphonate-silane in the reaction, relative to TMOS, the effect of ferroptosis on amino-acid-deprived MDA-MB-468 TNBCs at C’ dot concentrations of 15 is essentially switched off. Without wishing to be bound to any particular theory, this is due to the high affinity of iron to the phosphonate groups and related reduction of iron release once the iron-loaded particles are internalized by cells. In certain embodiments, phosphonate group bearing C’ dots effect ferroptosis and / or immune cell priming and / or activation. In certain embodiments, microwave plasma atomic emission spectroscopy is used to evaluate nanoparticle iron concentrations. These nanoparticles help delineate molecular mechanisms by which C’ dots induce ferroptosis and / or activation of immune cells.ExamplesExample 1 : Determination of structural properties of tunable immunomodulatory nanoparticle conjugates that enhance activation and cytotoxicity of MUC16-targeting, IL12-secreting (MUC16ecto / IL-12) CAR T cells and immunogenicity of MUC16ecto OC cells.

[0228] The present disclosure describes that structural variations in the described immunomodulatory nanoparticle conjugates can modulate cell death and pro-inflammatory changes. For example, the present example describes that biological activities of ultrasmall nanoparticles comprising silica (or silica nanoparticles) can be modulated by varying specific physicochemical parameters, such as core chemical composition and particle size. Without wishing to be bound to any theory, insights gained from these and other structural investigations with high precision can be leveraged to elucidate mechanisms of action and contributions of distinct particle compositional andstructural features that may maximize specific anti-tumor responses. In some embodiments, the present example describes intrinsic anti-cancer therapeutic properties of ultrasmall silica nanoparticles and related effects on ferroptosis induction and immune cell priming.

[0229] Compositional and / or structural parameters of silica nanoparticles (e.g., PEG-Cy5-C’ dots or C’ dots) responsible for observed modulation of biological properties and physicochemical characteristics can be altered and effects can be assessed on both ferroptosis and the tumor microenvironment (TME). For example, the following parameters have provided correlations with effects on both the immune TME and ferroptosis: (i) silica core size (e.g., ferroptosis-induced cell death substantially decreased as particle size increased from ~6 nm to ~10 nm); (ii) silica core composition (e.g., incorporation of more than 20 mol% of a phosphonate- silane led to a switch-off of ferroptosis- induced cell death in breast cancer cells, presumably by modulating / increasing affinity to iron); (iii) surface chemical heterogeneity / patchiness by working with differently charged dyes and employing HPLC (e.g., increasing surface chemical homogeneity increased ferroptotic cell death); (iv) targeting ligands (e.g., alpha melanocyte stimulating hormone (aMSH) peptide-decorated C’ dots (aMSH-PEG- C’ dots) induced ferroptosis and immune cell activation in a B16-F10 model.

[0230] Synthesis, purification and characterization of PEG-Cy5-C’ dots. Non-targeted PEG-Cy5-C’ dots as an exemplary base particle and89Zr labeled deferoxamine (DFO) functionalized89Zr-DFO-PEG-Cy5-C’ dots as exemplary radiotracers can be synthesized, purified, and characterized using established protocols and a combination of advanced characterization techniques as described herein. In particular, methodologies can include HPLC methods for characterization of such hybrid organic-inorganic core-shell nanoparticles as described herein.

[0231] Control of silica core composition. A set of exemplary PEG-Cy5-C’ dots with varying silica core compositions can be synthesized as described herein. Aside from pure silica precursors, monosodium salt of 3-(trihydroxysilyl)propyl-methylphosphonate (TPMP) can be used to introduce phosphonate groups into the silica core (0-20 mole percent) with higher affinity to iron than pure silica (it is noted that the silica core is microporous with pore sizes <2 nm, where metal ions from iron can be chelated, e.g., by surface Si-OH groups). Inductively coupled plasma (ICP) atomic emission spectroscopy can be used to quantify metal ion content.

[0232] Control of relative silica core and PEG shell size while maintaining overall particle size below 6-7 nm. Water-based synthesis of exemplary silica nanoparticles aide in control of silica core size at the level of a single atomic SiCh layer. The present example describes use of this exceptional degree of particle size control to generate a series of silica nanoparticles with overall particle size maintained below the cut-off for renal clearance (e.g., below 6-7 nm) to reduce unwanted off-target uptake, while varying sizes of core and shell (varying PEG-silane length), respectively.

[0233] Controlling hydrophobic particle surface patchiness. A series of exemplary PEG- Cy5-C’ dots with varying hydrophobic “patchiness” utilizing Cy5 dyes with different net charge (e.g., negatively charged sulfo-Cy5(-)-maleimide dye (GE) or positively charged Cy5(+)-maleimide dye (Lumiprobe)), as well as variations of synthesis parameters (e.g., pH). Hydrophobic patchiness, which can arise from Cy5 dyes ending up on C’ dot surfaces (e.g., C’ dots encapsulate up to 4 Cy5 dyes), can be verified by the number of peaks seen by HPLC (e.g., 1-4) using 150 mm Waters Xbridge BEH C4 protein separation columns (300 A pore size; 3.5pm particle size) and a water / acetonitrile mixture as mobile phase.

[0234] ‘Structural’ measurements. Exemplary C’ dots can be characterized in terms of their size (< 8 nm) and purity (>95%) prior to in vitro testing. For targeted particles, a number of scFv ligands per C’ dot can be about from one to two ligands.Example 2: Synthesis, characterization, and purification of MUC16-targeting C’ dots.

[0235] An exemplary 4H11 scFv amino acid sequence (269 amino acids in length; 28.6 kDa) is shown in SEQ ID NO: 1.MALPVTALLLPLALLLHAEVKLQESGGGFVKPGGSLKVSCAASGFTFSSYAMSWVRLSPEMRLEWVATI SSAGGYIFYSDSVQGRFTISRDNAKNTLHLQMGSLRSGDTAMYYCARQGFGNYGDYYAMDYWGQGTTVT VSSGGGGSGGGGSGGGGSDIELTQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNQLAWYQQKPGQSPE LLIYWASTRQSGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQQSYNLLTFGPGTKLEIKR ( SEQ ID NO : 1 )

[0236] A 4H11 scFv fragment for conjugation to a nanoparticle described herein was designed with three considerations: (1) the primary protein sequence of the variable light (VL) chain and variable heavy (VH) chain; (2) incorporation of a linker between the VL sequence and VH sequence; and (3) incorporation of a flexible C-terminal linker comprising a cysteine residue for conjugation. Production of 4H11 scFv from a full-length protein sequence is unpredictable. Low protein expression yields and / or protein misfolding are common problems related to scFv production. For example, initial attempts to generate a 4H11 scFv using a prokaryotic (E.coli) system failed due to protein misfolding and aggregation. However, suitable protein expression yields and proper protein folding was achieved as determined by specific binding assays to produce suitable 4H11 scFv fragments for use with embodiments of the present disclosure.

[0237] Linker type can also significantly impact conjugation of 4Hll-scFv conjugates to the described immunomodulatory nanoparticle conjugates. For example, a linker comprises a cysteineresidue. Moreover, linker length can also significantly impact conjugation of 4Hll-scFv conjugates to the described immunomodulatory nanoparticle conjugates. In some embodiments, a linker has a length of from about 5 amino acids to about 50 amino acids. In some embodiments, a linker has a length of from about 10 amino acids to about 40 amino acids. In some embodiments, a linker has a length of from about 5 amino acids to about 20 amino acids. In some embodiments, a linker has a length of from about 10 amino acids to about 20 amino acids. In some embodiments, a linker has a length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In some embodiments, a linker has a length of 6 amino acids. In some embodiments, a linker has a length of 7 amino acids. In some embodiments, a linker has a length of 10 amino acids. In some embodiments, a linker has a length of 15 amino acids. In some embodiments, a linker has a length of 20 amino acids.

[0238] An exemplary scFv conjugate amino acid sequence designed for conjugation to a nanoparticle described herein is shown in SEQ ID NO: 2.ME^SJWFLFFLSVTTGVHSEVKLQESGGGFVKPGGSLKVSCAASGFTFSSYAMSWVRLSPEMRLEWVAT ISSAGGYIFYSDSVQGRFTISRDNAKNTLHLQMGSLRSGDTAMYYCARQGFGNYGDYYAMDYWGQGTTV TVSSGGGGSGGGGSGGGGSDIELTQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNQLAWYQQKPGQSP ELLIYWASTRQSGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQQSYNLLTFGPGTKLEIKGGSCGSG EFL EQGHHHHHHHHHH* ( SEQ ID NO : 2 )Where: Signal Peptide, Variable Region, Linker, Tag (7 / A / TEV). Cysteine (for conjugation)

[0239] In some embodiments, a nucleic acid sequence encoding a 4Hll-scFv conjugate described herein is codon optimized for mammalian expression to express a protein sequence of at least 50 (e.g., at least 100; e.g., at least 150; e.g., at least 200; e.g., at least 250) amino acid sequences with at least 80% identity to a portion of SEQ ID NO:1 (e.g., wherein the portion is a variable region) (e.g., atleast 85% identity, e.g., at least 90% identity, e.g., at least 91% identity, e.g., at least 92% identity, e.g., at least 93% identity, e.g., at least 94% identity, e.g., at least 95% identity, e.g., at least 96% identity, e.g., at least 97% identity, e.g., at least 98% identity, e.g., at least 99% identity, e.g., 100% sequence identity to the portion of SEQ ID NO: 1 ).

[0240] In some embodiments, a nucleic acid sequence encoding a 4Hll-scFv conjugate described herein is codon optimized for mammalian expression to express a protein sequence of at least 50 (e.g., at least 100; e.g., at least 150; e.g., at least 200; e.g., at least 250) amino acid sequences with at least 80% identity to a portion of SEQ ID NO:2 (e.g., wherein the portion is a variable region) (e.g., at least 85% identity, e.g., at least 90% identity, e.g., at least 91% identity, e.g., at least 92% identity, e.g., at least 93% identity, e.g., at least 94% identity, e.g., at least 95% identity, e.g., at least 96% identity, e.g., at least 97% identity, e.g., at least 98% identity, e.g., at least 99% identity, e.g., 100% sequence identity to the portion of SEQ ID NO: 2).

[0241] Prior to particle attachment, 4Hll-scFv conjugates can be prepared by a two-step process: (i) reduction of the terminal cysteine with tris(2-carboxyethyl)phosphine (TCEP), to yield a cysteine with a free thiol; and (ii) site-specific conjugation of the electrophilic thiol with a maleimide functionalized construct. 4Hll-scFvs can be conjugated to C’ dots using a copper-free azide-alkyne click chemistry procedure. To that end, an azide-PEG3-maleimide linker can first be reacted with the reduced cysteine on the C-terminal region of the purified scFv, yielding an azide-containing 4H11- scFv. After purification, the product can be mixed with DBCO-functionalized C’ dots, forming a 4H11- scFv-C’ dot product, followed by final purification with HPLC to remove unreacted scFv.Example 3: Syngeneic Models of Murine Ovarian Peritoneal Carcinomatosis.

[0242] To understand how immunomodulatory nanoparticle conjugates can directly prime and activate the TME prior to administration of CAR T cells, two genetically distinct syngeneic murine models of hMUC16+ovarian peritoneal carcinomatosis were created using an ID8 model. Given the heterogeneity of human primary high-grade ovarian tumors and their metastatic (e.g., peritoneal, omental) lesions, differential pro-inflammatory and cytotoxic responses of these two metastatic models to immunomodulatory nanoparticle conjugates described herein, with and without CAR T cells, were examined. ID8 cells, ovarian epithelial cells, spontaneously transformed in vitro, acquire tumorigenic capacity and cause suppressive ascites fluid accumulation, a hallmark of late stage-advanced human ovarian carcinomatosis.

[0243] Orthotopic ID8 tumors were created in C57BL6 mice inoculated intraperitoneally (i.p.) with IxlO7ID8 cells for acquiring phenotypic and molecular signatures. Tumor growth was monitored by bioluminescence imaging (BLI) until detectable at about 2 weeks post-inoculation. The median survival time was about 47 days (data not shown), with formation of ascites at ~4 weeks post-injection (p.i.). Molecular characterization of tumor and splenic tissues was performed using quantitative reverse transcription polymerase chain reaction (RT-qPCR) over a 3-week period; data was measured as a fold change in expression relative to week 2, the earliest time point at which tumor tissue could be harvested. Gene expression analysis revealed that, as the tumor grew, pro-inflammatory cytokines (e.g., Ifny), chemokines associated with CD8+T cell recruitment (e.g., Cxcl9, CxcllO), and effector proteins (e.g., perforin, granzyme B) were downregulated (data not shown).Example 4: Cellular uptake, viability and gene expression modulation by tunable immunomodulatory nanoparticle conjugates in ID8 cells.

[0244] The present example also describes a custom-designed anti-hMUC16 scFv-conjugated ultrasmall nanoparticles for examining biological responses in ovarian cancer (OC) models (FIG. 3); the scFv was derived from the clonal sequence of the 4H11 CAR molecule (see, e.g., SEQ ID NO: 1 ). scFv ligand density per silica nanoparticle (e.g., C’ dot) can be optimized by varying the number of scFv moieties conjugated to the particle surface using methods described herein. Moreover, the extent to which such variations enhance immunogenicity and tumor cell kill in the TME can be determined, in addition to whether these improve upon results with non-targeted ultrasmall nanoparticles. Without wishing to be bound to any theory, the present disclosure contemplates that by optimizing the structural properties of non-targeted and MUC16-targeting ultrasmall nanoparticles (e.g., PEG-Cy5-C’ dots), and elucidating their biologic response correlations, improved CAR T cell cytotoxicity and efficacy, while limiting immune suppression, can be provided.

[0245] FIG. 3 shows concentration and time-dependent gene expression modulation by C’ dots in ID8 cells. A heatmap of gene expression profiles of ID8 cells incubated with increasing C’ dot concentrations (0, 0.1, 1, 15 pM) is shown over a 6-day period. Each quadrant represents an independent biological replicate.

[0246] In some embodiments, the immunomodulatory nanoparticle conjugates described herein regulates a pattern recognition receptor (e.g., a toll receptor, e.g., RIG pathway).

[0247] Cellular uptake of exemplary PEG-Cy5-C’ dots (without cellular receptor- and / or antigen- targeting ligands) in ID8 cells was found to rise in a concentration-dependent manner over a 24-hour period by flow cytometry (FC), expressed as median fluorescence intensity (MFI) (FIG. 4A).Further, exemplary C’ dot internalization by ID8 cells was observed over 24 hours using confocalmicroscopy (FIGs. 4B and 4C). Silica nanoparticles were well tolerated across a wide range of concentrations (IpM-lOpM) and incubation times (e.g., up to 6 days) without significant loss of viability (data not shown), as noted previously.

[0248] Cellular uptake studies in ID8 cells were preformed using a custom-designed anti- hMUC16 scFv-conjugated nanoparticle, by attaching single chain fragments (scFvs) to the particle surface to determine whether increased internalization amplifies anti-tumor immune responses and targeted cell kill. The protein sequence for C’ dot conjugation was derived from a previously described monoclonal antibody (clone 4H11) targeting the carboxy -terminal portion of MUC16 (see, e.g., SEQ ID NO: 1 ). Variable regions of the antibody were grafted onto a scFv scaffold, and the C- terminus was further modified by adding a single cysteine residue to allow for site-specific conjugation, a TEV (cysteine protease) cleavage site, and a hexa-histidine tag to aid purification (see, e.g., SEQ ID NO: 2). The gene was constructed and expressed in a mammalian expression system using CHO cells. The 4H11 scFv was successfully expressed and purified, with the terminal cysteine obtained in an oxidized state (e.g., cysteine). 4H11 scFv conjugates were then prepared for subsequent particle attachment as described herein. Final purification was performed with HPLC to remove unreacted scFv (FIG. 4D). Cellular uptake studies were then conducted in ID8 cells with the final product, anti-hMUC16 scFv-PEG-C’ dots, which showed substantially higher (factor of ~4) uptake than that found with unmodified C’ dots (FIGs. 4E and 4F).

[0249] Assess differential modulatory effects of non-targeted and MUC16-targeting C’ dots on the biological properties of MUC16ectoOC cells, immune cells, and CAR T cells. In vitro cell screening assays can be conducted using MUC16ecto+ ID8 cell lines, CAR T cells, bone marrow derived macrophages (BMDMs) and dendritic cells exposed to selected non-targeting and MUC16-targeting particles (as described herein) in order to identify lead candidates that enhance anti-tumor immune cell cytotoxicity and ferroptosis. These studies can serve to inform in vivo work, as well as yield mechanistic insights regarding distinct particle features driving pro-inflammatory and / or cell death responses.

[0250] Transduction and characterization of OC cells with MUC16ect0and GFP+ / luc+ retroviral constructs. GFP / luc+ MUC16ect0ID8 cells can be created by their sequential retroviral transduction using two SFG retroviral constructs encoding the (i) retained extracellular, transmembrane and cytoplasmic domains of the MUC16 antigen (MUC16ecto) and (ii) GFP / Lucif erase. Positive transduced cells will be enriched by FC following each transduction. Characterization of these OC cell lines will be performed using RT-qPCR.Example 5: Cellular uptake, viability, and gene expression modulation by tunable immunomodulatory nanoparticle conjugates in ID8 cells.

[0251] The present example confirms that an anti-MUC16 scFv-conjugated nanoparticle (e.g., C’ dot) probe can be used to produce biological responses in OC models (FIGs. 5A-5E); the scFv was derived from the clonal sequence of the 4H11 CAR molecule. The scFv ligand density per nanoparticle can be engineered by varying the number of scFv moieties conjugated to the particle surface using methods described herein, and can be determined the extent to which such variations enhance immunogenicity and tumor cell kill in the TME, in addition to whether these improve upon results with non-targeted C’ dots. Without wishing to be bound to any theory, the inventors hypothesize that by engineering the structural properties of non-targeted and MUC16-targeting PEG-Cy5-C’ dots, andelucidating their biologic response correlations, they can improve CAR T cell cytotoxicity and efficacy, while limiting immune suppression.

[0252] Cellular uptake, viability and expression profiling in particle-exposed OC cells.The present example also describes cellular uptake studies in MP cells using non-targeting PEG-Cy5- C’ dots (plain C’ dots) and a custom-designed anti-hMUC16 scFv-conjugated C’ dot. The latter was designed by attaching scFvs to the particle surface to determine whether increased internalization amplifies anti-tumor immune responses and targeted cell kill. As described herein, a protein sequence for C’ dot conjugation from a previously described monoclonal antibody (clone 4H11) targeting the carboxy-terminal portion of MUC16 (see, Dharma Rao T, Park KJ, Smith-Jones P, lasonos A, Linkov I, Soslow RA, Spriggs DR. Novel monoclonal antibodies against the proximal (carboxy-terminal) portions of MUC16. Appl Immunohistochem Mol Morphol. 2010;18(5):462-72. Epub 2010 / 05 / 11.Doi: 10.1097 / PAI.0b013e3181dbfcd2. PubMed PMID: 20453816; PMCID: PMC4388147, the contents of which is hereby incorporated by reference herein in its entirety). Variable regions of the antibody were grafted onto a scFv scaffold, and the C-terminus was further modified by adding a single cysteine residue to allow for site-specific conjugation, a TEV (cysteine protease) cleavage site, and a hexahistidine tag to aid purification. The gene was constructed and expressed in a mammalian expression system using CHO cells. The 4H11 scFv was successfully expressed and purified, with the terminal cysteine obtained in an oxidized state (i.e., cystine). 4H11 scFv conjugates were then prepared for subsequent particle attachment. Final purification with HPLC removed unreacted scFv (FIG. 5A).

[0253] Cellular uptake for both particle probes in MUC16+ and MUC16- MP cells was seen to increase in a concentration-dependent manner (0.1gM-3jjM) over a 24-hour (h) period by flow cytometry (FC), expressed as median fluorescence intensity (MFI) (FIG. 5B), but with significantlylower uptake observed in MUC16- MP cells. For MUC16+ MP cells, substantially higher uptake (factor 10-fold at lOOnM) was noted for anti-hMUC16 scFv-PEG-C’ dots relative to plain C’ dots, unlike that found for non-MUC16-expressing (MUC16-) MP cells. Furthermore, cellular internalization was greater for the targeted particle probe over a 24-h period by confocal microscopy (FIGs. 5C and 5D). No significant loss of viability was observed (data not shown), as demonstrated in our prior uptake studies using different cancer cell types. MUC16-targeting C’ dots also significantly enhanced representative proinflammatory cytokine / chemokine secretion over non-targeting C’ dots (FIG. 5E).

[0254] Syngeneic Models of Murine Ovarian Peritoneal Carcinomatosis. To understand how C’ dots can directly activate the TME prior to administration of CAR T cells, the present disclosure describes genetically distinct syngeneic murine models of MUC16+ ovarian peritoneal carcinomatosis: ID8 models exhibiting a spectrum of immune suppression (i.e., low (L) / high (H) VEGF levels; ID8(L) and ID8(H)) and genetically engineered Myc overexpressing (Myc+), p53null OC cell lines with or without mutant BRCA1 (Breast Cancer gene 1) - designated c-Myc-i- p53- / - (MP) and c-Myc-i- p53- / - BRCAl-mutant (homologous recombination (HR) deficient; MPB 1). Given the heterogeneity of human primary high-grade ovarian tumors and their metastatic (e.g., peritoneal, omental) lesions, the present example describes examination of differential pro-inflammatory and cytotoxic responses of these metastatic models to C’ dots, with and without CAR T cells. These cell lines cause suppressive ascites fluid accumulation within the peritoneal cavity, a hallmark of late stage- advanced human ovarian carcinomatosis.Example 6: Induction of pro-inflammatory, immune modulatory and cytotoxic responses of OC cells.

[0255] To examine intrinsic therapeutic properties of C’ dots and possible molecular mechanisms driving these activities, ID8(L), MP, and MPB1 cells were exposed to 15pM C’ dots (24h) for bulk RNA sequencing (RNA-seq) (FIGs. 6A-6B). Gene set enrichment analysis (KEGG in FIG.6A; Reactome in FIG. 6B) revealed significant genotype- specific regulation of pro-inflammatory, immunogenic, and cytotoxic signatures. Relative to ID8(L) cells, MP and / or MPB1 cells revealed upregulation of TN Fa, cytokine / chemokine signaling, protein transcription and translation, and cell cycle checkpoint pathways. Induction of metabolic and ferroptotic (cytotoxic) pathways was significantly more pronounced in ID8(L) cells.

[0256] On the basis of the described bulk RNA-seq analyses, genes representing five major functional classes (cytokines, antigen presentation, iron metabolism and ferroptosis, damage-associated molecular patterns (DAMPS) and cell cycle) were selected for expression profiling in cell lines.Expression levels were measured as fold changes over the non-particle exposed group and normalized using the 2-AACTmethod. Overall, genotype-and time-dependent significant upregulation (e.g., greater than 2) of multiple transcripts within these classes over a 72-h period were observed (FIG. 7A).Moreover, genes related to iron metabolism were upregulated in ID8 and MP cells e.g., ferritin heavy chain, Fthl). Further, Slc3a2 and Slc7all, two subunits of the cystine / glutamate antiporter system, which may act as a compensatory mechanism to states of increased oxidative stress, were upregulated by C’ dots. Of note, genes related to antigen presentation were significantly upregulated in all lines (e.g., TAPI, TAP2), while pronounced changes in DAMPS (e.g., Hsp70al a / al b) were observed in both ID8 and MP cells at 24h. Mechanistically, without wishing to be bound to any theory, C’ dots mayinduce a cell stress response in OC cells, for example, through ferroptotic induction, as evidenced by a significant increase in Hsp70 mRNA in vitro and in vivo (see FIG. 7A; FIG. 10B). Sustained transcriptional activation of cytokines correlated with increased cytokine secretion profiles (FIG. 7B), which were significantly upregulated in MP and MPB1 cells. These expression profiles highlight pronounced and sustained proinflammatory and immunogenic responses, particularly for MP and MPB1 cells. Importantly, C’ dot treatment resulted in G2 / M cell cycle arrest in a genotype-independent manner (FIG. 7C), which correlated with an upregulation of the cell cycle inhibitor Cdknla (or p21, FIG. 7A). This finding may implicate upregulation of a DNA damage response mechanism or senescence induction. Similar responses were observed with human OC lines, UWB 1.289 + / -BRCA1 (data not shown).

[0257] Next, in order to demonstrate that C’ dots can synergize with MUC16-targeting, IL- 12 secreting CAR T cells (referred to hereafter simply as “CAR T cells” although the present disclosure recognizes that any engineered immune cells, e.g., engineered T cells, beyond the exemplified MUC16- targeting, IL- 12 secreting CAR T cells may be used in combination with the provided immunomodulatory nanoparticle conjugates) and increase anti-tumor cytotoxicity, a series of co-culture experiments were performed with CAR T cells and luciferase ( / wc+j-expressing OC cells, the latter with or without particle pre- treatment. Luc+ OC cells were incubated with 15|iM C’ dots for 24h prior to CAR T cell exposure for an additional 24h using an effector-to-target cell ratio of 1:1 and a luciferase-based assay for measuring viability. All cell groups were assayed at the same time. Despite the fact that particles were non-targeted and non-optimized, combination treatment demonstrated statistically significant cell viability losses after normalizing to results of a control group (i.e., media without exposure to C’ dots or CAR T cells), which synergized with CAR T cells (FIG. 8A; FIG. 8B).By contrast, no appreciable decrease in viability was observed when OC cells were pre-incubated with15pM C’ dots using a CD19-targeted CAR T cell control, relative to a control group without CAR T cells (data not shown). FIG. 8C shows competitive binding and / or viability of luc+ ID8(L) 24h after incubation following the same treatments as in (a), except using MUC16-targeting C’ dots.Interestingly, significant viability losses were noted for HR-deficient MPB1 cells relative to the other cell lines examined, an area under active investigation. In line with our cytotoxicity data, significant changes were also found in TNFa, IFNy, and IL-2 levels, greatest with the combination therapy (FIGs. 8D-8F, boxed). Finally, CD19-targeted CAR T cell controls showed no appreciable decrease in viability when ID8 cells were pre-incubated with 15gM C’ dots (data not shown). Together, these findings suggest that non-targeted / non-optimized C’ dots favorably modulate immunogenicity of all OC cell lines tested to significantly increase effector cell kill, with the potential to activate the TME in orthotopic metastatic OC models prior to CAR T cell therapy. OC cell viability losses may be considerably higher for a fully optimized and targeted particle probe.Example 7 : Immune modulation and induction of cell death by immunomodulatory nanoparticle conjugates to maximize anti-tumor responses to CAR T cells.

[0258] Serial monitoring of tumor burden and organ-specific particle distributions using optical imaging. Monitoring of luc+ OC cells via bioluminescence imaging (BLI) can be performed as early as one-week post-injection (p.i.), before macroscopic lesions can be identified. BLI can be used to confirm tumor engraftment prior to treatment studies, and for monitoring treatment responses. In vivo BLI (FIG. 9A) in the ID8 model (i) facilitates serial tracking of metastatic disease and (ii) guides time-dependent harvesting of critical tumor-bearing tissues within the peritoneal cavity. Cy5-encapsulating C’ dots were able to be detected via whole-body imaging (FIG. 9B) and within major organs using correlative ex-vivo fluorescence imaging (FIG. 9F), as compared with saline vehicle (FIG. 9E). High Cy5 fluorescence signal-to-background ratios within harvested organs and ascites (FIGs. 9F and 9H) also correlated well with harvested C’ dot-treated tissues on BLI (FIG. 9D); saline vehicle-treated tissues are also shown (FIG. 9C). After i.v. injection of a single particle dose (12 nmoles), Cy5 fluorescence imaging can be acquired and compared with BLI using the IVIS SpectrumCT, before and after i.p. luciferin administration, in all animals to assess particle localization to tumor-infiltrated tissues. Imaging techniques described herein can also be conducted using the immunomodulatory nanoparticle conjugates (e.g., MUC16-targeting nanoparticles) described herein.

[0259] Screening PK / biodistribution and PET studies.89Zr radiolabeling of particle probes can be performed for screening PK / biodistribution (n=5 mice / C’ dot tracer; ~20 pGi / mouse) and via serial PET imaging (n=5 mice / C’ dot tracer; ~200 pCi / mouse). Animals can be scanned up to 72 hours after i.v. -injection of89Zr-DFO-PEG-C’ dots94or89Zr-DFO-scFv-PEG-C’ dots (e.g., MUC16-targeting nanoparticles) in mice implanted with -IxlO7OC cells (n=5 / cohort; $). Blood and urine specimens (n=3 / cohort) can be collected and analyzed using radio-iTLC to confirm stability of the product. Data (FIG. 9G) demonstrate a high tumor-to-background ratio on maximum intensity projection (M1P) PET imaging of ID8 tumor-bearing mice (3 wks post-inoculation) 24 hours p.i. of89Zr-DFO-PEG-C dots.

[0260] Particle-driven cellular and molecular profiles and suppressive activities in the ID8 model can be used as a means of maximizing anti-tumor responses and reducing off-target toxicity to inform suitable treatment strategies for c-Myc-i- p53- / - and c-Myc-i- p53- / - BRCAl-mutant GEM models. For example, design of new precision treatment paradigms in high-grade OC models will rest on detailed knowledge of cellular and molecular profiles of nanoparticle immunoconjugates that alterthe tumor-immune landscape. These candidates, along with optimized concentrations and incubation times, can elucidate abilities of the described nanoparticles and nanoparticle conjugates to modulate / activate specific immune cell populations, pro-inflammatory phenotypes, and cell death in the ID8 (and then in more clinically relevant MP and MPB1 models). Particle dose escalation procedures (up to 6 doses) can be implemented, as needed, at levels that induce the foregoing responses (e.g., 12 nmoles / dose, 60 M stock): (i) maximum fold changes (i.e., >2) for specific genes (e.g., cytokines and DAMPs), (ii) increased cytotoxic T cell activity (j'CD8+ / Treg), (iii) reduced immune suppression (JM1 / M2, J, percentage (%) of Tregs, MDSCs in tumors), and (iv) enhanced cytotoxicity will serve as the initial dosing level for MP and MPB 1 models with the aim of maximizing efficacy, as described herein.

[0261] Transcriptome-wide profiling and cytokine release assays in biological specimens. In vivo pilot studies with non-targeted / non-optimized C’ dots in adult C57BL6 mice, inoculated with IxlO7ID8(L) cells, were assigned to one of four treatment arms ( / z=6 mice / arm): (i) vehicle control (CT), (ii) non-targeting C’ dots (3 doses; 12 nmoles / each, 3 days apart), (iii) CAR T cells (5xl05), or (iv) combination therapy, with CAR T cells given 4 days after the final C’ dot dose. Vehicle replaced CAR T cells in the remaining treatment arms. All animals were sacrificed at the same time point - 10 days (lOd) post CAR T cell or vehicle treatment. RT-qPCR of harvested tumor tissues showed significant upregulation of multiple expression signatures (i.e., fold changes extending beyond horizontal black bars in FIG. 10B and FIG. 10C). Even 14d / 10d post last C’ dot / CAR T dose, a sustained C’ dot-driven upregulation of multiple proinflammatory cytokines, DAMPs, and immune markers (i.e., Type I IFN) were seen, notably with combinatorial treatment without adverse events, as compared with CAR T cells alone (FIG. 10B). Relative to monotherapies, the combination treatmentalso yielded an expanded spectrum of proinflammatory responses. Importantly, a similar trend was not seen in the spleen (FIG. IOC), suggesting that C’ dot modulatory effects were localized to target tissues. Ascites specimens from the combination group compared with the CAR T alone group, revealed a substantial increase in concentrations of key inflammatory cytokines driving T cell recruitment and cytotoxicity, including TN Fa (FIG. 10D). Finally, the percentage of CAR T cells persisting in the peripheral blood neared significance in the combination arm relative to the CAR T cell arm itself (FIG. 10E), suggesting that C’ dots can enhance persistence over time. Similar- techniques can also be conducted using the immunomodulatory nanoparticle conjugates (e.g., MUC16-targeting nanoparticles) described herein.

[0262] Immunophenotyping and transcriptomics. Changes in gene expression that translate into phenotypic changes in the TME can be confirmed using the methods described herein, and dosing frequency established for evaluating nanoparticle immunoconjugate can be determined.Immunophenotyping can be performed on tumor, ascites, and splenic tissue specimens harvested from ID8 models for both particle- and vehicle-treated cohorts. A multiparametric How cytometry (MPFC) panel can be used to analyze and compare populational changes in immune cell infiltrates. To evaluate effective anti-tumor immune responses among cohorts, the following endpoints can be used: (i) innate immune responses, (ii) cytotoxic T cell activity ()CD8+ / Trcg), and (iii) reduced immune suppression ( M1 / M2, J, Tregs, MDSC) within tumors.

[0263] Immunophenotyping data in the ID8(L) model using the same four treatment arms, dosing schedules, and time points are shown in FIG. 10A. Tumor tissue specimens obtained (FIGs. 11A-11E, FIG. 12A-12F) showed statistically significant changes in CD8+ T cells, CAR T cells, CXCR3+CAR T cells, Tregs, and the CD8+ / Treg ratio, greatest for the combination arm (FIGs. 11A-HE). A roughly 7-fold increase was found for CXCR3+CAR T cells (i.e. , activated T cells).Surprisingly, the CD8+ / Treg ratio increased 100-fold, largely due to a near complete loss of Tregs over this time period. This was accompanied by significant and sustained increases in co-stimulatory molecules (i.e., CD40 and CD86) on Ml macrophages and the M 1 / M2 ratio, as well as an acute increase in the overall DC population. Factor of 3 decreases in MDSCs were also seen over this 10-day period (FIG. 12A-12F). These changes - induced by non-optimized / non-targeted particles - suggest that C’ dots have the potential to substantially activate anti-tumor immune responses in the TME without off-target effects - effects that may be further amplified once particles arc further engineered or if utilizing a targeted probe.

[0264] Single-cell transcriptomics analyses (scRNA sequencing, scRNA-seq) can be conducted to investigate transcriptional changes and identify specific cell populations responsive to particle treatment (e.g., lineage differentiation of Tregs; polarization spectrum of macrophages). In preliminary scRNA-seq data (FIGs. 13A-13H), an unsupervised clustering analysis on ascites specimens harvested from ID8 tumor-bearing mice was performed 4 days after three i.v. -injections of C’ dots (12 nanomoles / dose; 6O|1M stock). Six clusters (FIG. 13A) were identified: one tumor and five immune cell subpopulations. Gene set enrichment analysis (GSEA) for particle- versus saline vehicle-exposed tumor cells revealed 3 distinct pathways (FIGs. 13B, 13F-13H) - two upregulated (pro -inflammatory response and cell death) and one downregulated (anti-inflammatory / pro-tumorigenic wound healing). Importantly, GSEA also identified significant gene enrichment of pathways regulating responses to external and biotic stimuli, as exemplified by the Hsp protein family (data not shown), lending support to the mechanism proposed for C’ dot upregulation of IFN pathways (FIG. 8C). Finally, in agreement with our immunophenotyping data (FIGs. 11A-11E, FIGs. 12A-12F), scRNA-seq analysis showedsignificant upregulation of proinflammatory phenotypes in macrophages, DCs and T cells (i.e., Slamf7,Cxcl9, CxcllO) and downregulation of immune suppressive phenotypes (e.g., Argl, Cd206, Cdl63, Trem2, Mif, Ccl8, Cd83, Sgkl, Nr4a2) (FIGs. 13C-13E).

[0265] FIGs. 11A-11E show single cell dissociates of ID8(L) tumor specimens stained for surface markers at 4 and 10 days after CAR T cell or vehicle administration: CD8+ T cells (FIG. 11A), CAR T cells (FIG. 11B), Tregs (FIG. 11C), CXCR3+CAR T cells (FIG. 11D), and the CD8+ / Treg ratio (FIG. HE). Data was computed as mean+ / -s.e.m. *=p<.05, **=p<.01, ***=p<.005, ****=p< .001 (1-way ANOVA with Tukey test for multiple comparisons).

[0266] Using the same sample cohorts and analytical procedures as FIGs. 11A-11E, analyses were performed by FC for assessing Ml CD40+, Ml CD86+, M1 / M2 ratio, DCs, MDSCs, and M2+ cells at indicated times. *=p<.05, **=p<.01, ***=p<.005, ****=p<.001 (1-way ANOVA with Tukey test). FIG. 12A shows %CDllb+F4 / 80+CD40+ for CT, C’ dots, CAR T, and Combo treatments. FIG. 12B shows %iNOS+ of CDllb+F4 / 80+ for CT, C’ dots, CAR T, and Combo treatments. FIG. 12C shows M1 / M2 for CT, C’ dots, CAR T, and Combo treatments. FIG. 12D shows %Ly6C+Ly6G+ of CDllb-i- for CT, C’ dots, CAR T, and Combo treatments. FIG. 12E shows %Argl+ of CDllb+F4 / 80+ for CT, C’ dots, CAR T, and Combo treatments. FIG. 12F shows %CDllc+MHCll+ for CT, C’ dots, CAR T, and Combo treatments.Example 8: Nanoparticle immunoconjugates drive enhanced anti-tumor immunity.

[0267] Orthotopic ovarian cancer (OC) models exhibiting a spectrum of immune suppression highlights issues of intratumoral heterogeneity which pose a major hurdle to effective treatment planning in the clinic. Methods and compositions described herein can be used for treating the TME ofMP and MPB1 models, in turn, enabling investigation of differential immunomodulatory effects and cytotoxicity across a range of OC models.

[0268] Transcription profiling and cytokine release screening assays in biological specimens. Using particle dosing strategies as described herein for treating ID8 tumors, the procedures described herein can also be performed using nanoparticle immunoconjugates as described herein.Bulk RNA-seq can be used to screen and quantify fold changes in gene expression with inoculated MUC16+ MP and MPB1 cells (n=64 mice total). Changes in cytokine expression can be validated by Luminex-based assays. Tumor growth can be monitored and animals can be euthanized for harvesting tissue specimens at study endpoints to conduct biological assays and histopathology.

[0269] Immunophenotyping and transcriptomics of tumor-infiltrating cells. Procedures described herein for immunophenotyping (IP) analyses in MP / MPB1 models (same collection time points) can be repeated using the same nanoparticle immunoconjugates and dosing frequencies as in the present example as described herein. scRNA-seq analyses for harvested samples exposed to either particles or vehicle at the same collection time points can also be performed as described herein (n=64 mice total for combined studies).

[0270] Ferroptotic Induction. The extent to which observed changes in immune TME parameters can be attributed to ferroptotic induction versus other cell death processes (e.g., immune- related) can be examined by repeating experiments with and without liproxstatin-114. ID8, MP, and MPB1 tumor models ($; 6-8 weeks old) can be i.v.-injected with three, 12 nmol doses (60pM stock) of particles (e.g., nanoparticle immunoconjugates as described herein) or saline vehicle. For each model, changes in tumor growth will be monitored in 3 cohorts of mice (n=6 mice / cohort): (i) C’ dots without and (ii) with liproxstatin- 1 and (iii) vehicle (control). Tumor burden can be monitored in situ andassessed ex-vivo by optical imaging (see Il.b.l) over at least 10 days. H&E staining of tumor tissues can be performed to assess the degree of necrosis using automated image analysis (Qupath). RT-qPCR can also be performed to assay genes related to ferroptosis, iron metabolism, and antigen presentation. Transcriptional changes in key genes can be confirmed at the protein level by western blots. To evaluate effective anti-tumor immune responses in the Tumor Microenvironment (TME), the following endpoints can be used: (i) Efficacy: scFv-C’ dots, C’ dots are more effective than vehicle alone; (ii) j'T cell activity (CD8+ / Treg), (iii) reverse immune suppression (i.e., '['M1 / J.M2, j,Tregs, |MDSCs, J, TAMS, T IFNs, | antigen presentation); (iv) max tumor %ID / g / min off-target.

[0271] In case the MP and MPB1 models are less immune-responsive in vivo than the ID8 model using an optimized dosing strategy, particle concentrations can be increased from 60 pM to lOOpM and dosing frequencies can be re-evaluated (i.e., 1, 3, 6 doses). Preliminary studies (e.g., FIG. 5) show that particle concentration is one of the major drivers of ferroptosis and immunomodulation. Accordingly, increasing particle concentration can further augment these effects to potentially improve treatment of immune suppressive TMEs.

[0272] Animal studies described herein that involve longitudinal outcomes (PK, tumor burden, etc.) can be reported per time point. Each animal’s profile can be characterized by a summary statistic (such as area under the time-concentration profile) and compared using a rank-based test. Gene expression changes can be conducted using a log-transform. Point estimates and confidence intervals can be back- transformed, and reported as fold-change using the delta method. For scRNA / TCR-seq experiments, the Bioconductor workflow can be followed for pre-processing, and clustering and lineage inference can be performed. Pathway and gene set overdispersion analysis (PAGODA) can be used to detect transcriptional heterogeneity within a cell population, and a weighted principalcomponent analysis (PCA) will be conducted for each gene set defining tumor and immune populations and states.Example 9: Assess differential anti-tumor immune responses of CAR T cells in combination with C’ dots to maximize efficacy and expand the window for CAR T cell therapy.

[0273] Determine timing window for CAR T cell administration in orthotopic models. An injection time of CAR T cells can be determined in order to achieve a statistically significant survival benefit in mice inoculated with MP and MPB1 cells. 5xl05CAR T cells can be i.p. injected at two time points (days 17 and 24 post implantation) to assess a timing interval that results in the best median survival (n= 10 mice / cohort).

[0274] Assess anti-tumor cytotoxic effects of CAR T cells for particle-exposed and nonexposed tumors. Particle-driven immunomodulation of the TME can improve outcomes for CAR T cell therapies in solid tumors. To evaluate potential synergies, combination strategies can utilize a single nanoparticle immunoconjugate prior to CAR T cell treatment (Schema, FIG. 10A). Three responses can be evaluated: (i) tumor burden by ex vivo BLI, (ii) increased anti-tumor immunity by IP, and (iii) median survival times.

[0275] Treatment strategies combining CAR T cells and particles. Particle dosing strategies that maximize anti-tumor responses in OC models as described herein can be applied to MP and MPB 1 mice, with and without subsequent CAR T cell therapy for IP and scRNA-seq. For each model, five cohorts of mice (n=4 mice / cohort) can be imaged and treated as follows: (i) IL 12+ CAR T cells; (ii) particle alone; (iii) combination C’ dots / IL12+ CAR T cells; (iv) saline vehicle, and (v) IL12- CAR T cells. For the MP and MPB1 models, CAR T cells can be injected post-inoculation using an optimal timing window as described herein. Tumor, spleen, and ascites specimens can be harvested andassessed for treatment endpoints. Model conditions meeting these endpoints can undergo transcriptomics analysis (n=2 cohorts, 4 animals / cohort). IP studies can be conducted in NOD-SCID gamma (NSG) mice to elucidate the contribution of endogenous immunity to treatment responses.Confirmation of a role for particle-mediated Treg decreases (see FIGs. i 1 A- HE) can be investigated by conducting IP and Treg transfer studies in depleted regulatory T cell (DEREG) mice. This can allow for a distinction between Treg loss of phenotypic / functional lineage commitment and selective depletion of Treg cells.

[0276] Survival studies. Survival studies can be performed in the MP and MPB1 tumor models using the first four treatment arms as described above (n=10 mice / cohort). Histopathology can be performed at the study endpoint.

[0277] Induction of ferroptosis for improving antigenic heterogeneity in MP and MPB1 tumors. One major barrier to effective immunotherapy targeting is antigenic heterogeneity, limiting recognition by cellular Its. To assess for antigen variants, post-treatment changes were monitored (FIG. 14) in both homogeneous (pink curve; “CAR T + C’ dot - ID8”) and heterogeneous (black curve, ‘Mixed’ MUC16+ / -; “CAR T + C’ dot - Mixed”) ID8(L) models. Pilot data showed significant and nearly equivalent tumor volume losses in these models over saline (blue curve; “Saline - Mixed”) after i.v. -injection of dual non-optimized MUC16-targeting C’ dots and CAR T cells. Significant differences in tumor volumes were also seen between bilateral MUC16+ and MUC16- flank tumors and relative to vehicle control (inset). Findings indicate successful treatment of heterogeneous tumors without antigen escape variants, as they maintain equivalent levels of antigen (data not shown). No significant weight loss nor significantly elevated cytokines (e.g., IL-6, TNF-a, IFN-y) were found for any cohort to suggest toxicity (data not shown). Importantly, particle-induced cell death programs, such asferroptosis, may target cells independently of their antigenic profile through a spreadable activity and enhance effectiveness of CAR T cell therapy. To investigate this, the same procedures as described above can be used for both OC models, utilizing four cohorts (n=5 / cohort): (i) nanoparticle immunoconjugate; (ii) CAR T cells; (iii) combination therapy; and (iv) vehicle. Transcriptomics studies can be repeated, but can incorporate T cell repertoire analysis to evaluate clonality.Example 10: CpG-targeting nanoparticle immunoconjugates and uses thereof.

[0278] CpG-targeting C’ dots'. Despite their promise as cancer immunomodulatory agents, STING and Toll-like receptors (TLR) agonists (e.g., unmethylated cytosine-phosphate-guanine oligodeoxynucleotides (CpG-ODNs)), designed to stimulate the immune system, have failed to demonstrate sufficient antitumor efficacy and often exhibit poor cellular uptake and are prone to degradation. Non-specific interactions with proteases, nucleases and immune cells can diminish their immune modulatory activities and lead to adverse reactions, thereby compromising their safety.Ongoing clinical trials in melanoma patients using locally- or in tratumorally -injected CpG-ODNs, as monotherapies or in combination with ICBs have provided evidence of tolerability and improved immune responses. However, no single approved product has yet emerged, highlighting a critical need for newer, more potent, delivery agents.

[0279] To this end, and to address key limitations of TLR9 agonists, a first-in-class ultrasmall and renally clearable TLR9-targeting agents was constructed from a FDA Investigational New Drug (IND)-cleared fluorescent core-shell silica base NP, Cornell prime dots (C’ dots), for i.v. injection. Conjugation of multiple CpG-ODN ligands to the C’ dot surface (FIGS. 15A-15C) can substantially augment their immunomodulatory properties and other biological activities over that of the base nanoparticle. Detailed structural investigations after conjugating two different nuclease-resistantazide-modified CpG-ODN ligands (e.g., CpG Class B (CpG-B)) to the surface of dibenzocyclooctyne (DBCO) modified C’ dots were performed to maximally amplify pro-inflammatory responses and augment innate immunity, alone or with ICB. CpG motifs vary in their molecular structures, patterns of plasmacytoid dendritic (pDC) and B cell activation, and TLR9 signaling, leading to upregulation of proinflammatory genes (e.g., TNFa, Type I IFNs) and downstream activation of interferon regulatory factor (IRF) and nuclear factor kappa-B (NFKB) signaling. Without wishing to be bound to any theory, CpG-ODNs may also inhibit suppressive activities of MDSCs and TAMs that mediate anti-tumor responses in weakly immunogenic tumors. CpG-B monomers strongly induce pDC maturation, B cell proliferation, and the production of IL-6, IFNoc, and TNFa.

[0280] TLR9 Tarsetins and Uptake of CpG-C’ dots. CpG 1826 (CpG-B), 5’-TCCATGACGTTCCTGACGTT-3’, was custom- synthesized and adapted at its 5’ end with a 9-carbon spacer (e.g., SpCo), and an azide group (Integrated DN A Technology, Coralville, Iowa)) for conjugation to 15 .M DBCO-PEG-Cy5-C’ dots using a click chemistry approach. Based on UV absorbance spectra, -7-10 ODNs per C’ dot, on average, were detected. Prior to conducting in vitro binding studies, we confirmed Tlr9 expression on both murine cancer cells and APCs by expression profiling (data not shown). Bone marrow-derived dendritic cells (BMDCs), derived from the B16-GM model and exposed to CpG (L)-C’ dots for 48 hours (h), showed a significant increase in the overall percentage (%) of DCs (FIG. 16A), % MHC-II-positive (+) DCs (FIG. 16B), MHC-II protein expression (FIG. 16C), and Tlr9+ DCs (FIG. 16D) by flow cytometry, relative to controls (CT: PEG-C’ dots, media). Marked increases in specific DC maturation / co- stimulatory pathway markers (FIG. 16E) and anti-tumor cytokine production (IL12p70, IL-6) were also observed by expression profiling, accompanied by nosignificant changes in immune suppressive markers (FIG. 16F). Structured illumination microscopy ofB16-GM cells incubated with CpG-C’ dots (red) and FITC-dextran (green), a lysosomal marker, showed co-localization (FIG. 16G). Finally, significantly increased phagocytotic activity was observed relative to controls when CpG-C’ dot-exposed DCs were incubated with B16-GM cells (FIG. 16H).

[0281] Treatment Efficacy and Immunophenotyping: Intrinsic therapeutic activities of C’ dots were investigated in a myeloid cell-rich syngeneic murine melanoma model that allowed for exploration of resistance mediated by infiltrating myeloid cells. B16-GMCSF (referred to as B16- GM), a genetically-engineered mouse model (GEMM) that is a highly immune suppressive model that exhibits decreased sensitivity to checkpoint inhibition, was treated as described herein. Cohorts of B16-GM tumor-bearing mice were treated with a multiple, high-dose ( lOpM, 6 nmoles total) regimen of i.v. -injected CpG-PEG-C’ dots and PEG-C’ dots when tumors reached 50mm (FIG. 17), and compared with vehicle-treated mice. A statistically significant (p<0.0001) and marked drop of nearly 93% in tumor volumes (n=3 mice, bottom curve with triangle points) was observed relative to control values (n=3 mice, top curve with square points) after CpG-C’ dot administration ~2 weeks postimplantation. Additionally, a statistically significant volume drop (p<0.01) was noted between CpG-C’ dot and C’ dot-treated animals (FIG. 17). To investigate contributions to these responses from various cell populations in the TME, immunophenotyping was performed to examine both myeloid and T cell fractions, with results depicted in FIGS. 18A-18E. A statistically significant decrease in numbers of suppressive tumor-associated macrophages, designated M2 (CDllb+F480+CD206+) macrophages (FIG. 18A), was found in the TME after CpG-C’ dot treatment relative to vehicle control, as well as after administration of PEG-C’ dots, 2 weeks post-implantation (e.g., study termination). In addition, an Increase was observed in the anti-tumor (Ml -like) macrophage population (CD1 lb+F480+iNOS+)(FIG. 18B) in tumors treated with CpG-C’ dots relative to vehicle control. Together, these findings resulted in increased M1:M2 ratios in CpG-C’ dot treated mice relative to those treated with either PEG-C’ dots or saline FIG. 18C). For the T cell panel, we observed significant drops in regulatory T cells (Tregs), as well as the ratio of cytotoxic (CD8+) T cells to Tregs, for both particle treatments relative to saline.Example 11: Induction of particle-driven cell death in OC models (FIGS. 19A-19B).

[0282] C’ dot induction of ferroptosis has been observed in multiple human cancer cell lines and is one of the iron-driven cell death programs by which C’ dots can inhibit tumor growth and / or promote regression. Identification of lead candidates and assessment of conditions yielding maximal cell death induction are key objectives. MUC16ectoOC cells will be incubated over a range of concentrations (lOOnM - 15pM) and incubation times (up to 72h), with and without an iron chelator (i.e., deferoxamine, DFO, lOOpM) as a control. Induction of cell death can be assessed by time-lapse microscopy, as well as gene expression and cell cycle analyses for viability, cell cycle phase, oxidative stress markers, and iron metabolism signatures as described herein. Results can be compared with those found for OC cells treated with other known inducers (Erastin) or specific inhibitors (liproxstatin- 1) of ferroptosis. In FIG. 19B, a statistically significant drop of nearly 70% in tumor volumes (bottom triangle curve) to control values (top diamond curve) is shown following a multiple, high-dose (36 nmoles; 60pM) regimen of i.v.-injected aMSH-PEG-C’ dots in ID8(L) mice when tumors reached 50mm3. After intraperitoneal (i.p.) injection of liproxstatin- 1 , this effect was substantially attenuated (middle square curve), confirming that ferroptotic induction significantly contributes to overall antitumor cell kill in this model.Equivalents

[0283] It is to be understood that while the disclosure has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the claims. Other aspects, advantages, and modifications are within the scope of the claims.

[0284] This written description uses examples to disclose the invention, including the best mode, and also to enable any person skilled in the art to practice the present embodiments, including making and using any compositions, nanoparticles, and therapies, and performing any incorporated methods. The patentable scope of the present embodiments is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they include structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal languages of the claims.SEQUENCESSEQ ID NO: 1MALPVTALLLPLALLLHAEVKLQESGGGFVKPGGSLKVSCAASGFTFSSYAMSWVRLSPEMRLEWVATISSAGGYIFYSDSVQGRFTISRDNAKNTLHLQMGSLRSGDTAMYYCARQGFGNYGDYYAMDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIELTQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNQLAWYQQKPGQSPELLIYWASTRQSGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQQSYNLLTFGPGTKLEIKRSEQ ID NO: 2MEWSWVFLFFLSVTTGVHSEVKLQESGGGFVKPGGSLKVSCAASGFTFSSYAMSWVRLSPEMRLEWVATISSAGGYIFYSDSVQGRFTISRDNAKNTLHLQMGSLRSGDTAMYYCARQGFGNYGDYYAMDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIELTQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNQLAWYQQKPGQSPELLIYWASTRQSGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQQSYNLLTFGPGTKLEIKGGSCGSGENLYFQGHHHHHHHHHHSEQ ID NO: 3GGGGSGGGGSGGGGSSEQ ID NO: 4TCCATGACGTTCCTGACGTT

Claims

CLAIMSWhat is claimed is:1 A method of treating a disease or condition, the method comprising: administering to a subject a first pharmaceutical composition comprising: an immunomodulatory nanoparticle conjugate, wherein the immunomodulatory nanoparticle conjugate comprises:(1) a nanoparticle; and(2) a plurality of cellular receptor- and / or antigen- targeting ligands, wherein the nanoparticle has a diameter no greater than 20 nanometers, wherein the subject also receives a second pharmaceutical composition comprising engineered cells.2 A method of treating a disease or condition, the method comprising: administering to a subject(1) a first pharmaceutical composition comprising(a) an immunomodulatory nanoparticle conjugate, wherein the immunomodulatory nanoparticle conjugate comprises:(i) a nanoparticle; and(ii) a plurality of cellular receptor- and / or antigen- targeting ligands, wherein the nanoparticle has a diameter no greater than 20 nanometers; and(2) a second pharmaceutical composition comprising engineered immune cells .

3. A method of treating a disease or condition, the method comprising: administering to a subject a first pharmaceutical composition comprising engineered immune cells, wherein the subject also receives a second pharmaceutical composition comprising an immunomodulatory nanoparticle conjugate, wherein the immunomodulatory nanoparticle conjugate comprises:(1) a nanoparticle; and(2) a plurality of cellular receptor- and / or antigen- targeting ligands, wherein the nanoparticle has a diameter no greater than 20 nanometers.4 The method of any one of the preceding claims, wherein the plurality of cellular receptor- and / or antigen- targeting ligands comprises one or more species of targeting ligands.5 The method of any one of the preceding claims, wherein the plurality of cellular receptor- and / or antigen- targeting ligands targets domains that are expressed on the surface of tumor cells.6 The method of any one of the preceding claims, wherein the plurality of cellular receptor- and / or antigen- targeting ligands comprises a plurality of immunomodulatory / stimulatory ligands (of the same species or of differing species).

7. The method of any one of the preceding claims, wherein the plurality of immunomodulatory / stimulatory ligands comprises or is a plurality of oligodeoxynucleotides (ODNs).8 The method of any one of the preceding claims, wherein the plurality of ODNs comprises or is a plurality of ODNs containing an unmethylated cytosine-phosphate-guanine (CpG) motif (or CpG ODNs)).9 The method of any one of the preceding claims, wherein the plurality of ODNs bind to Toll-like receptor 9 (TLR9).10 The method of any one of the preceding claims, wherein the plurality of cellular receptor- and / or antigen- targeting ligands is a member selected from the set consisting of a recombinant antibody fragment (Fabs), a single chain variable fragment (scFv), and a single domain antibody (sdAb) fragment.11 The method of any one of the preceding claims, wherein the plurality of cellular receptor- and / or antigen- targeting ligands is a single chain variable fragment (scFv).12 The method of any one of the preceding claims, wherein the plurality of cellular receptor- and / or antigen- targeting ligands comprises an anti-MUC16 antibody fragment.

13. The method of claim 11 or 12, wherein the antibody fragment or scFv comprises a first linker sequence.

14. The method of claim 13, wherein the first linker sequence comprises a sequence having at least 80% identity to GGGGSGGGGSGGGGS (SEQ ID NO: 3).

15. The method of any one of claims 11-14, wherein the scFv comprises a second linker sequence attached to the C-terminus end of the scFv.

16. The method of claim 15, wherein the second linker sequence comprises a cysteine residue.

17. The method of claim 15 or 16, wherein the second linker sequence comprises a sequence having at least 85% identity to GGSCGSG (SEQ ID NO: 3).

18. The method of any one the preceding claims, wherein the plurality of cellular receptor- and / or antigen- targeting ligands comprises species having a sequence of at least 50 amino acid sequences with at least 80% identity to a portion of SEQ ID NO: 1.

19. The method of any one of the preceding claims, wherein the plurality of cellular receptor- and / or antigen- targeting ligands comprises a species having a sequence of at least 50 amino acid sequences with at least 80% identity to a portion of SEQ ID NO:2.

20. The method of any one of the preceding claims, wherein the plurality of cellular receptor- and / or antigen- targeting ligands is covalently or non-covalently bonded to the nanoparticle.21 . The method of any one of the preceding claims, wherein the nanoparticle is coated with an organic polymer.

22. The method of any one of the preceding claims, wherein the plurality of cellular receptor- and / or antigen- targeting ligands comprises from 1 to 75 cellular receptor- and / or antigen- targeting ligands conjugated to the nanoparticle.

23. The method of any one of the preceding claims, wherein the immunomodulatory nanoparticle conjugate comprises a radiotherapeutic agent.

24. The method of any one of the preceding claims, wherein the nanoparticle comprises silica.

25. The method of any one of the preceding claims, wherein the nanoparticle comprises a silica core.

26. The method of any one of the preceding claims, wherein the nanoparticle comprises a dye- encapsulated core and a silica shell surrounding the core.

27. The method of any one of the preceding claims, wherein the nanoparticle comprises a fluorescent compound within the core.

28. The method of any one of the preceding claims, wherein the average nanoparticle diameter is from 1 to 20 nm.

29. The method of any one of the preceding claims, wherein the immunomodulatory nanoparticle conjugate has an average diameter no greater than 50 nm.

30. The method of any one of the preceding claims, wherein the nanoparticle comprises a silica composition such that ferroptosis is not induced.

31. The method of any one of the preceding claims, wherein the nanoparticles have a silica composition such that ferroptosis may be induced.

32. The method of any one of the preceding claims, wherein the immunomodulatory nanoparticle conjugate further comprises a therapeutic agent.

33. The method of any one of the preceding claims, wherein the immunomodulatory nanoparticle conjugate further comprises a radiotherapeutic agent.

34. The method of any one of the preceding claims, wherein the nanoparticle further comprises a targeting ligand.

35. The method of any one of the preceding claims, wherein the first pharmaceutical composition and / or second pharmaceutical composition further comprises a carrier.

36. The method of any one of the preceding claims, wherein the engineered immune cells comprises CAR T cells.

37. The method of any one of the preceding claims, wherein the engineered cells are MUC16- targeting engineered cells.

38. The method of any one of the preceding claims, wherein the engineered cells secrete IL- 12.

39. The method of any one of the preceding claims, wherein the engineered cells are or comprise MUC16-targeting, IL- 12 secreting CAR T cells.

40. The method of any one of the preceding claims, wherein the first pharmaceutical composition is being used to enhance activity of the second pharmaceutical composition.

41. The method of any one of the preceding claims, wherein the first pharmaceutical composition activates anti-tumor immune responses in the tumor microenvironment (TME).

42. The method of any one of the preceding claims, wherein the first pharmaceutical composition is administered before, after, or at the same time as the second pharmaceutical composition.

43. The method of any one of the preceding claims, wherein the method increases CD8+ / Tregs ratio at least about 5-fold.

44. The method of any one of the preceding claims, wherein the method increases co-stimulatory molecules on Ml macrophages and / or (overall) dendritic cell (DC) population.

45. The method of any one of the preceding claims, wherein the method results in a M1 / M2 ratio greater than 0.5.

46. The method of any one of the preceding claims, wherein the method decreases myeloid-derived suppressor cells (MDSCs) by a factor of at least 2.

47. An immunomodulatory nanoparticle conjugate comprising: a nanoparticle; and a plurality of cellular receptor- and / or antigen- targeting ligands conjugated to the nanoparticle, wherein the nanoparticle has a diameter no greater than 20 nanometers, for use with an engineered cellular therapy.

48. A composition comprising an immunomodulatory nanoparticle conjugate and engineered cells for use in engineered cellular therapy.

49. The composition of claim 47, wherein the composition is formulated at a therapeutically effective dose.

50. A treatment comprising:(a) a therapeutically effective amount of an immunomodulatory nanoparticle conjugate for use in combination with engineered cells; or(b) a therapeutically effective amount of engineered cells for use in combination with an immunomodulatory nanoparticle conjugate, for use in a method of treating a disease or condition or preventing disease or condition occurrence or recurrence in a subject.

51. A radiotherapeutic composition comprising an immunomodulatory nanoparticle conjugate and engineered cells for use in engineered cellular therapy, wherein the immunomodulatory nanoparticle conjugate activates anti-tumor immune responses in the tumor microenvironment (TME) and wherein the nanoparticle conjugate comprises a radiotherapeutic agent.

52. The radiotherapeutic composition of claim 51, wherein the composition is formulated at a therapeutically effective dose.

53. The radiotherapeutic composition of claim 52, wherein the therapeutically effective dose is at least 0.27 microcuries / kg relative to a microenvironment control.

54. An immunomodulatory nanoparticle conjugate comprising: a nanoparticle; and a plurality of immunomodulatory / stimulatory ligands (of the same species or of differing species) conjugated to the nanoparticle, wherein the nanoparticle has a diameter no greater than 20 nanometers.

55. The immunomodulatory nanoparticle conjugate of claim 54, wherein the plurality of immunomodulatory ligands comprises or is a plurality of pattern recognition receptors (PRR) agonist moieties.

56. The immunomodulatory nanoparticle conjugate of claim 55, wherein the plurality of PRR agonist moieties is or comprises toll-like receptors (TLRs) agonist moieties, RIG-1 like receptors (RLRs) agonist moieties, NOD-like receptors (NLRs) agonist moieties, C-type lectin receptors (CLRs) agonist moieties, or cytosolic DNA sensors.

57. The immunomodulatory nanoparticle conjugate of any one of claims 54-56, wherein the plurality of immunomodulatory ligands comprises or is a plurality of oligodeoxynucleotides (ODNs).

58. The immunomodulatory nanoparticle conjugate of claim 57, wherein the plurality of CpG ODNs bind to Toll-like receptor 9 (TLR9).

59. The immunomodulatory nanoparticle conjugate of any one of claims 57 or 58, wherein the plurality of ODNs comprises or is a plurality of ODNs containing an unmethylated cytosine-phosphate- guanine (CpG) motif (or CpG ODNs), and wherein the CpG motif comprises or is a sequence according to 5’-TCCATGACGTTCCTGACGTT-3’ (SEQ ID NO: 4).

60. The immunomodulatory nanoparticle conjugate of any one of claims 54-59, wherein the plurality of immunomodulatory / stimulatory ligands is covalently or non-covalently bonded to the nanoparticle.

61. The immunomodulatory nanoparticle conjugate of any one of claims 54-60, wherein the nanoparticle is coated with an organic polymer.

62. The immunomodulatory nanoparticle conjugate of any one of claims 54-61, wherein the plurality of immunomodulatory / stimulatory ligands comprises from 1 to 75 immunomodulatory / stimulatory ligands conjugated to the nanoparticle.

63. The immunomodulatory nanoparticle conjugate of any one of claims 54-62, wherein the immunomodulatory nanoparticle conjugate comprises a radiolabel.

64. The immunomodulatory nanoparticle conjugate of any one of claims 54-63, wherein the nanoparticle comprises silica.

65. The immunomodulatory nanoparticle conjugate of any one of claims 54-64, wherein the nanoparticle comprises a silica core.

66. The immunomodulatory nanoparticle conjugate of any one of claims 54-65, wherein the nanoparticle comprises a dye-encapsulating core and a silica shell surrounding the core.

67. The immunomodulatory nanoparticle conjugate of any one of claims 54-66, wherein the nanoparticle comprises a fluorescent compound within the core.

68. The immunomodulatory nanoparticle conjugate of any one of claims 54-67, further comprising a therapeutic agent.

69. The immunomodulatory nanoparticle conjugate of any one of claims 54-68, wherein the plurality of immunomodulatory / stimulatory ligands conjugated to the nanoparticle enhance immunomodulatory properties of the nanoparticle.

70. The immunomodulatory nanoparticle conjugate of any one of claims 54-69, wherein the immunomodulatory ligands are immunostimulatory.

71. A method of treating a disease or condition, the method comprising administering to a subject a pharmaceutical composition comprising the immunomodulatory nanoparticle conjugate of any one of claims 54-70.

72. The method of claim 71, wherein the immunomodulatory nanoparticle conjugate modulates the tumor microenvironment.

73. The method of claim 72, wherein the nanoparticle does or does not modulate the tumor microenvironment.

74. The method of any one of claims 71-73, wherein the plurality of immunomodulatory / stimulatory ligands does or does not modulate the tumor microenvironment.

75. The method of any one of claims 71-74, wherein the plurality of immunomodulatory / stimulatory ligands activate one or more pattern recognition receptors (PRR), augment proinflammatory responses, and / or innate and adaptive immune responses beyond that of the particle itself.

76. The method of any one of claims 71-74, wherein the PRR is or comprises toll-like receptors (TLRs), RIG-1 like receptors (RLRs), NOD-like receptors (NLRs), C-type lectin receptors (CLRs), and cytosolic DNA sensors, such as the Stimulator of Interferon Genes (STING).

77. The method of any one of claims 71-76, comprising administering a therapeutic radioisotope.

78. The method of any one of claims 71 -77, wherein the pharmaceutical composition further comprises a carrier.

Images