Compositions and methods for penetration, distribution and response of targeting particles in malignant brain tumors

Abstract

To provide compositions and methods for penetration, distribution and response of targeting particles in malignant brain tumors.SOLUTION: Described herein are nanoparticle conjugates that demonstrate enhanced penetration into tumor tissue (e.g., brain tumor tissue) and diffusion within the tumor interstitium, e.g., for treatment of cancer. Further described are methods of targeting tumor-associated macrophages, microglia, and / or other cells in a tumor microenvironment using such nanoparticle conjugates. Moreover, diagnostic, therapeutic, and theranostic (diagnostic and therapeutic) platforms featuring such nanoparticle conjugates are described for treating targets in both the tumor and surrounding microenvironment, thereby enhancing the efficacy of cancer treatment. Use of the nanoparticle conjugates described herein with other conventional therapies, including chemotherapy, radiotherapy, immunotherapy, and the like, is also envisaged.SELECTED DRAWING: None

Description

[Technical field]

[0001] This application claims the benefit of U.S. Application No. 62 / 330,029, filed April 29, 2016, the disclosure of which is incorporated herein by reference in its entirety.

[0002] This invention relates generally to nanoparticle conjugates for the treatment of cancer, as well as imaging and treatment methods using such nanoparticle conjugates.

[0003] This invention was made with Government support under Grant No. CA199081 awarded by the National Institutes of Health (NIH). The Government has certain rights in this invention. [Background technology]

[0004] One of the current challenges in treating patients harboring epidermal growth factor receptor mutant (EGFRmt+) and platelet-derived growth factor B (PDGFB)-driven malignant brain tumors is the limited CNS penetration of standard daily dosages of EGFR and PDGFR small molecule inhibitors (SMIs), such as gefitinib and dasatinib (das), respectively. The most common cancer that metastasizes to the brain is non-small cell lung cancer (NSCLC), while glioblastoma multiforme (GBM) is the most common primary malignant brain tumor. As an attractive candidate molecule for targeted cancer therapy, the epidermal growth factor receptor (EGFR) exhibits activating mutations in 25% of metastatic NSCLC and 40-50% of primary GBM. These mutations are associated with high response rates to EGFR tyrosine kinase inhibitors (TKIs), such as gefitinib. However, approximately one-third of patients develop central nervous system (CNS) metastases after response to TKIs. This is due to the lower SMI concentration in the brain or CSF, which is inadequate for killing EGFRmt+ tumor cells.Even though high doses of treatment are administered intermittently, the CNS response of patients with EGFR-mutant (EGFRmt+) NSCLC has only been partially successful.Currently, it remains a challenge to achieve sufficient EGFR inhibitor concentration in brain tissue to maximize the treatment of primary malignancies or metastatic disease, or in cerebrospinal fluid (CSF) to treat leptomeningeal metastases.

[0005] As another example, GBM requires aggressive local therapy and adjuvant chemotherapy to target the widespread microscopic disease penetration. However, such treatment combinations only confer short-term survival benefits, and alternative therapeutic strategies utilizing small molecule inhibitors (SMIs) such as dasatinib (BMS-354825) are increasingly being incorporated into treatment planning protocols. Dasatinib, a highly potent second-generation ATP-competitive inhibitor of multiple protein tyrosine kinases including PDGFR and Src family kinases (SFKs), is known to reduce tumor cell survival, as well as proliferation and metastatic activity in vitro, but most clinical trials using dasatinib and other SMIs as monotherapy have failed to demonstrate survival benefit in unselected malignant glioma patient populations.

[0006] There are therapeutic attempts to modulate the tumor microenvironment. For example, recent therapeutic attempts have attempted to reeducate stromal cells within the tumor microenvironment with antitumorigenic effects (see "Microenvironmental regulation of tumor progression and metastasis," Daniela Quail and Johanna Joyce, Nature Medicine, Vol. 19, No. 11, November 2013). Previous studies have used inhibitors of colony-stimulating factor-1 (CSF-1) receptor (CSF-1R) to target the tumor microenvironment in a mouse proneural GBM model, significantly prolonging survival and regressing established tumors (see "CSF-1R Inhibition Alters Macrophage Polarization And Blocks Glioma Progression," Pyonteck et al., Nature Medicine, Vol. 19, No. 10, October 2013). Furthermore, current strategies against genomically defined metastatic disease to the brain are limited by variable and inefficient delivery across the blood-brain barrier, resulting in low tumor penetration even at tolerated systemic doses. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] "Microenvironmental regulation of tumor progression and metastasis," Daniela Quail and Johanna Joyce, Nature Medicine, Volume 19, Issue 11, November 2013 [Non-Patent Document 2] "CSF-1R Inhibition Alters Macrophage Polarization And Blocks Glioma Progression," Pyonteck et al., Nature Medicine, Vol. 19, No. 10, October 2013 Summary of the Invention [Problem to be solved by the invention]

[0008] These findings highlight the need to develop new drug delivery approaches and further highlight important factors that may limit treatment responses to EGFR inhibitors and other TKIs, such as bioavailability, permeability, serum protein binding, drug-specific properties, and non-specific tissue uptake. Furthermore, there remains a need for non-invasive and quantifiable vehicles that enhance drug delivery to primary and metastatic tumors (e.g., brain tumors), as well as a need to improve tumor delivery and therapeutic index of existing drugs in the treatment of primary and metastatic tumors. [Means for solving the problem]

[0009] For example, nanoparticle conjugates are described herein that exhibit enhanced penetration into tumor tissue (e.g., brain tumor tissue) and diffusion within the tumor stroma for the treatment of cancer (e.g., primary and metastatic brain tumors). Additionally, methods are described for targeting tumor-associated macrophages, microglia, and / or other cells in the tumor microenvironment using such nanoparticle conjugates. Additionally, diagnostic, therapeutic, and theranostic (diagnostic and therapeutic) platforms featuring such nanoparticle conjugates are described for treating targets in both the tumor and surrounding microenvironments, thereby enhancing the efficacy of cancer treatment. It is also envisioned to use the nanoparticle conjugates described herein with other conventional therapies, such as chemotherapy, radiation therapy, and immunotherapy.

[0010] To overcome treatment resistance, combinations of multi-targeted kinase inhibitors and single-targeted kinase inhibitors have been developed.Importantly, the multi-modality combination of targeting agents, including particle-based probes designed with SMI, chemotherapeutic agents, radiotherapy labels, and / or immunotherapy, can enhance the efficacy of treatment and / or improve treatment regimens for malignant brain tumors.Combined with molecular imaging labels, these vehicles can then monitor the delivery, accumulation, and retention of drugs, which can provide optimal therapeutic index.

[0011] Furthermore, the use of radiolabels and / or fluorescent markers bound to (or incorporated into or on or otherwise associated with) nanoparticles allows for quantitative assessment of particle uptake and monitoring of treatment response. In various embodiments, modular linkers are described for incorporating targeting ligands to develop drug delivery systems with controlled pharmacological properties. The described platform determines the effect of targeting on nanoparticle penetration and accumulation, thereby establishing a platform that can be adapted to improve the delivery of various tractable SMIs, for example, to primary and metastatic brain tumors.

[0012] In one aspect, the present invention is directed to a method of treating cancer comprising administering to a subject a pharmaceutical composition comprising a nanoparticle drug conjugate (NDC), the nanoparticle drug conjugate comprising a nanoparticle having an average diameter of 20 nm or less, a linker moiety, and a drug moiety, wherein the drug moiety and the linker moiety form a cleavable linker-drug construct attached (e.g., covalently and / or non-covalently bound) to the nanoparticle, and wherein the NDC is readily diffused within the tumor interstitium.

[0013] In certain embodiments, the cancer comprises a member selected from the group consisting of malignant brain tumor, metastatic brain tumor, non-small cell lung cancer (NSCLC) and glioblastoma multiforme (GBM).

[0014] In certain embodiments, the methods provide for accumulation and / or (more uniform) distribution of the drug moiety in tissues sufficient to treat primary malignancies or metastatic disease, hi certain embodiments, the methods provide for accumulation and / or (more uniform) distribution of the drug moiety in cerebrospinal fluid sufficient to treat leptomeningeal metastases.

[0015] In certain embodiments, the nanoparticles have an average diameter of 3 to 8 nm.

[0016] In certain embodiments, the linker moiety comprises a cleavable linker and / or a biocleavable linker. In certain embodiments, the linker moiety comprises a member selected from the group consisting of a peptide, a hydrazone, a PEG, and a moiety comprising one or more amino acids (natural and / or unnatural amino acids). In certain embodiments, the linker moiety comprises an enzyme-sensitive linker moiety.

[0017] In certain embodiments, the drug moiety comprises a member selected from the group consisting of a small molecule inhibitor (SMI), a tyrosine kinase inhibitor (TKI), an EGFR inhibitor (e.g., gefitinib), and a PDGFR inhibitor (e.g., dasatinib).

[0018] In certain embodiments, a nanoparticle drug conjugate comprises one or more targeting moieties (e.g., targeting peptides) (e.g., a tumor targeting moiety, e.g., an RGD-containing moiety, e.g., cRGDY, which targets an integrin (integrin receptor), and / or a microenvironment targeting moiety, e.g., αMSH, which targets the melanocortin-1 receptor) (e.g., for delivery of a drug moiety (e.g., a small molecule inhibitor, SMI) (e.g., to an integrin- and / or melanocortin-1 (MC1)-expressing cell (e.g., tumor, macrophage)). In certain embodiments, a nanoparticle drug conjugate comprises 1 to 20 separate targeting moieties (e.g., of the same type or of different types).

[0019] In certain embodiments, the method includes administering a nanoparticle drug conjugate (e.g., multiple NDCs of the same or similar composition) having a first portion for delivering and targeting the drug moiety to the tumor and an NDC having a second portion for delivering and targeting the drug moiety to the microenvironment surrounding the tumor.

[0020] In certain embodiments, the first and second moieties may be on the same or different NDCs that are administered to the subject in one or more compositions.

[0021] In certain embodiments, the NDC comprises (e.g., within the nanoparticle, attached to the nanoparticle (directly or via a linker), and / or attached to a drug moiety) a radioisotope (e.g., a PET tracer), e.g., 89 Zr, 64 Cu, and / or 124 In certain embodiments, the radioisotope comprises: 99m Tc, 111 In, 64 Cu, 67 Ga, 68 Ga, 67 Cu, 123 I, 124 I, 125 I,11 C. 13 N, 15 O. 18 F, 186 Re, 188 Re, 153 Sm, 166 Ho, 177 Lu, 149 Pm, 90 Y, 213 Bi, 103 Pd, 109 Pd, 159 Gd, 140 La, 198 Au, 199 Au, 169 Yb, 175 Yb, 165 Dy, 166 Dy, 105 Rh, 111 Ag, 89 Zr, 225 Ac, and 192 Ir.

[0022] In certain embodiments, the drug moiety comprises an SMI (e.g., CSF-1R, dasatinib) or a chemotherapeutic agent (e.g., sorafenib, paclitaxel, docetaxel, MEK162, etoposide, lapatinib, nilotinib, crizotinib, fulvestrant, vemurafenib, bexorotene, and / or camptothecin).

[0023] In certain embodiments, the nanoparticle drug conjugate comprises an immune modulator and / or an anti-inflammatory agent. In certain embodiments, the immune modulator and / or anti-inflammatory agent comprises αMSH.

[0024] In certain embodiments, the methods include administration of an antibody or antibody fragment (eg, for immunotherapy).

[0025] In certain embodiments, the composition comprises an NDC conjugated with an antibody and / or an antibody fragment.

[0026] In certain embodiments, the method comprises administration of an NDC conjugated with an antibody fragment, wherein the antibody fragment is a member selected from the set consisting of a recombinant antibody fragment (fAb), a single chain variable fragment (scFv), and a single domain antibody (sdAb) fragment.

[0027] In certain embodiments, the antibody fragment is a single chain variable fragment (scFv). In certain embodiments, the antibody fragment is a single domain (sdAb) fragment.

[0028] In certain embodiments, the pharmaceutical composition comprises nanoparticles targeted to cancer cells such that the nanoparticles accumulate in a concentration sufficient to induce ferroptosis of the cancer cells.

[0029] In certain embodiments, the nanoparticles comprise silica (e.g., where the nanoparticles comprise, e.g., a fluorescent compound bound to and / or incorporated within the nanoparticles). In certain embodiments, the nanoparticles comprise a silica-based core and a silica shell surrounding at least a portion of the core (e.g., where the nanoparticles comprise a fluorescent compound within the core).

[0030] In certain embodiments, the pharmaceutical composition includes a carrier.

[0031] In another aspect, the invention provides a method for in vivo diagnosis and / or staging of cancer, comprising the steps of: delivering to a subject a pharmaceutical composition, the pharmaceutical composition comprising a nanoparticle drug conjugate (NDC), the nanoparticle drug conjugate comprising a nanoparticle having an average diameter of 20 nm or less; a linker moiety; a drug moiety, the drug moiety and the linker moiety forming a cleavable linker-drug construct attached (e.g., covalently and / or non-covalently attached) to the nanoparticle, wherein the NDC readily diffuses within the tumor interstitium; and a radioisotope (e.g., a PET tracer), e.g., 89 Zr, 64 Cu, and / or 124The present invention is directed to methods comprising the steps of: including I (e.g., within the nanoparticle, attached to the nanoparticle (directly or via a linker) and / or attached to the drug moiety); and detecting the radioisotope in the subject (e.g., by PET, x-ray, MRI, CT, etc.).

[0032] In certain embodiments, the NDC comprises one or more targeting moieties (e.g., targeting peptides) (e.g., a tumor targeting moiety, e.g., an RGD-containing moiety, e.g., cRGDY, which targets an integrin receptor), and / or a microenvironment targeting moiety, e.g., αMSH (which targets MC1-R), for delivering, e.g., a drug moiety (e.g., SMI).

[0033] In certain embodiments, the cancer comprises a member selected from the group consisting of malignant brain tumor, metastatic brain tumor, non-small cell lung cancer (NSCLC) and glioblastoma multiforme (GBM).

[0034] In certain embodiments, the methods provide for accumulation and / or (more uniform) distribution of the drug moiety in tissues sufficient to treat primary malignancies or metastatic disease, hi certain embodiments, the methods provide for accumulation and / or (more uniform) distribution of the drug moiety in cerebrospinal fluid sufficient to treat leptomeningeal metastases.

[0035] In certain embodiments, the nanoparticles have an average diameter of 3 to 8 nm.

[0036] In certain embodiments, the radioisotope is 99m Tc, 111 In, 64 Cu, 67 Ga, 68 Ga, 67 Cu, 123 I, 124 I, 125 I, 11 C. 13 N, 15 O. 18 F, 186 Re, 188 Re,153 Sm, 166 Ho, 177 Lu, 149 Pm, 90 Y, 213 Bi, 103 Pd, 109 Pd, 159 Gd, 140 La, 198 Au, 199 Au, 169 Yb, 175 Yb, 165 Dy, 166 Dy, 105 Rh, 111 Ag, 89 Zr, 225 Ac, and 192 Ir.

[0037] In certain embodiments, the linker moiety comprises a cleavable linker and / or a biocleavable linker. In certain embodiments, the linker moiety comprises a member selected from the group consisting of a peptide, a hydrazone, a PEG, and a moiety comprising one or more amino acids (natural and / or unnatural amino acids). In certain embodiments, the linker moiety comprises an enzyme-sensitive linker moiety.

[0038] In certain embodiments, the drug moiety comprises a member selected from the group consisting of a small molecule inhibitor (SMI), a tyrosine kinase inhibitor (TKI), an EGFR inhibitor (e.g., gefitinib), and a PDGFR inhibitor (e.g., dasatinib).

[0039] In certain embodiments, the method includes mapping the concentration of a radioisotope in a subject, e.g., in 2D or 3D, and optionally detecting fluorescence from a fluorescent compound (e.g., a fluorescent compound bound to and / or incorporated within a nanoparticle of the NDC).

[0040] In certain embodiments, the step of detecting / mapping the radioisotope is part of a treatment for cancer.

[0041] In certain embodiments, the method is a theranostic method.

[0042] In another aspect, the present invention is directed to a pharmaceutical composition for use in a method of treating cancer comprising administering to a subject a pharmaceutical composition comprising a nanoparticle drug conjugate, the pharmaceutical composition comprising a nanoparticle drug conjugate (NDC), the nanoparticle drug conjugate comprising a nanoparticle having an average diameter of 20 nm or less; a linker moiety; and a drug, wherein the drug moiety and the linker moiety form a cleavable linker-drug construct attached (e.g., covalently and / or non-covalently attached) to the nanoparticle, and wherein the NDC readily diffuses into the tumor stroma.

[0043] In certain embodiments, the NDC comprises one or more targeting moieties (e.g., targeting peptides) (e.g., a tumor targeting moiety, e.g., an RGD-containing moiety, e.g., cRGDY, which targets integrins (integrin receptors), and / or a microenvironment targeting moiety, e.g., αMSH, which targets the melanocortin-1 receptor), for example, delivering a drug moiety (e.g., a small molecule inhibitor, SMI) (e.g., to integrin-expressing cells and / or melanocortin-1 (MC1)-expressing cells (e.g., tumors, macrophages)).

[0044] In certain embodiments, the NDC is a radioisotope (e.g., a PET tracer), e.g., 89 Zr, 64 Cu, and / or 124 I (e.g., within the nanoparticle, attached to the nanoparticle (directly or via a linker), and / or attached to a drug moiety).

[0045] In certain embodiments, the cancer comprises a member selected from the group consisting of malignant brain tumor, metastatic brain tumor, non-small cell lung cancer (NSCLC) and glioblastoma multiforme (GBM).

[0046] In certain embodiments, the methods of treating cancer provide for accumulation and / or (more uniform) distribution of the drug moiety in tissues sufficient to treat primary malignancies or metastatic disease, hi certain embodiments, the methods of treating cancer provide for accumulation and / or (more uniform) distribution of the drug moiety in cerebrospinal fluid sufficient to treat leptomeningeal metastases.

[0047] In certain embodiments, the nanoparticles have an average diameter of 3 to 8 nm.

[0048] In certain embodiments, the radioisotope is 99m Tc, 111 In, 64 Cu, 67 Ga, 68 Ga, 67 Cu, 123 I, 124 I, 125 I, 11 C. 13 N, 15 O. 18 F, 186 Re, 188 Re, 153 Sm, 166 Ho, 177 Lu, 149 Pm, 90 Y, 213 Bi, 103 Pd, 109 Pd, 159 Gd, 140 La, 198 Au, 199 Au, 169 Yb, 175 Yb, 165 Dy, 166 Dy, 105 Rh, 111 Ag, 89 Zr, 225 Ac, and 192 Ir.

[0049] In certain embodiments, the linker moiety comprises a cleavable linker and / or a biocleavable linker. In certain embodiments, the linker moiety comprises a member selected from the group consisting of a peptide, a hydrazone, a PEG, and a moiety comprising one or more amino acids (natural and / or unnatural amino acids). In certain embodiments, the linker moiety comprises an enzymatically cleavable linker.

[0050] In certain embodiments, the drug moiety comprises a member selected from the group consisting of a small molecule inhibitor (SMI), a tyrosine kinase inhibitor (TKI), an EGFR inhibitor (e.g., gefitinib), and a PDGFR inhibitor (e.g., dasatinib).

[0051] In certain embodiments, the pharmaceutical composition includes a carrier.

[0052] In one aspect, the present invention provides a pharmaceutical composition for use in a method for in vivo diagnosis and / or staging of cancer, comprising a nanoparticle drug conjugate (NDC), the nanoparticle drug conjugate comprising a nanoparticle having a mean diameter of 20 nm or less; a linker moiety; and a drug moiety, wherein the NDC readily diffuses within the tumor stroma, and wherein the in The invention is directed to pharmaceutical compositions where in vivo diagnosis and / or staging comprises the steps of delivering the composition to a subject and detecting the radioisotope in the subject (e.g., by PET, x-ray, MRI, CT, etc.).

[0053] In certain embodiments, the NDC comprises one or more targeting moieties (e.g., targeting peptides) (e.g., a tumor targeting moiety, e.g., an RGD-containing moiety, e.g., cRGDY, which targets integrins (integrin receptors), and / or a microenvironment targeting moiety, e.g., αMSH, which targets the melanocortin-1 receptor), for example, delivering a drug moiety (e.g., a small molecule inhibitor, SMI) (e.g., to integrin-expressing cells and / or melanocortin-1 (MC1)-expressing cells (e.g., tumors, macrophages)).

[0054] In certain embodiments, the NDC is a radioisotope (e.g., a PET tracer), e.g., 89 Zr, 64 Cu, and / or 124 I (e.g., within the nanoparticle, attached to the nanoparticle (directly or via a linker), and / or attached to a drug moiety).

[0055] In certain embodiments, the cancer comprises a member selected from the group consisting of malignant brain tumor, metastatic brain tumor, non-small cell lung cancer (NSCLC) and glioblastoma multiforme (GBM).

[0056] In certain embodiments, the methods provide for accumulation and / or (more uniform) distribution of the drug moiety in tissues sufficient to treat primary malignancies or metastatic disease, hi certain embodiments, the methods provide for accumulation and / or (more uniform) distribution of the drug moiety in cerebrospinal fluid sufficient to treat leptomeningeal metastases.

[0057] In certain embodiments, the nanoparticles have an average diameter of 3 to 8 nm.

[0058] In certain embodiments, the radioisotope is 99m Tc, 111 In, 64 Cu, 67 Ga, 68 Ga, 67 Cu, 123 I, 124 I, 125 I, 11 C. 13 N, 15 O. 18 F, 186 Re, 188 Re, 153 Sm, 166 Ho, 177 Lu, 149 Pm, 90 Y, 213 Bi, 103 Pd, 109 Pd, 159 Gd,140 La, 198 Au, 199 Au, 169 Yb, 175 Yb, 165 Dy, 166 Dy, 105 Rh, 111 Ag, 89 Zr, 225 Ac, and 192 Ir.

[0059] In certain embodiments, the linker moiety comprises a cleavable linker and / or a biocleavable linker. In certain embodiments, the linker moiety comprises a member selected from the group consisting of a peptide, a hydrazone, a PEG, and a moiety comprising one or more amino acids (natural and / or unnatural amino acids). In certain embodiments, the linker moiety comprises an enzyme-sensitive linker.

[0060] In certain embodiments, the drug moiety comprises a member selected from the group consisting of a small molecule inhibitor (SMI), a tyrosine kinase inhibitor (TKI), an EGFR inhibitor (e.g., gefitinib), and a PDGFR inhibitor (e.g., dasatinib).

[0061] In certain embodiments, the method includes mapping the concentration of a radioisotope in a subject, e.g., in 2D or 3D, and, optionally, detecting fluorescence from a fluorescent compound (e.g., a fluorescent compound bound to and / or incorporated within the nanoparticles of the NDC).

[0062] In certain embodiments, the step of detecting / mapping the radioisotope is part of a treatment for cancer.

[0063] In certain embodiments, the method is a theranostic method.

[0064] In certain embodiments, the pharmaceutical composition includes a carrier.

[0065] In another aspect, the present invention is directed to a pharmaceutical composition comprising a nanoparticle drug conjugate (NDC), the nanoparticle drug conjugate comprising a nanoparticle having an average diameter of 20 nm or less, a linker moiety, and a drug moiety, wherein the NDC readily diffuses into the tumor interstitium.

[0066] In certain embodiments, the NDC comprises one or more targeting moieties (e.g., targeting peptides) (e.g., a tumor targeting moiety, e.g., an RGD-containing moiety, e.g., cRGDY, which targets integrins (integrin receptors), and / or a microenvironment targeting moiety, e.g., αMSH, which targets the melanocortin-1 receptor), for example, delivering a drug moiety (e.g., a small molecule inhibitor, SMI) (e.g., to integrin-expressing cells and / or melanocortin-1 (MC1)-expressing cells (e.g., tumors, macrophages)).

[0067] In certain embodiments, the NDC is a radioisotope (e.g., a PET tracer), e.g., 89 Zr, 64 Cu, and / or 124 I (e.g., within the nanoparticle, attached to the nanoparticle (directly or via a linker), and / or attached to a drug moiety).

[0068] In certain embodiments, the tumor comprises a member selected from the group consisting of malignant brain tumor, metastatic brain tumor, non-small cell lung cancer (NSCLC), and glioblastoma multiforme (GBM).

[0069] In certain embodiments, the NDC provides sufficient accumulation and / or (more uniform) distribution of the drug moiety in tissues to treat primary malignancies or metastatic disease, hi certain embodiments, the NDC provides sufficient accumulation and / or (more uniform) distribution of the drug moiety in cerebrospinal fluid to treat leptomeningeal metastases.

[0070] In certain embodiments, the nanoparticles have an average diameter of 3 to 8 nm.

[0071] In certain embodiments, the pharmaceutical composition comprises: 99m Tc, 111 In, 64 Cu, 67 Ga, 68 Ga, 67 Cu, 123 I, 124 I, 125 I, 11 C. 13 N, 15 O. 18 F, 186 Re, 188 Re, 153 Sm, 166 Ho, 177 Lu, 149 Pm, 90 Y, 213 Bi, 103 Pd, 109 Pd, 159 Gd, 140 La, 198 Au, 199 Au, 169 Yb, 175 Yb, 165 Dy, 166 Dy, 105 Rh, 111 Ag, 89 Zr, 225 Ac, and 192 Ir.

[0072] In certain embodiments, the linker moiety comprises a cleavable linker and / or a biocleavable linker. In certain embodiments, the linker moiety comprises a member selected from the group consisting of a peptide, a hydrazone, a PEG, and a moiety comprising one or more amino acids (natural and / or unnatural amino acids). In certain embodiments, the linker moiety comprises an enzyme-sensitive linker.

[0073] In certain embodiments, the drug moiety comprises a member selected from the group consisting of a small molecule inhibitor (SMI), a tyrosine kinase inhibitor (TKI), an EGFR inhibitor (e.g., gefitinib), and a PDGFR inhibitor (e.g., dasatinib).

[0074] In another aspect, the present invention is directed to a method of manipulating (e.g., regulating, controlling) cellular behavior in a tumor microenvironment, comprising administering to a subject a pharmaceutical composition comprising a nanoparticle conjugate comprising: nanoparticles having an average diameter of 20 nm or less; a linker moiety (e.g., a cleavable linker, e.g., a biocleavable linker, e.g., a moiety comprising a peptide, a hydrazone, PEG, and / or one or more amino acids (natural and / or unnatural amino acids)); and a modulator moiety, wherein the nanoparticle conjugate readily diffuses into the tumor interstitium.

[0075] In certain embodiments, the nanoparticle conjugates include one or more targeting moieties (e.g., targeting peptides) (e.g., a tumor targeting moiety, e.g., an RGD-containing moiety, e.g., cRGDY, which targets integrins (integrin receptors), and / or a microenvironment targeting moiety, e.g., αMSH, which targets the melanocortin-1 receptor), for delivering the modulator moiety (e.g., to integrin-expressing cells and / or melanocortin-1 (MC1)-expressing cells (e.g., tumors, macrophages)).

[0076] In certain embodiments, the nanoparticle conjugate is a radioisotope (e.g., a PET tracer), e.g., 89 Zr, 64 Cu, and / or 124 I (e.g., within the nanoparticle, attached to the nanoparticle (directly or via a linker), and / or attached to a drug moiety).

[0077] In certain embodiments, the tumor comprises a member selected from the group consisting of malignant brain tumor, metastatic brain tumor, non-small cell lung cancer (NSCLC), and glioblastoma multiforme (GBM).

[0078] In certain embodiments, the nanoparticles have an average diameter of 3 to 8 nm.

[0079] In certain embodiments, the radioisotope is 99m Tc, 111 In, 64 Cu, 67 Ga, 68 Ga, 67 Cu, 123 I, 124 I, 125 I, 11 C. 13 N, 15 O. 18 F, 186 Re, 188 Re, 153 Sm, 166 Ho, 177 Lu, 149 Pm, 90 Y, 213 Bi, 103 Pd, 109 Pd, 159 Gd, 140 La, 198 Au, 199 Au, 169 Yb, 175 Yb, 165 Dy, 166 Dy, 105 Rh, 111 Ag, 89 Zr, 225 Ac, and 192 Ir.

[0080] In certain embodiments, the linker moiety comprises a cleavable linker and / or a biocleavable linker. In certain embodiments, the linker moiety comprises a member selected from the group consisting of a peptide, a hydrazone, a PEG, and a moiety comprising one or more amino acids (natural and / or unnatural amino acids). In certain embodiments, the linker moiety comprises an enzyme-sensitive linker.

[0081] In certain embodiments, the cell comprises a member selected from the group consisting of a macrophage, a tumor-associated macrophage and / or microglia (TAM), a dendritic cell, and a T cell.

[0082] In certain embodiments, the tumor microenvironment is an in vivo tumor microenvironment in the treatment of cancer, brain cancer, malignant cancer, and / or malignant brain cancer.

[0083] In certain embodiments, the modulator portion comprises an inhibitor of colony stimulating factor-1 (CSF-1R) for targeting TAMs, and the modulator portion and linker portion form a cleavable linker-modulator construct attached (e.g., covalently and / or non-covalently attached) to the nanoparticle. In certain embodiments, the modular portion comprises an immune modulator (αMSH), and the modular portion comprises an inhibitor of colony stimulating factor-1 (CSF-1R) for targeting TAMs. The modulator moiety and linker moiety form a cleavable linker-modulator construct that is attached (eg, covalently and / or non-covalently attached) to the nanoparticle.

[0084] It is contemplated that details and features described with respect to one embodiment of the invention may be applied to other embodiments of the invention. definition

[0085] In order that this disclosure may be more readily understood, certain terms are first defined below. Further definitions for the following terms, as well as other terms, are set forth throughout the specification.

[0086] In this application, the use of "or" means "and / or" unless otherwise stated. As used in this application, the term "comprise" and variations of this term, such as "comprising" and "comprises," are not intended to exclude other additives, ingredients, integers, or steps. As used in this application, the terms "about" and "approximately" are used as equivalents. Any numerical values ​​used in this application, whether about / approximately or not, are meant to encompass any normal variation recognized by one of ordinary skill in the art. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​that is within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction of the stated reference value (greater than or less than the reference value), unless otherwise stated or otherwise apparent from the context (except where such number exceeds 100% of possible values).

[0087] "Administration": The term "administration" refers to introducing a substance into a subject. Generally, any route of administration may be utilized, including, for example, parenteral (e.g., intravenous), oral, topical, subcutaneous, intraperitoneal, intra-arterial, inhalation, vaginal, rectal, nasal, introduction into the cerebrospinal fluid, or instillation into a body segment. In certain embodiments, administration is oral. Additionally or alternatively, in certain embodiments, administration is parenteral. In certain embodiments, administration is intravenous.

[0088] "Antibody": As used herein, the term "antibody" refers to a polypeptide that contains sufficient canonical immunoglobulin sequence elements to confer specific binding to a particular target antigen. Naturally occurring intact antibodies are tetrameric agents of approximately 150 kD, consisting 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 contains at least four domains, each about 110 amino acids long - an amino-terminal variable (VH) domain (located at the tip of the Y structure), followed by three constant domains: CH 1 , C.H. 2 , and the carboxy-terminal C-H 3 (located at the base of the stem of the Y). A short region known as the "switch" connects the heavy chain variable and constant regions. The "hinge" connects the CH to the rest of the antibody. 2 and C.H. 3 The hinge region connects the heavy chain polypeptides of the intact antibody to each other. Two disulfide bonds in this hinge region connect the two heavy chain polypeptides of the intact antibody to each other. Each light chain is composed of two domains separated from each other by another "switch" - an amino-terminal variable (VL) domain followed by a carboxy-terminal constant (CL) domain. Intact antibody tetramers have heavy and light chains linked to each other by a single disulfide bond, and two other disulfide bonds connect the heavy chain hinge regions to each other, so that the dimers are connected to each other and form a tetramer. Naturally occurring antibodies also typically have a CH 2The variable domains are glycosylated at the nucleosomes and the nucleosomes. 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) bundled against each other in a compressed antiparallel beta barrel. Each variable domain contains three hypervariable loops (CDR1, CDR2, and CDR3) known as "complementarity determining regions" and four somewhat invariant "framework" regions (FR1, FR2, FR3, and FR4). When a natural antibody folds, the FR regions form beta sheets that provide a structural framework for the domain, and the CDR loop regions from both the heavy and light chains are assembled together in three-dimensional space to produce a single hypervariable antigen-binding moiety located at the tip of a Y structure. The Fc region of a naturally occurring antibody binds to elements of the complement system and to receptors on effector cells, including, for example, effector cells that mediate cytotoxicity. The affinity and / or other binding properties of the Fc region for the Fc receptor can be modulated by glycosylation or other modifications. In certain embodiments, the antibodies produced and / or utilized according to the present invention comprise a glycosylated Fc domain, including Fc domains that have been modified or engineered such as by glycosylation. For purposes of the present invention, in certain embodiments, any polypeptide or complex of polypeptides that comprises a full immunoglobulin domain sequence as found in a natural antibody can be referred to and / or used as an "antibody," regardless of whether such polypeptide is produced naturally (e.g., produced by an organism in response to an antigen) or produced by recombinant engineering, chemical synthesis, or other artificial systems or methodologies. In certain embodiments, the antibody is polyclonal, and in certain embodiments, the antibody is monoclonal. In certain embodiments, the antibody has constant region sequences characteristic of mouse, rabbit, primate, or human antibodies. In certain embodiments, the antibody sequence elements are humanized, primatized, chimeric, etc., as known in the art.Moreover, the term "antibody," as used herein, in appropriate embodiments (unless otherwise stated or clear from the context), can refer to any of the constructs or formats known or developed in the art for utilizing the structural and functional features of antibodies in alternative presentations. For example, in embodiments, antibodies utilized in accordance with the present invention may include, but are not limited to, intact IgG, IgE and IgM, bispecific or multispecific antibodies (e.g., Zybodies®, etc.), single chain Fv, polypeptide-Fc fusions, Fab, cameloid antibodies, masked antibodies (e.g., Probodies®), small modular immunopharmaceuticals (Small Modular Immunopharmaceuticals), and the like. ImmunoPharmaceuticals ("SMIPs™"), single chain or tandem diabodies (TandAb®), VHH, Anticalins®, Nanobodies®, minibodies, BiTEs®, ankyrin repeat proteins or DARPINs®, Avimers®, DART, TCR-like antibodies, Adnectins®, Affilins®, Trans-bodies®, Affibodies®, TrimerX®, MicroProteins, Fynomers®, Centyrins®, and KALBITOR®. In certain embodiments, the antibody may lack covalent modifications (e.g., glycan attachment) that it has when produced in nature. In certain embodiments, the antibodies may contain covalent modifications (e.g., attachment of a glycan, a payload (e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.) or other pendant group (e.g., polyethylene glycol, etc.).

[0089] "Antibody fragment": As used herein, an "antibody fragment" includes a portion of an intact antibody, such as an antigen-binding region or a variable region of an antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; triabodies; tetrabodies: linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. For example, antibody fragments include isolated fragments consisting of the variable regions of the heavy and light chains, "Fv" fragments, recombinant single-chain polypeptide molecules in which the light and heavy chain variable regions are connected by a peptide linker ("ScFv protein"), and minimal recognition units consisting of amino acid residues that mimic the hypervariable region. In many embodiments, an antibody fragment contains sufficient sequence of the parent antibody for the fragment to bind to the same antigen as the parent antibody binds, and in certain embodiments, the fragment binds to the antigen with an affinity comparable to that of the parent antibody and / or competes with the parent antibody for binding to the antigen. Examples of antigen-binding fragments of antibodies include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, scFv fragments, Fv fragments, dsFv diabodies, dAb fragments, Fd' fragments, Fd fragments, and isolated complementarity determining region (CDR) regions. Antigen-binding fragments of antibodies may be produced by any means. For example, antigen-binding fragments of antibodies may be produced enzymatically or chemically by fragmentation of intact antibodies and / or recombinantly from genes encoding partial antibody sequences. Alternatively or additionally, antigen-binding fragments of antibodies may be produced synthetically, in whole or in part. Antigen-binding fragments of antibodies optionally include single-chain antibody fragments. Alternatively or additionally, antigen-binding fragments of antibodies may include multiple chains linked together, for example, by disulfide linkages. Antigen-binding fragments of antibodies may optionally include multimolecular complexes. Functional single domain antibody fragments are in the range of about 5 kDa to about 25 kDa, e.g., about 10 kDa to about 20 kDa, e.g., about 15 kDa; functional single chain fragments are about 10 kDa to about 50 kDa, e.g., about 20 kDa to about 45 kDa, e.g., about 25 kDa to about 30 kDa; and functional fab fragments are about 40 kDa to about 80 kDa, e.g., about 50 kDa to about 70 kDa, e.g., about 60 kDa.

[0090] "Associated": As used herein, the term "associated" typically refers to two or more entities that are in physical proximity to each other, directly or indirectly (e.g., via one or more additional entities that act as linking agents), such that the entities form a structure that is sufficiently stable to be in physical proximity under relevant conditions, e.g., physiological conditions. In certain embodiments, the associated moieties are linked to each other by covalent bonds. In certain embodiments, the associated entities are linked by non-covalent bonds. In certain embodiments, the associated entities are linked to each other by specific non-covalent interactions (e.g., by interactions between interacting ligands that distinguish between the interaction partner and the other entity present in the context of use, such as, for example, streptavidin / avidin interactions, antibody / antigen interactions, etc.). Alternatively or additionally, a sufficient number of weaker non-covalent interactions can provide sufficient stability for the moieties to remain associated. Exemplary non-covalent interactions include, but are not limited to, electrostatic interactions, hydrogen bonding, affinity, metal coordination, physical adsorption, host-guest interactions, hydrophobic interactions, pi-stacking interactions, van der Waals interactions, magnetic interactions, electrostatic interactions, dipole-dipole interactions, and the like.

[0091] "Biocompatible": The term "biocompatible" as used herein is intended to describe a material that does not induce a substantial adverse response in vivo. In certain embodiments, a material is "biocompatible" if it is not toxic to cells. In certain embodiments, a material is "biocompatible" if its addition to cells in vitro results in less than or equal to 20% cell death and / or if its administration in vivo does not induce inflammation or other such adverse effects. In certain embodiments, a material is biodegradable.

[0092] "Biodegradable": As used herein, a "biodegradable" material is a material that, when introduced into a cell, is broken down by cellular mechanisms (e.g., enzymatic degradation) or by hydrolysis into components that the cell can reuse or process without significant toxic effects on the cell. In certain embodiments, the components resulting from the degradation of the biodegradable material do not induce inflammation and / or other adverse effects in vivo. In certain embodiments, the biodegradable material is degraded enzymatically. Alternatively or additionally, in certain embodiments, the biodegradable material is degraded by hydrolysis. In certain embodiments, biodegradable polymeric materials are degraded into their component polymers. In certain embodiments, the degradation of biodegradable materials (e.g., including biodegradable polymeric materials) involves hydrolysis of ester bonds. In certain embodiments, the degradation of materials (e.g., including biodegradable polymeric materials) involves cleavage of urethane linkages.

[0093] "Carrier": As used herein, "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water or aqueous solutions Saline solutions and aqueous solutions of dextrose and glycerol are preferably used as carriers, particularly for injection solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin.

[0094] "Cancer": As used herein, the term "cancer" refers to a disease, disorder, or condition in which cells exhibit relatively abnormal, uncontrolled, and / or spontaneous growth, resulting in cells exhibiting an abnormally elevated proliferation rate and / or an abnormal growth phenotype characterized by a significant loss of control of cell proliferation. In certain embodiments, the cancer may be characterized by one or more tumors. In certain embodiments, the cancer is a malignant brain tumor, a metastatic brain tumor, non-small cell lung cancer (NSCLC) or glioblastoma multiforme (GBM).Those skilled in the art will be familiar with the various types of cancers that may be treated with the present invention, including, for example, adrenocortical carcinoma, astrocytoma, basal cell carcinoma, carcinoid, cardiac, cholangiocarcinoma, chordoma, chronic myeloproliferative neoplasms, craniopharyngioma, ductal intraepithelial carcinoma, ependymoma, intraocular melanoma, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), gestational trophoblastic disease, glioma, histiocytosis, leukemia (e.g., acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), hairy cell leukemia, myelogenous leukemia, myeloid leukemia, myeloid leukemia, myelogenous ... leukemia), lymphoma (e.g., Burkitt's lymphoma [non-Hodgkin's lymphoma], cutaneous T-cell lymphoma, Hodgkin's lymphoma, mycosis fungoides, Sézary syndrome, AIDS-related lymphoma, follicular lymphoma, diffuse large B-cell lymphoma), melanoma, Merkel cell carcinoma, mesothelioma, myeloma (e.g., multiple myeloma), myelodysplastic syndrome, papillomatosis, paraganglioma, pheochromacytoma, pleuropulmonary blastoma, retinoblastoma, sarcoma (e.g., Ewing's sarcoma, Kaposi's sarcoma, osteosarcoma, rhabdomyosarcoma, uterine sarcoma, angiosarcoma), Wilms' tumor, and / or Recognizing various types of cancer, including cancer of the adrenal cortex, anus, appendix, bile duct, bladder, bone, brain, breast, bronchus, central nervous system, cervix, colon, endometrium, esophagus, eye, fallopian tube, gallbladder, gastrointestinal tract, germ cell, head and neck, heart, intestine, kidney (e.g., Wilms tumor), larynx, liver, lung (e.g., non-small cell lung cancer, small cell lung cancer), mouth, nasal cavity, oral cavity, ovary, pancreas, rectum, skin, stomach, testis, throat, thyroid, penis, pharynx, peritoneum, pituitary gland, prostate, rectum, salivary gland, ureter, urethra, uterus, vagina, vulva, malignant brain tumor, metastatic brain tumor, non-small cell lung cancer (NSCLC), and / or glioblastoma multiforme (GBM).

[0095] "Chemotherapeutic Agent" or "Drug": As used herein, the term "chemotherapeutic agent" or "drug" (e.g., anti-cancer drug) is understood in the art to be meant to refer to one or more proapoptotic, cytostatic and / or cytotoxic agents, including, for example, agents that are utilized and / or recommended for use in treating one or more diseases, disorders or conditions associated with unwanted cell proliferation, among others. In many embodiments, chemotherapeutic agents are useful in the treatment of cancer. In some embodiments, the chemotherapeutic agent may be or may include one or more alkylating agents, one or more anthracyclines, one or more cytoskeletal disrupting agents (e.g., microtubule targeting agents such as taxanes, maytansine and its analogs), 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 platinum-based 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., sharing suitable antiproliferative activity): In certain embodiments, the chemotherapeutic agent is actinomycin, all-trans retinoic acid, auristatin, azacitidine, azathioprine, The therapeutic agent may be or include one or more of 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 its analogs (e.g., DM1) mechlorethamine, mercaptopurine, methotrexate, mitoxantrone, maytansinoids, oxaliplatin, paclitaxel, pemetrexed, teniposide, thioguanine, topotecan, valrubicin, vinblastine, vincristine, vindesine, vinorelbine, and combinations thereof. In some embodiments, a chemotherapeutic agent may be utilized in conjunction with the antibody-drug conjugate. In some embodiments, the chemotherapeutic agent may be hLL1-doxorubicin, hRS7-SN-38, hMN-14-SN-38, hLL2-SN-38, hA20-SN-38, hPAM4-SN-38, hLL1-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-P-Dox, hLL1-Pro-2-P-Dox, P4 / D10-doxorubicin, gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, glembatumomab vedotin, SAR3419, SAR566658, BIIB015, BT 062, SGN-75, SGN-CD19A, AMG-172, AMG-595, BAY-94-9343, ASG-5ME, ASG-22ME, ASG-16M8F, MDX-1203, MLN-0264, anti-PSMA ADCs, 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, the chemotherapeutic agent may be or include one or more of farnesylthiosalicylic acid (FTS), 4-(4-chloro-2-methylphenoxy)-N-hydroxybutanamide (CMH), estradiol (E2), tetramethoxystilbene (TMS), δ-tocotrienol, salinomycin, or curcumin.

[0096] "Imaging Agent": As used herein, "imaging agent" refers to any element, molecule, functional group, compound, fragment or portion thereof that facilitates detection of an agent to which it is conjugated (e.g., a polysaccharide nanoparticle). Examples of imaging agents include, but are not limited to, various ligands, radionuclides (e.g., 3 H, 14 C. 18 F, 19 F, 32 P, 35 S, 135 I, 125 I, 124 I, 123 I, 131 I, 64 Cu, 68 Ga, 187 Re, 111 In, 90 Y, 99m Tc, 177 Lu, 89 Zr) fluorescent dyes (see below for certain exemplary fluorescent dyes), chemiluminescent agents (e.g., acridinium esters, stable dioxetanes, etc.), bioluminescent agents, spectrally resolved inorganic fluorescent agents, These include optical semiconductor nanocrystals (i.e., quantum dots), metal nanoparticles (e.g., gold, silver, copper, platinum, etc.) nanoclusters, paramagnetic metal ions, enzymes (see below for specific examples of enzymes), colorimetric labels (such as, e.g., dyes, colloidal gold, etc.), biotin, dioxygenin, haptens, and proteins for which antisera or monoclonal antibodies are available. Radionuclides may be attached, for example, by click chemistry reactions.

[0097] "Nanoparticle": As used herein, the term "nanoparticle" refers to a particle having a diameter of less than 1000 nanometers (nm). In some embodiments, the nanoparticle has a diameter of less than 300 nm as defined by the National Science Foundation. In some embodiments, the nanoparticle has a diameter of less than 100 nm as defined by the National Institutes of Health. In certain embodiments, very small nanoparticles are used, e.g., nanoparticles having an average diameter of 20 nm or less, e.g., 15 nm or less, e.g., 10 nm or less, e.g., 3 nm to 8 nm (e.g., with a size distribution such that at least 85 wt% (e.g., at least 85 wt%, at least 90 wt%, at least 95 wt%, at least 98 wt%, or at least 99 wt%) of the nanoparticles are 20 nm or less, e.g., 15 nm or less, e.g., 10 nm or less, e.g., 3 nm to 8 nm). In some embodiments, the nanoparticles are typically micelles that include an encapsulated compartment separated from the bulk solution by a micellar membrane composed of amphiphilic entities that surround and encapsulate a space or compartment (e.g., to define a cavity). In some embodiments, the micellar membrane is composed of at least one polymer, such as a biocompatible and / or biodegradable polymer.

[0098] "Peptide" or "Polypeptide": The term "peptide" or "polypeptide" refers to a string of at least two (e.g., at least three) amino acids linked together by peptide bonds. In certain embodiments, a polypeptide comprises naturally occurring amino acids; alternatively, or in addition, in certain embodiments, a polypeptide comprises one or more unnatural amino acids (i.e., compounds that do not occur in nature but that can be incorporated into a polypeptide chain: see, for example, http: / / www.cco.caltech.edu / ~dadgrp / Unnatstruct.gif, which presents structures of unnatural amino acids that have been successfully incorporated into functional ion channels) and / or may substitute amino acid analogs known in the art. In certain embodiments, one or more amino acids in a protein may be modified by the addition of a chemical entity, such as, for example, a carbohydrate group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification.

[0099] "Pharmaceutical composition": As used herein, the term "pharmaceutical composition" refers to an active agent formulated together with one or more pharma- ceutically acceptable carriers. In certain embodiments, the active agent is present in a unit dosage suitable for administration in a therapeutic regimen that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to an appropriate population. In certain embodiments, the pharmaceutical compositions may be specifically formulated for administration in solid or liquid form, including: oral administration, e.g., drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., buccal, sublingual, and those targeted for systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, e.g., subcutaneous, intramuscular, intravenous, or epidural injections, or sustained release formulations, as sterile solutions or suspensions; topical application, e.g., creams, ointments, or controlled release patches or sprays applied to the skin, lungs, or oral cavity; vaginally or rectally, e.g., as pessaries, creams, or foams; sublingually; ophthalmically; transdermally; or nasally, to the lungs, as well as other mucosal surfaces.

[0100] "Radiolabel" or "radioisotope": As used herein, a "radiolabel" or "radioisotope" refers to a moiety that contains at least one radioactive isotope of an element. Examples of suitable radiolabels include, but are not limited to, those described herein. In certain embodiments, the radiolabel is one used in positron emission tomography (PET). In certain embodiments, the radiolabel is one used in single photon emission computed tomography (SPECT). In certain embodiments, the radioisotope is: 99m Tc, 111 In, 64 Cu, 67 Ga, 68 Ga, 67 Cu, 123 I, 124 I, 125 I, 11 C. 13 N, 15 O. 18 F, 186Re, 188 Re, 153 Sm, 166 Ho, 177 Lu, 149 Pm, 90 Y, 213 Bi, 103 Pd, 109 Pd, 159 Gd, 140 La, 198 Au, 199 Au, 169 Yb, 175 Yb, 165 Dy, 166 Dy, 67 Cu, 105 Rh, 111 Ag, 89 Zr, 225 Ac, 82 Rb, and 192 Contains Ir.

[0101] "Subject": As used herein, the term "subject" includes humans and mammals (e.g., mice, rats, pigs, cats, dogs, and horses). In many embodiments, the subject is a mammal, particularly a primate, especially a human. In certain embodiments, the subject is a livestock animal, such as, for example, cows, sheep, goats, cows, pigs; poultry, such as, for example, chickens, ducks, geese, turkeys; and domestic animals, particularly pets, such as dogs and cats. In certain embodiments (particularly in the research context), the subject mammal is, for example, a rodent (e.g., mice, rats, hamsters), rabbits, primates, or pigs, such as crossbred pigs.

[0102] "Substantially": As used herein, the term "substantially" refers to a quantitative state that indicates a total or near total degree or degree of a feature or characteristic of interest. Those skilled in the art of biology will understand that biological and chemical phenomena rarely, if ever, proceed to completion and / or completeness, or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of inherent completeness in many biological and chemical phenomena.

[0103] "Therapeutic Agent," "Drug," "Pharmaceutical Composition": As used herein, the terms "therapeutic agent," "drug," and "pharmaceutical composition" refer to any agent that has a therapeutic effect and / or induces a desired biological and / or pharmacological effect when administered to a subject.

[0104] "Therapeutically effective amount": As used herein, "therapeutically effective amount" refers to an amount that produces a desired effect when 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 according to a therapeutic dosing regimen for treating the disease, disorder, and / or condition. In certain embodiments, a therapeutically effective amount is an amount that reduces the incidence and / or severity of and / or delays the onset of one or more symptoms of a disease, disorder, and / or condition. Those skilled in the art will appreciate that the term "therapeutically effective amount" does not in fact require successful treatment to be achieved in a particular individual. Rather, a therapeutically effective amount may be an amount that, when administered to a patient in need of such treatment, results in a particular desired pharmacological response in a substantial number of subjects. In certain embodiments, reference to a therapeutically effective amount may be a reference to an amount measured in one or more specific tissues (e.g., tissues suffering from a disease, disorder, or condition) or bodily fluids (e.g., blood, saliva, serum, sweat, tears, urine, etc.). Those of skill in the art will recognize that in certain embodiments, a therapeutically effective amount of a particular agent or therapy may be formulated and / or administered in a single dose, while in certain embodiments, a therapeutically effective agent may be formulated and / or administered in multiple doses, e.g., as part of a dosing regimen.

[0105] "Treatment": As used herein, the term "treatment" (also "treat" or "treating") refers to any administration of a substance that partially or completely alleviates, ameliorates, relieves, inhibits, delays the onset, reduces the severity, and / or reduces the incidence of one or more symptoms, features, and / or causes of a particular disease, disorder, and / or condition. Such treatment may be treatment of subjects who do not exhibit symptoms of the associated disease, disorder, and / or condition and / or who exhibit only early symptoms of the disease, disorder, and / or condition. Alternatively or additionally, such treatment may be treatment of subjects who exhibit one or more established symptoms of the associated disease, disorder, and / or condition. In certain embodiments, treatment may be treatment of subjects who have been diagnosed as suffering from the associated disease, disorder, and / or condition. In certain embodiments, treatment may be treatment of subjects who are known to have one or more susceptibility factors that are statistically correlated with an increased risk of developing the associated disease, disorder, and / or condition. The present invention provides, for example, the following items. (Item 1) 1. A method of treating cancer, comprising administering to a subject a pharmaceutical composition comprising a nanoparticle drug conjugate (NDC), the nanoparticle drug conjugate comprising: Nanoparticles with an average diameter of 20 nm or less, a linker moiety, and Drug portion wherein the drug moiety and the linker moiety form a cleavable linker-drug construct attached (e.g., covalently and / or non-covalently attached) to the nanoparticle, and the NDC is readily diffused within the tumor interstitium. (Item 2) 2. The method of claim 1, wherein the cancer comprises a member selected from the group consisting of malignant brain tumor, metastatic brain tumor, non-small cell lung cancer (NSCLC) and glioblastoma multiforme (GBM). (Item 3) 3. The method according to item 1 or 2, which achieves accumulation and / or (more uniform) distribution of the drug moiety in tissues sufficient to treat a primary malignant tumor or metastatic disease. (Item 4) 4. The method according to any one of items 1 to 3, which achieves an accumulation and / or (more uniform) distribution of the drug moiety in the cerebrospinal fluid sufficient to treat leptomeningeal metastases. (Item 5) 5. The method according to any one of items 1 to 4, wherein the nanoparticles have an average diameter of 3 to 8 nm. (Item 6) 6. The method according to any one of items 1 to 5, wherein the linker moiety comprises a cleavable linker and / or a biocleavable linker. (Item 7) 7. The method of any one of items 1 to 6, wherein the linker moiety comprises a member selected from the group consisting of a peptide, a hydrazone, a PEG, and a moiety comprising one or more amino acids (natural and / or unnatural amino acids). (Item 8) 7. The method of any one of items 1 to 6, wherein the linker moiety comprises an enzyme-sensitive linker moiety. (Item 9) The method of any one of claims 1 to 8, wherein the drug moiety comprises a member selected from the group consisting of a small molecule inhibitor (SMI), a tyrosine kinase inhibitor (TKI), an EGFR inhibitor (e.g., gefitinib), and a PDGFR inhibitor (e.g., dasatinib). 10. The method of any one of items 1 to 9, wherein the nanoparticle drug conjugate comprises one or more targeting moieties. (Item 11) 11. The method of claim 10, wherein the nanoparticle drug conjugate comprises 1 to 20 separate targeting moieties (e.g., of the same type or of different types). (Item 12) The method of any one of the preceding items, comprising administering a nanoparticle drug conjugate having a first portion for delivering and targeting the drug moiety to a tumor, and a NDC having a second portion for delivering and targeting the drug moiety to the microenvironment surrounding the tumor. (Item 13) 13. The method of claim 12, wherein the first and second portions can be on the same or different NDCs administered to the subject in one or more compositions. (Item 14) The method of any one of the preceding items, wherein the NDC comprises a radioisotope. (Item 15) The radioisotope is 99m Tc, 111 In, 64 Cu, 67 Ga, 68 Ga, 67 Cu, 123 I, 124 I, 125 I, 11 C. 13 N, 15 O. 18 F, 186 Re, 188 Re, 153 Sm, 166 Ho, 177 Lu, 149 Pm, 90 Y, 213 Bi, 103 Pd, 109 Pd, 159 Gd, 140 La, 198 Au, 199 Au, 169 Yb, 175 Yb, 165 Dy, 166 Dy, 105 Rh, 111 Ag, 89 Zr, 225 Ac, and 192 15. The method of claim 14, comprising one or more members selected from the group consisting of: Ir. (Item 16) The method of any one of the preceding items, wherein the drug moiety comprises a small molecule inhibitor SMI (e.g., CSF-1R, dasatinib) or a chemotherapeutic agent. (Item 17) The method of any one of the preceding items, wherein the nanoparticle drug conjugate comprises an immune modulator and / or an anti-inflammatory agent. (Item 18) 18. The method of claim 17, wherein the immune modulator and / or anti-inflammatory agent comprises alpha MSH. (Item 19) The method of any one of the preceding items comprising administration of an antibody or antibody fragment (e.g., for immunotherapy). (Item 20) 20. The method of claim 19, wherein the composition comprises an NDC conjugated with an antibody and / or an antibody fragment. (Item 21) 2. The method of any one of the preceding items, comprising administration of an NDC having an antibody fragment attached thereto, wherein the antibody fragment is a member selected from the set consisting of a recombinant antibody fragment (fAb), a single chain variable fragment (scFv), and a single domain antibody (sdAb) fragment. (Item 22) 22. The method of claim 21, wherein the antibody fragment is a single-chain variable fragment (scFv). (Item 23) 23. The method of claim 21 or 22, wherein the antibody fragment is a single domain (sdAb) fragment. (Item 24) 2. The method of any one of the preceding items, wherein the pharmaceutical composition comprises nanoparticles targeted to cancer cells such that the nanoparticles accumulate in a concentration sufficient to induce ferroptosis of the cancer cells. (Item 25) 2. The method of any one of the preceding items, wherein the nanoparticles comprise silica. (Item 26) 13. The method of any one of the preceding items, wherein the nanoparticles comprise a silica-based core and a silica shell surrounding at least a portion of the core. (Item 27) The method of any one of the preceding items, wherein the pharmaceutical composition comprises a carrier. (Item 28) A method for in vivo diagnosis and / or staging of cancer, comprising: Delivering to a subject a pharmaceutical composition, the pharmaceutical composition comprising a nanoparticle drug conjugate (NDC), the nanoparticle drug conjugate comprising: Nanoparticles with an average diameter of 20 nm or less, A linker portion, The drug portion; and radioactive isotope Including, wherein the drug moiety and the linker moiety form a cleavable linker-drug construct attached (e.g., covalently and / or non-covalently attached) to the nanoparticle, and the NDC readily diffuses into the tumor interstitium. Steps and detecting the radioisotope in the subject; A method comprising: (Item 29) 29. The method of claim 28, wherein the NDC comprises one or more targeting moieties. (Item 30) 30. The method of claim 28 or 29, wherein the cancer comprises a member selected from the group consisting of malignant brain tumor, metastatic brain tumor, non-small cell lung cancer (NSCLC) and glioblastoma multiforme (GBM). (Item 31) 31. The method according to any one of items 28 to 30, which achieves accumulation and / or (more uniform) distribution of the drug moiety in tissues sufficient to treat a primary malignant tumor or metastatic disease. (Item 32) 32. The method according to any one of items 28 to 31, which achieves an accumulation and / or (more uniform) distribution of the drug moiety in the cerebrospinal fluid sufficient to treat leptomeningeal metastases. (Item 33) 33. The method according to any one of items 28 to 32, wherein the nanoparticles have an average diameter of 3 to 8 nm. (Item 34) The radioisotope is 99m Tc, 111 In, 64 Cu, 67 Ga, 68 Ga, 67 Cu, 123 I, 124 I, 125 I, 11 C. 13 N, 15 O. 18 F, 186 Re, 188 Re, 153 Sm, 166 Ho, 177 Lu, 149 Pm, 90 Y, 213 Bi, 103 Pd, 109 Pd, 159 Gd, 140 La, 198 Au, 199 Au, 169 Yb, 175 Yb, 165 Dy, 166 Dy, 105 Rh, 111 Ag, 89 Zr, 225 Ac, and 192 34. The method of any one of items 28 to 33, comprising one or more members selected from the group consisting of: Ir. (Item 35) 35. The method according to any one of items 28 to 34, wherein the linker moiety comprises a cleavable linker and / or a biocleavable linker. (Item 36) 36. The method of any one of items 28 to 35, wherein the linker moiety comprises a member selected from the group consisting of a peptide, a hydrazone, a PEG, and a moiety comprising one or more amino acids (natural and / or unnatural amino acids). (Item 37) 36. The method of any one of items 28 to 35, wherein the linker moiety comprises an enzyme-sensitive linker moiety. (Item 38) 38. The method of any one of items 28 to 37, wherein the drug moiety comprises a member selected from the group consisting of a small molecule inhibitor (SMI), a tyrosine kinase inhibitor (TKI), an EGFR inhibitor, and a PDGFR inhibitor. (Item 39) The method of any one of items 28 to 38, comprising a step of mapping the concentration of the radioisotope in the subject, e.g., in 2D or 3D, and optionally detecting fluorescence from a fluorescent compound (e.g., a fluorescent compound bound to and / or incorporated within the nanoparticles of the NDC). (Item 40) 40. The method of claim 39, wherein the step of detecting / mapping the radioisotope is part of a treatment for the cancer. (Item 41) The method according to item 40, which is a theranostic method. (Item 42) 1. A pharmaceutical composition for use in a method of treating cancer comprising administering to a subject a pharmaceutical composition comprising a nanoparticle drug conjugate, the pharmaceutical composition comprising a nanoparticle drug conjugate (NDC), the nanoparticle drug conjugate comprising: Nanoparticles with an average diameter of 20 nm or less, A linker portion, Drugs wherein the drug moiety and the linker moiety form a cleavable linker-drug construct attached (e.g., covalently and / or non-covalently attached) to the nanoparticle, and wherein the NDC readily diffuses into the tumor interstitium. (Item 43) 43. The pharmaceutical composition of claim 42, wherein the NDC comprises one or more targeting moieties. (Item 44) 44. The pharmaceutical composition according to item 42 or 43, wherein the NDC comprises a radioisotope. (Item 45) 45. The pharmaceutical composition of any one of items 42 to 44, wherein the cancer comprises a member selected from the group consisting of malignant brain tumor, metastatic brain tumor, non-small cell lung cancer (NSCLC) and glioblastoma multiforme (GBM). (Item 46) 46. ​​The pharmaceutical composition according to any one of items 42 to 45, wherein said method of treating cancer achieves accumulation and / or (more uniform) distribution of the drug moiety in tissues sufficient to treat primary malignant tumors or metastatic disease. (Item 47) 47. The pharmaceutical composition according to any one of items 42 to 46, wherein said method of treating cancer achieves an accumulation and / or (more uniform) distribution of the drug moiety in the cerebrospinal fluid sufficient to treat leptomeningeal metastases. (Item 48) 48. The pharmaceutical composition according to any one of items 42 to 47, wherein the nanoparticles have a mean diameter of 3 to 8 nm. (Item 49) The radioisotope is 99m Tc, 111 In, 64 Cu, 67 Ga, 68 Ga, 67 Cu, 123 I, 124 I, 125 I, 11 C. 13 N, 15 O. 18 F, 186 Re, 188 Re, 153 Sm, 166 Ho, 177 Lu, 149 Pm, 90 Y, 213 Bi, 103 Pd, 109 Pd, 159 Gd, 140 La, 198 Au, 199 Au, 169 Yb, 175 Yb, 165 Dy, 166 Dy, 105 Rh, 111 Ag, 89Zr, 225 Ac, and 192 49. The pharmaceutical composition according to any one of items 44 to 48, comprising one or more members selected from the group consisting of Ir. (Item 50) 50. The pharmaceutical composition according to any one of items 42 to 49, wherein the linker moiety comprises a cleavable linker and / or a biocleavable linker. (Item 51) 51. The pharmaceutical composition according to any one of items 42 to 50, wherein the linker moiety comprises a member selected from the group consisting of a peptide, a hydrazone, a PEG, and a moiety comprising one or more amino acids (natural and / or unnatural amino acids). (Item 52) 51. The pharmaceutical composition according to any one of items 42 to 50, wherein the linker moiety comprises an enzymatically cleavable linker. (Item 53) 53. The pharmaceutical composition of any one of items 42 to 52, wherein the drug moiety comprises a member selected from the group consisting of a small molecule inhibitor (SMI), a tyrosine kinase inhibitor (TKI), an EGFR inhibitor (e.g., gefitinib), and a PDGFR inhibitor (e.g., dasatinib). (Item 54) 54. A pharmaceutical composition according to any one of items 42 to 53, comprising a carrier. (Item 55) 1. A pharmaceutical composition for use in a method for in vivo diagnosis and / or staging of cancer, comprising a nanoparticle drug conjugate (NDC), the nanoparticle drug conjugate comprising: Nanoparticles with an average diameter of 20 nm or less, A linker portion, Drug portion wherein the NDC readily spreads within the tumor stroma, and wherein the in vivo diagnosis and / or staging comprises: delivering the composition to a subject; detecting a radioisotope in the subject; 13. A pharmaceutical composition comprising: (Item 56) 56. The pharmaceutical composition of claim 55, wherein the NDC comprises one or more targeting moieties. (Item 57) The NDC may be a radioisotope (e.g., a PET tracer), e.g., 89 Zr, 64 Cu, and / or 124 57. The pharmaceutical composition according to item 55 or 56, comprising I (e.g. within the nanoparticle, attached to the nanoparticle (directly or via a linker), and / or attached to the drug moiety). (Item 58) 58. The pharmaceutical composition of any one of items 55 to 57, wherein the cancer comprises a member selected from the group consisting of malignant brain tumor, metastatic brain tumor, non-small cell lung cancer (NSCLC) and glioblastoma multiforme (GBM). (Item 59) 59. The pharmaceutical composition according to any one of items 55 to 58, wherein said method achieves accumulation and / or (more uniform) distribution of the drug moiety in tissues sufficient to treat a primary malignant tumor or a metastatic disease. (Item 60) 60. The pharmaceutical composition according to any one of items 55 to 59, wherein said method achieves an accumulation and / or (more uniform) distribution of the drug moiety in the cerebrospinal fluid sufficient to treat leptomeningeal metastases. (Item 61) 61. The pharmaceutical composition according to any one of items 55 to 60, wherein the nanoparticles have a mean diameter of 3 to 8 nm. (Item 62) The radioisotope is 99m Tc, 111 In, 64 Cu, 67 Ga, 68 Ga, 67 Cu, 123 I, 124 I, 125 I, 11 C. 13 N, 15 O. 18 F, 186 Re, 188 Re, 153 Sm,166 Ho, 177 Lu, 149 Pm, 90 Y, 213 Bi, 103 Pd, 109 Pd, 159 Gd, 140 La, 198 Au, 199 Au, 169 Yb, 175 Yb, 165 Dy, 166 Dy, 105 Rh, 111 Ag, 89 Zr, 225 Ac, and 192 62. The pharmaceutical composition according to any one of items 55 to 61, comprising one or more members selected from the group consisting of Ir. (Item 63) 63. The pharmaceutical composition according to any one of items 55 to 62, wherein the linker moiety comprises a cleavable linker and / or a biocleavable linker. (Item 64) 64. The pharmaceutical composition according to any one of items 55 to 63, wherein the linker moiety comprises a member selected from the group consisting of a peptide, a hydrazone, a PEG, and a moiety comprising one or more amino acids (natural and / or unnatural amino acids). (Item 65) 64. The pharmaceutical composition according to any one of items 55 to 63, wherein the linker moiety comprises an enzyme-sensitive linker. (Item 66) 66. The pharmaceutical composition of any one of items 55 to 65, wherein the drug moiety comprises a member selected from the group consisting of a small molecule inhibitor (SMI), a tyrosine kinase inhibitor (TKI), an EGFR inhibitor (e.g., gefitinib), and a PDGFR inhibitor (e.g., dasatinib). (Item 67) 67. The pharmaceutical composition of any one of items 55 to 66, comprising a step of mapping the concentration of the radioisotope in the subject, e.g., in 2D or 3D, and optionally detecting fluorescence from a fluorescent compound (e.g., the fluorescent compound bound to and / or incorporated within the nanoparticles of the NDC). (Item 68) 68. The pharmaceutical composition according to any one of items 55 to 67, wherein the step of detecting / mapping the radioisotope is part of a treatment for the cancer. (Item 69) 69. The pharmaceutical composition according to item 68, wherein the method is a theranostic method. (Item 70) 70. The pharmaceutical composition according to any one of items 55 to 69, comprising a carrier. (Item 71) 1. A pharmaceutical composition comprising a nanoparticle drug conjugate (NDC), the nanoparticle drug conjugate comprising: Nanoparticles with an average diameter of 20 nm or less, a linker moiety, and Drug portion wherein the NDC readily diffuses into the tumor interstitium. (Item 72) 72. The pharmaceutical composition of claim 71, wherein the NDC comprises one or more targeting moieties. (Item 73) 73. The pharmaceutical composition according to item 71 or 72, wherein the NDC comprises a radioisotope. (Item 74) 74. The pharmaceutical composition of any one of items 71 to 73, wherein the tumor comprises a member selected from the group consisting of malignant brain tumor, metastatic brain tumor, non-small cell lung cancer (NSCLC), and glioblastoma multiforme (GBM). (Item 75) 75. The pharmaceutical composition according to item 71 or 74, wherein said NDC provides sufficient accumulation and / or (more uniform) distribution of the drug moiety in tissues to treat a primary malignant tumor or metastatic disease. (Item 76) 75. The pharmaceutical composition according to any one of items 71 to 74, wherein said NDC provides sufficient accumulation and / or (more uniform) distribution of the drug moiety in the cerebrospinal fluid to treat leptomeningeal metastases. (Item 77) 77. The pharmaceutical composition according to any one of items 71 to 76, wherein the nanoparticles have a mean diameter of 3 to 8 nm. (Item 78) 99m Tc, 111 In, 64 Cu, 67 Ga, 68 Ga, 67 Cu, 123 I, 124 I, 125 I, 11 C. 13 N, 15 O. 18 F, 186 Re, 188 Re, 153 Sm, 166 Ho, 177 Lu, 149 Pm, 90 Y, 213 Bi, 103 Pd, 109 Pd, 159 Gd, 140 La, 198 Au, 199 Au, 169 Yb, 175 Yb, 165 Dy, 166 Dy, 105 Rh, 111 Ag, 89 Zr, 225 Ac, and 192 78. The pharmaceutical composition according to any one of items 71 to 77, comprising one or more members selected from the group consisting of: Ir. (Item 79) 79. The pharmaceutical composition according to any one of items 71 to 78, wherein the linker moiety comprises a cleavable linker and / or a biocleavable linker. (Item 80) 80. The pharmaceutical composition according to any one of items 71 to 79, wherein the linker moiety comprises a member selected from the group consisting of a peptide, a hydrazone, a PEG, and a moiety comprising one or more amino acids (natural and / or unnatural amino acids). (Item 81) 80. The pharmaceutical composition according to any one of items 71 to 79, wherein the linker moiety comprises an enzyme-sensitive linker. (Item 82) 82. The pharmaceutical composition of any one of items 71 to 81, wherein the drug moiety comprises a member selected from the group consisting of a small molecule inhibitor (SMI), a tyrosine kinase inhibitor (TKI), an EGFR inhibitor (e.g., gefitinib), and a PDGFR inhibitor (e.g., dasatinib). (Item 83) 1. A method of manipulating cellular behavior in a tumor microenvironment, comprising administering to a subject a pharmaceutical composition comprising a nanoparticle conjugate, the nanoparticle conjugate comprising: Nanoparticles with an average diameter of 20 nm or less, a linker moiety, and Modulator section wherein the nanoparticle conjugate readily diffuses into the tumor interstitium. (Item 84) 84. The method of claim 83, wherein the nanoparticle conjugate comprises one or more targeting moieties. (Item 85) 85. The method of claim 83 or 84, wherein the nanoparticle conjugate comprises a radioisotope. (Item 86) 86. The method of claim 85, wherein the tumor comprises a member selected from the group consisting of malignant brain tumors, metastatic brain tumors, non-small cell lung cancer (NSCLC) and glioblastoma multiforme (GBM). (Item 87) Item 88. The method of item 85 or 86, wherein the nanoparticles have an average diameter of 3 to 8 nm. The radioisotope is 99m Tc, 111 In, 64 Cu, 67 Ga,68 Ga, 67 Cu, 123 I, 124 I, 125 I, 11 C. 13 N, 15 O. 18 F, 186 Re, 188 Re, 153 Sm, 166 Ho, 177 Lu, 149 Pm, 90 Y, 213 Bi, 103 Pd, 109 Pd, 159 Gd, 140 La, 198 Au, 199 Au, 169 Yb, 175 Yb, 165 Dy, 166 Dy, 105 Rh, 111 Ag, 89 Zr, 225 Ac, and 192 88. The method of any one of items 85 to 87, comprising one or more members selected from the group consisting of: Ir. (Item 89) 89. The method according to any one of items 85 to 88, wherein the linker moiety comprises a cleavable linker and / or a biocleavable linker. (Item 90) 90. The method of any one of items 85 to 89, wherein the linker moiety comprises a member selected from the group consisting of a peptide, a hydrazone, a PEG, and a moiety comprising one or more amino acids (natural and / or unnatural amino acids). (Item 91) 91. The method of any one of items 83 to 90, wherein the linker moiety comprises an enzyme-sensitive linker. (Item 92) 92. The method of any one of paragraphs 83 to 91, wherein the cells comprise a member selected from the group consisting of macrophages, tumor-associated macrophages and / or microglia (TAM), dendritic cells, and T cells. (Item 93) 93. The method of any one of items 83 to 92, wherein the tumor microenvironment is an in vivo tumor microenvironment in the treatment of cancer, brain cancer, malignant cancer, and / or malignant brain cancer. (Item 94) 94. The method of any one of items 83 to 93, wherein the modulator portion comprises an inhibitor of colony stimulating factor-1 (CSF-1R) for targeting TAM, wherein the modulator portion and the linker portion form a cleavable linker-modulator construct bound (e.g., covalently and / or non-covalently bound) to the nanoparticle. (Item 95) The modular portion comprises an immune modulator (αMSH), 94. The method of any one of items 83 to 93, wherein the modulator moiety and the linker moiety form a cleavable linker-modulator construct attached (e.g., covalently and / or non-covalently attached) to the nanoparticle.

[0106] The drawings are presented herein for purposes of illustration and not limitation.

[0107] The foregoing, other objects, aspects, features, and advantages of the present disclosure will become more apparent and will be better understood with reference to the following description taken in conjunction with the accompanying drawings. [Brief description of the drawings]

[0108] [Figure 1]1A-1B are exemplary cleavable linker-drug constructs attached to ultra-small particles (e.g., average particle sizes of 20 nm or less, 15 nm or less, or 10 nm or less are exemplified). The figures demonstrate that protease-mediated drug release occurs intracellularly by detachment of the drug moiety at the enzyme cleavage site after the nanoparticle-drug conjugate reaches the desired location. The figures show ultra-small silica nanoparticles for delivery of small molecule inhibitors according to exemplary embodiments of the present invention. For example, the nanoparticles deliver small molecule inhibitors (SMIs) to primary and metastatic brain tumors with improved therapeutic index.

[0109] [Diagram 2] Figures 2-6 are images from experiments on a mouse model of high-grade glioma driven by platelet-derived growth factor B (PDGFB). [Diagram 3] Figures 2-6 are images from experiments on a mouse model of high-grade glioma driven by platelet-derived growth factor B (PDGFB). [Figure 4] Figures 2-6 are images from experiments on a mouse model of high-grade glioma driven by platelet-derived growth factor B (PDGFB). [Diagram 5] Figures 2-6 are images from experiments on a mouse model of high-grade glioma driven by platelet-derived growth factor B (PDGFB). [Figure 6] Figures 2-6 are images from experiments on a mouse model of high-grade glioma driven by platelet-derived growth factor B (PDGFB).

[0110] [Figure 7] Figures 7-9 are images from experiments demonstrating integrin expression and particle uptake in the RCAS-PDGFB glioma model. [Figure 8] Figures 7-9 are images from experiments demonstrating integrin expression and particle uptake in the RCAS-PDGFB glioma model. [Figure 9]Figures 7-9 are images from experiments demonstrating integrin expression and particle uptake in the RCAS-PDGFB glioma model.

[0111] [Figure 10] Figure 10 is a chart showing the use of the RCAS-PDGFB mouse glioma model to study C'-dot distribution with simultaneous vital staining. The figure shows that mice were given in vivo injections of RGD-targeted C'-dots and sacrificed at 3 or 96 hours. FITC-labeled dextran of 70 kDa served as a surrogate marker for blood-brain barrier (BBB) ​​breakdown. Hoeschst staining was used to demonstrate nuclear localization.

[0112] [Figure 11] FIG. 11 shows images from an ex vivo study of cRGD-Cy5-C′-dot distribution in RCAS tumor-bearing mice.

[0113] [Figure 12] FIG. 12 shows images from an ex vivo study of 124I-RGD-Cy5-C'-dot distribution in RCAS tumor-bearing mice.

[0114] [Figure 13] 13A-13B are images from an in vivo baseline study using the base particle probe (i.e., FDA-IND approved cRGDY-PEG-C'dots) in conjunction with a time-dependent vital staining method that provides an initial assessment of blood-brain barrier permeability, intratumoral penetration and particle distribution kinetics as a function of integrin targeting (versus non-integrin targeting using cRGDY-PEG-C'dots).

[0115] [Figure 14]Figure 14 shows images taken at 96 hours showing that nanoparticles with RGD showed greater diffusion in the tumor than those with RAD. Figure 14 also shows images of 70 kDa FITC-labeled dextran 3 hours after administration as a reference tracer of similar size to the nanoparticle conjugates, suggesting intracellular localization of Cy5-C' dots at least as early as 3 hours after treatment.

[0116] [Figure 15] Figures 15A-15F are triple fluorescent labeling images of FITC-dextran as a fiducial tracer of similar size to the nanoparticle conjugates of Figures 13A-13B. As explained above, the data suggest intracellular localization of Cy5-C' dots, at least as early as 3 hours after treatment.

[0117] [Figure 16] FIG. 16 shows MRI-PET and histological images of 124I-cRGDY-PEG-C' dots in brain tumors.

[0118] [Figure 17] Figures 17 and 18 are Western blot, fluorescent, and microscopic images demonstrating that gefitinib-C'-dots retain potency in H1650 cells comparable to free drug (or improved). RGD-C'-dots are internalized into the lysosomes of H1650 cells, and describe the optimization of delivery and release of small molecule inhibitors (SMIs) from nanoparticle drug conjugates (NDCs) (e.g., Yoo et al. 2015, Bioorg Med Chem). For example, drug levels in the CNS limit the clinical use of SMIs, even for sensitive brain tumors. Gefitnib can be used as a tool to evaluate nanoparticle-drug potency and kinetics. [Figure 18]Figures 17 and 18 are Western blot, fluorescent, and microscopic images demonstrating that gefitinib-C'-dots retain potency in H1650 cells comparable to free drug (or improved). RGD-C'-dots are internalized into the lysosomes of H1650 cells, and describe the optimization of delivery and release of small molecule inhibitors (SMIs) from nanoparticle drug conjugates (NDCs) (e.g., Yoo et al. 2015, Bioorg Med Chem). For example, drug levels in the CNS limit the clinical use of SMIs, even for sensitive brain tumors. Gefitnib can be used as a tool to evaluate nanoparticle-drug potency and kinetics.

[0119] [Figure 19] Figure 19 is an image of H1650 flank xenografts treated with Gef-NDC. The images show particle-specific fluorescence and achieve pEGFR inhibition in a time-dependent manner, which is relevant for determining drug delivery and efficacy of NDC in NSCLC tumor-bearing mice.

[0120] [Figure 20] Figures 20 and 21 show experimental results for characterization of gefitinib and gef-NDC responses in a patient-derived EGFR L858R NSCLC line (ECLC26). [Figure 21] Figures 20 and 21 show experimental results for characterization of gefitinib and gef-NDC responses in a patient-derived EGFR L858R NSCLC line (ECLC26).

[0121] FIG. 20 shows the viability of ECLC26 versus gefitinib (1 nM to 1 μM) at 72 hours.

[0122] FIG. 21 shows phosphor-EGFR inhibition in ECLC26 by gefitinib and type II gef-NDC.

[0123] [Figure 22]FIG. 22 shows exemplary linker chemistries relevant to the development and testing of dasatinib NDCs for investigation in the RCAS-PDGF glioma model.

[0124] [Diagram 23] Figures 23A-23F are images demonstrating that "pulsed" high dose erlotinib improves CNS penetration for NSCLC metastases. Response of CNS metastases to pulsed erlotinib in three patients is shown. Grommes et al., Neuro Oncol., 2011 Dec 13(12):1364-9. Response is clear, but even at high doses, response is unpredictable.

[0125] 23A and 23B are contrast (gadolinium)-enhanced axial T1 MRI sequences of patient number 3 with leptomeningeal metastasis (arrows) 6 months before (FIG. 23A) and after (FIG. 23B) treatment.

[0126] Figures 23C and 23D are images taken of patient number 6 with coexisting brain metastases (large arrows) and leptomeningeal metastases (not shown) 5 months before (Figure 23C) and after (Figure 23D) treatment.

[0127] Figures 23E and 23F are images of patient number 8 with coexisting brain metastases (arrowheads) and leptomeningeal metastases (not shown) before (Figure 23E) and after (Figure 23F) two months of treatment.

[0128] [Figure 24] FIG. 24 is MRI-PET and histological imaging of 124I-cRGDY-PEG-C' dots in brain tumors.

[0129] [Diagram 25]Figure 25 shows images from an ex vivo study of cRGD-C'-dot distribution in mouse gliomas. Triple fluorescent labeling of RAD-nanoparticles (NPs) at 3 hours demonstrated no difference between Cy5 and FITC signals, suggesting that non-targeted particles do not significantly diffuse across areas of disrupted blood-brain barrier at this time point.

[0130] [Figure 26] FIG. 26 provides imaging and quantitative data for RGD vs. RAD compared at 96 hours.

[0131] [Figure 27] Figures 27 and 28 are images and data showing distribution analysis by pixel correlation. High power imaging of focal areas of tumors treated with targeted and non-targeted nanoparticles. RCAS-tva tumor-bearing mice were treated with either radiolabeled RGD-targeted or RAD nanoparticles, injected with FITC-dextran 3 hours before sacrifice, and sacrificed 96 hours after treatment. Frozen sections were analyzed for fluorescent signal using high power imaging of representative areas. The data show that areas of BBB disruption as marked by FITC closely overlap with areas of RAD nanoparticle signal, compared to the more diffuse pattern of Cy5 signal over FITC hotspots in tumors treated with RGD nanoparticles. Each animal has an average of 4 to 5 areas per section (N=4 mice). [Figure 28]Figures 27 and 28 are images and data showing distribution analysis by pixel correlation. High power imaging of focal areas of tumors treated with targeted and non-targeted nanoparticles. RCAS-tva tumor-bearing mice were treated with either radiolabeled RGD-targeted or RAD nanoparticles, injected with FITC-dextran 3 hours before sacrifice, and sacrificed 96 hours after treatment. Frozen sections were analyzed for fluorescent signal using high power imaging of representative areas. The data show that areas of BBB disruption as marked by FITC closely overlap with areas of RAD nanoparticle signal, compared to the more diffuse pattern of Cy5 signal over FITC hotspots in tumors treated with RGD nanoparticles. Each animal has an average of 4 to 5 areas per section (N=4 mice).

[0132] [Figure 29] Figure 29 is an image of a Western blot showing that dasatinib-NDC achieves PDGFR inhibition in a dose-dependent manner at levels similar to free drug. TS543 cells (a neurosphere tumor line) harboring a constitutively activating mutation of PDGFRA Δ8,9 were treated with the indicated drugs for 4 hours followed by 20 ng / ml of PDGF-BB for 10 minutes.

[0133] [Diagram 30] FIG. 30 is an image of dasatinib-NDC distribution in tumors 3 and 96 hours after treatment.

[0134] [Diagram 31] FIG. 31 is an H&E and fluorescence image of the comparable distribution of fluorescent signal in targeted and non-targeted nanoparticle drug conjugates compared to targeted and non-targeted particles alone.

[0135] [Diagram 32]Figure 32 shows H&E images showing that gefitinib-NDC achieves p-EGFR target inhibition 18 hours after treatment. ECLC26 tumor-bearing mice were treated with gefitinib-NDC, gefitinib PO (150mg / kg), or oral saline vehicle and sacrificed 18 hours after treatment. Tumors were embedded in paraffin, sectioned, and stained with p-EGFR and H&E.

[0136] [Diagram 33] FIG. 33 shows data from multiple dose treatment of ECLC26 flank tumor-bearing mice resulting in robust tumor control.

[0137] [Diagram 34] FIG. 34 is a western blot image showing ECLC26 growth following treatment with an NDC provided herein.

[0138] [Diagram 35] Figure 35 is a histological image showing that 45 μM RGD-Das-NDC effectively inhibited the target in primary gliomas compared to untreated controls after 24 hours. 24 hours after iv injection of nanoparticle drug conjugates, brain tissue was harvested and stained for expression of phosphore s6 ribosomal protein. Growth factors and mitogens induce activation of p70 S6 kinase and subsequent phosphorylation of S6 ribosomal protein. Phosphorylation of S6 ribosomal protein correlates with increased translation of mRNA transcripts containing oligopyrimidine tracts in the 5' untranslated region. These particular mRNA transcripts (5'TOP) code for proteins involved in cell cycle progression, as well as ribosomal proteins and elongation factors required for translation. The phosphorylation site of S6 ribosomal protein includes several residues (Ser235, Ser236, Ser240, and Ser244) located within the small carboxy-terminal region of the S6 protein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0139] Throughout this specification where compositions are described as having, including, or comprising particular components, or methods are described as having, including, or comprising particular steps, it is further contemplated that there are compositions of the invention that consist essentially of or consist of the recited components, and that there are methods of the invention that consist essentially of or consist of the recited processing steps.

[0140] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the invention remains operable. Moreover, two or more steps or actions may be conducted simultaneously.

[0141] For example, the reference herein to any publication 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 to be a statement of prior art with respect to any claim.

[0142] Various embodiments described herein utilize ultra-small, i.e., 10 nm or smaller, FDA-IND approved fluorescent organosilica particles (C-dots), and / or ultra-small poly(ethylene glycol)-coated (PEGylated) near infrared (NIR) fluorescent silica nanoparticles, referred to as C'-dots. For example, in certain embodiments, C-dots or C'-dots are surface-fitted with one or more PET radiolabels and one or more targeting ligands (e.g., integrin-targeting peptide cyclo-(Arg-Gly-Asp-Tyr) (cRGDY)). For further details regarding C-dots, see U.S. Pat. No. 8,298,677 B2, "Fluorescent silica-based nanoparticles," U.S. Patent Application Publication No. 2013 / 0039848A1, "Fluorescent silica-based nanoparticles," U.S. Patent Application Publication No. 2014 / 0248210A1, "Multimodal silica-based nanoparticles," U.S. Patent Application Publication No. 2015 / 0248210A1, "Multimodal silica-based nanoparticles," and U.S. Patent Application Publication No. 2016 / 0248210A1, the contents of which are incorporated herein by reference in their entireties. No. 2015 / 0366995A1 "Mesoporous oxide nanoparticles and methods of making and using the same” and U.S. Patent Application Publication No. 2016 / 0018 This is described in No. 404A1 "Multilayer fluorescent nanoparticles and methods of making and using same."

[0143] C-dots (or C'-dots) offer a unique platform for drug delivery due to their physical properties and demonstrated in vivo characteristics in humans. These particles are ultra-small and benefit from the EPR effect in the tumor microenvironment while retaining the desired clearance and pharmacokinetic properties. To achieve this goal, in certain embodiments, a nanoparticle drug delivery system is described herein in which drug constructs are covalently attached to C-dots (or other nanoparticles). C-dot-based (or C'-dot-based) NDCs for drug delivery provide good biostability, minimize premature drug release, and exhibit controlled release of bioactive compounds. In certain embodiments, peptide-based linkers are used for NDC applications. These linkers, with highly predictable release kinetics that rely on enzyme-catalyzed hydrolysis by lysosomal proteases, are stable both in vitro and in vivo in association with antibodies and polymers. For example, cathepsin B is a protease highly expressed in lysosomes and can be utilized to facilitate the release of drugs from macromolecules. By incorporating short, protease-sensitive peptides between the macromolecular backbone and the drug molecule, controlled release of the drug can be obtained in the presence of enzymes.

[0144] In certain embodiments, the NDCs are ultra-small (e.g., have an average diameter of about 5 nm to about 10 nm (e.g., about 6 nm)) and utilize enzyme-sensitive linkers, e.g., where the release of the drug is catalyzed by a protease. In one example, gefitinib, an important epidermal growth factor receptor mutant (EGFRmt+)-tyrosine kinase inhibitor (TKI) cancer drug, was modified and incorporated onto the particles. The resulting NDCs showed excellent in vitro stability, solubility, and were found to be active in EGFRmt+-expressing NSCLC cells.

[0145] In certain embodiments, the NDC comprises one or more targeting moieties, for example, to target a particular tissue type (e.g., a particular tumor). The NDC with the targeting moiety enhances the internalization of the drug in the tumor cell (e.g., the targeting ligand binds to a receptor on the tumor cell and / or delivers the drug to the tumor cell (e.g., by increasing the permeability)). For example, silica nanoparticles are added to a mixture of cRGDY-PEG conjugate and maleimide bifunctional PEG to create a particulate therapeutic with an additional targeting moiety (e.g., cRGD). The maleimide bifunctional PEG supports the further attachment of the drug-linker conjugate to create theranostic products.

[0146] In some embodiments, the ultra-small particles may be associated with PET labels and / or optical probes. The nanoparticles may be observed in vivo (e.g., by PET) to assess the accumulation of the drug at the target site. For example, nanoparticles with PET labels (e.g., without drug substances) may be administered first. Then, by analyzing the in vivo PET images of the nanoparticles, the concentration and accumulation rate of the drug (e.g., conjugated with nanoparticles) in the tumor may be estimated. Based on the obtained estimates, the dose may be determined to provide personalized medicine (e.g., tumor size rather than patient weight). In some embodiments, the radiolabeled drug may be traced in vivo. High concentrations of chemotherapy agents are potentially dangerous if they are not targeted. In some embodiments, nanoparticles with optical probes (e.g., fluorophores) may be used for intraoperative imaging of tumors (e.g., where the surface of the tissue / tumor is exposed) and / or biopsy.

[0147] The therapeutic agent and the nanoparticles can be separately radiolabeled or optically labeled, allowing for independent monitoring of the therapeutic agent and the nanoparticles. In one embodiment, radiofluorinated (i.e. 18F) Dasatinib was coupled to a PEG-3400 moiety attached to the nanoparticles via an NHS ester linkage. The radioactive fluorine is important because it allows independent monitoring of time-dependent changes in drug distribution and release from radioiodized C24I) fluorescent (Cy5) nanoparticles. In this way, the prodrug (dasatinib) and nanoparticles can be monitored. This allows for optimization of prodrug design compared to prior art methods where the dual labeling approach is not used. In another embodiment, iodine molecules (e.g., 131 I), or other therapeutic gamma or alpha emitters, are conjugated to PEG via a maleimide functional group, where the therapeutic agent cannot dissociate from PEG in vivo.

[0148] In various embodiments, the NDC is a drug compound covalently attached to a C-dot nanoparticle (or other nanoparticle (e.g., C'-dot)) via a molecular linker. In certain embodiments, the linker incorporates a peptide (e.g., dipeptide) sequence that is sensitive to trypsin (the enzyme of interest) and / or cathepsin B, enzymes found primarily in the lysosomes of cells. In certain embodiments, a class of linker chemistries incorporates an amide bond between the linker and the drug. In certain embodiments, a class of linker chemistries utilizes a degradable moiety between the linker and the drug. In some embodiments, the linker is designed to release the drug from the nanoparticle (e.g., C-dot, e.g., C'-dot) under certain conditions, e.g., under conditions of proteolytic hydrolysis.

[0149] Examples of drugs that can be used include RTK inhibitors, such as dasatinib and gefitinib, which can target either platelet-derived growth factor receptor (PDGFR) or EGFRmt+ expressed by primary tumor cells of human or mouse origin (e.g., genetically engineered mouse models of high-grade gliomas, neurospheres derived from brain tumor explants of human patients), and / or tumor cell lines of non-neuronal origin.Dasatinib and gefitinib analogs can be synthesized to allow covalent attachment to several linkers without disrupting the underlying chemical structure that defines the active binding site.

[0150] The synthetic approach was validated and the desired linker-drug constructs and NDCs were obtained as described in International Application No. PCT / US2015 / 032565 (published as WO2015 / 183882 on December 3, 2015), the contents of which are incorporated herein by reference in their entirety.

[0151] C-dots or C'-dots allow the coupling of antibody fragments to therapeutic radiolabels (e.g. 177 Lu, 225 Ac, 90 Y, 89 Zr) to act as a highly specific and effective multi-therapeutic targeting particle probe. Alternatively, coupling of targeting peptides known to be immunomodulatory and anti-inflammatory in nature, such as alpha MSH, with C-dots or C'-dots can also be combined with C-dots or C'-dots radiotherapy (and / or other particle-based) platforms to achieve enhanced efficacy. In certain embodiments, the concentration of radioisotopes and / or antibody fragments is higher in therapeutic applications compared to diagnostic applications.

[0152] Molecular therapeutics (e.g., antibodies) can modulate the immune system for antitumor activity by manipulating immune checkpoints (e.g., the monoclonal antibody ipilimumab inhibits CTLA4, a negative regulatory molecule that inhibits immune system function). The rationale is to trigger a pre-existing but dormant antitumor immune response. Other molecules and pathways act as immune switches. PD-1, another negative regulatory receptor expressed on T cells, has also been targeted. Switching a single immune checkpoint may not be sufficient to induce an antitumor response, explaining in part the failure of targeting a single immune regulatory checkpoint like PD-1 or CTLA4. However, without wishing to be bound by any theory, in some cases treatment can be aided by adding RT, which is believed to have immune-modulating properties. In these cases, tumors outside the RT treatment field have been found to shrink as a result of a presumed systemic inflammation or immune response elicited by RT, highlighting the potential for radiation to trigger a systemic antitumor immune response. Increasing immune activity may also enhance the local effects of RT.

[0153] By increasing the concentration of these immunoconjugates alone, the disease can be treated. To further treat the disease, a therapeutic radiolabel can also be added. In certain embodiments, the immunoconjugates act as therapeutic agents at high concentrations and without a therapeutic radiolabel. In certain embodiments, the radiolabels are attached to the same nanoparticles in an all-in-one multi-therapeutic platform. Alternatively, the therapeutic radiolabels can be administered independently. Further details are provided in International Application No. PCT / US16 / 26434 (published as WO2016 / 164578 on October 13, 2016), the contents of which are incorporated herein by reference in their entirety.

[0154] In contrast to other multimodal platforms, immunoconjugates can include different moieties attached to the nanoparticle itself. For example, in certain embodiments, a radioisotope is attached to the nanoparticle and an antibody fragment is attached to the nanoparticle, i.e., in these embodiments, the radiolabel is not attached to the antibody fragment itself. As another example, an immunoconjugate can include a targeting ligand attached to the nanoparticle, a radioisotope attached to the nanoparticle, and an antibody fragment attached to the nanoparticle. The stoichiometric ratio of the different moieties attached to the C-dot can affect the biodistribution of the nanoparticle immunoconjugate.

[0155] In certain embodiments, the nanoparticles comprise silica, polymers (e.g., poly(lactic-co-glycolic acid) (PLGA)), biologics (e.g., protein carriers), and / or metals (e.g., gold, iron). In certain embodiments, the nanoparticles are "C-dots," as described in U.S. Patent Application Publication No. 2013 / 0039848A1 by Bradbury et al., which is incorporated herein by reference.

[0156] In certain embodiments, the nanoparticles are spherical. In certain embodiments, the nanoparticles are not spherical. In certain embodiments, the nanoparticles are or include a material selected from the group consisting of metal / metalloid / nonmetal, metal / metalloid / nonmetal oxide, metal / metalloid / nonmetal sulfide, metal / metalloid / nonmetal carbide, metal / metalloid / nonmetal nitride, liposome, semiconductor, and / or combinations thereof. In certain embodiments, the metal is selected from the group consisting of gold, silver, copper, and / or combinations thereof.

[0157] Nanoparticles are made of metal / semimetal / nonmetal oxides, e.g., silica (SiO 2 ), titania (TiO 2 ), Alumina (Al 2 O 3 ), zirconia (ZrO 2 ), Germania (GeO 2 ), Tantalum Pentoxide (Ta 2 O 5 ), NbO2 and / or non-oxides, such as metal / semi-metal / non-metal borides, carbides, sulfides and nitrides, such as titanium and combinations thereof (Ti, TiB 2 , TiC, TiN, etc.

[0158] The nanoparticles may comprise one or more polymers, such as one or more polymers approved for human use by the U.S. Food and Drug Administration (FDA) under 21 CFR 177.2600, including, but not limited to, polyesters (e.g., polylactic acid, lactic acid-co-glycolic acid, polycaprolactone, polyvalerolactone, poly(1,3-dioxan-2-one)); polyanhydrides (e.g., poly(sebacic anhydride)); polyethers (e.g., polyethylene glycol); polyurethanes; polymethacrylates; polyacrylates; polycyanoacrylates; and copolymers of PEG and poly(ethylene oxide) (PEO).

[0159] The nanoparticles may include one or more degradable polymers, such as certain polyesters, polyanhydrides, polyorthoesters, polyphosphazenes, polyphosphoesters, certain polyhydroxy acids, polypropyl fumerates, polycaprolactones, polyamides, poly(amino 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, polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (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 ester), which may be suitable for use according to the present application.

[0160] In certain embodiments, nanoparticles can have or be modified to have one or more functional groups. Such functional groups (in nanoparticles or on the surface of nanoparticles) can be used to associate with any agent (e.g., detectable entity, targeting entity, therapeutic entity, or PEG). In addition to changing the charge of the surface by introducing or modifying surface functional groups, 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.

[0161] In certain embodiments, the nanoparticles comprise (e.g., bind to) one or more targeting ligands, such as, but not limited to, small molecules (e.g., folate, dyes, etc.), aptamers (e.g., A10, AS1411), polysaccharides, small biomolecules (e.g., iodine, galactose, bisphosphonates, biotin), oligonucleotides, and / or proteins (e.g., (poly)peptides (e.g., αMSH, RGD, octreotide, AP peptides, epidermal growth factor, chlorotoxin, transferrin, etc.), antibodies, antibody fragments, proteins, etc.). In certain embodiments, the nanoparticles comprise 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.).

[0162] In certain embodiments, a PET (positron emission tomography) tracer is used as the imaging agent. In certain embodiments, the PET tracer is 89 Zr, 64 Cu, 18 F]fluorodeoxyglucose. In certain embodiments, the nanoparticles comprise these and / or other radiolabels.

[0163] In certain embodiments, the nanoparticles include one or more fluorophores. Fluorophores include fluorescent dyes, fluorescent dye quencher molecules, any organic or inorganic dyes, metal chelates, or any fluorescent enzyme substrates, including enzyme substrates capable of activating proteases. In certain embodiments, the fluorophores include long-chain carbophilic cyanines. In other embodiments, the fluorophores include DiI, DiR, DiD, and the like. Fluorophores include far-red and near-infrared fluorescent dyes (NIRF). Fluorophores include, but are not limited to, carbocyanine fluorescent dyes and indocyanine fluorescent dyes. In certain embodiments, the imaging agent may be, but is not limited to, 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); and ADS780WS, ADS830WS, and ADS832WS (American Dye Source) and Kodak Commercially available fluorescent dyes include X-SIGHT 650, Kodak X-SIGHT 691, and Kodak X-SIGHT 751 (Carestream Health).

[0164] In certain embodiments, the nanoparticles include (e.g., have attached thereto) one or more targeting ligands, for example, for targeting to cancer tissues / cells of interest.

[0165] In certain embodiments, the nanoparticles comprise between 1 and 20 distinct targeting moieties (e.g., of the same type or of different types), where the targeting moieties bind to receptors on tumor cells (e.g., the nanoparticles have an average diameter of 15 nm or less, e.g., 10 nm or less, e.g., about 5 nm to about 7 nm, e.g., about 6 nm). In certain embodiments, the 1 to 20 targeting moieties comprise alpha-melanocyte-stimulating hormone (αMSH). In certain embodiments, the nanoparticles comprise a targeting moiety (e.g., αMSH).

[0166] In certain embodiments, the compositions and methods described herein induce cell death via ferroptosis by ingestion of nanoparticles.Furthermore, the present disclosure describes administering high concentrations of ultra-small (e.g., having a diameter of 20 nm or less, e.g., 15 nm or less, e.g., 10 nm or less) nanoparticles multiple times over the course of treatment in combination with a nutrient-depleted environment, thereby modulating cellular metabolic pathways that induce cell death by the mechanism of ferroptosis.Ferroptosis involves iron, reactive oxygen species, and a synchronized mode of cell death execution.Further details are provided in International Application No. PCT / US16 / 34351 (published as WO2016 / 196201 on December 8, 2016), the contents of which are incorporated herein by reference in their entirety.

[0167] Cancers that may be treated include, for example, prostate cancer, breast cancer, testicular cancer, cervical cancer, lung cancer, colon cancer, bone cancer, glioma, glioblastoma, multiple myeloma, sarcoma, small cell carcinoma, melanoma, renal cancer, liver cancer, head and neck cancer, esophageal cancer, thyroid cancer, lymphoma, pancreatic cancer (e.g., BxPC3), lung cancer (e.g., H1650), and / or leukemia.

[0168] In certain embodiments, the nanoparticles comprise a therapeutic agent, e.g., a drug moiety (e.g., a chemotherapeutic agent) and / or a therapeutic radioisotope. As used herein, "therapeutic agent" refers to any agent that has a therapeutic effect and / or induces a desired biological and / or pharmacological effect when administered to a subject.

[0169] In certain embodiments, for example, when combination therapy is used, the treatment methods of the embodiments include administration of nanoparticles and administration of one or more drugs (e.g., separately or conjugated to the nanoparticles), such as sorafenib, paclitaxel, docetaxel, MEK162, etoposide, lapatinib, nilotinib, crizotinib, fulvestrant, vemurafenib, bexorotene, and / or camptothecin.

[0170] The surface chemistry, coating uniformity (if a coating is present), surface charge, composition, concentration, administration frequency, shape, and / or size of the nanoparticles can be tailored to produce the desired therapeutic effect.

[0171] For example, nanoparticle conjugates are described herein that exhibit enhanced penetration into tumor tissue (e.g., brain tumor tissue) and diffusion within the tumor interstitium, for the treatment of cancer. Additionally, methods are described for targeting tumor-associated macrophages, microglia, and / or other cells in the tumor microenvironment using such nanoparticle conjugates. Additionally, diagnostic, therapeutic, and theranostic (diagnostic and therapeutic) platforms featuring such nanoparticle conjugates are described for treating targets in both the tumor and the surrounding microenvironment, thereby enhancing the efficacy of cancer treatment. It is also envisioned to use the nanoparticle conjugates described herein with other conventional therapies, such as chemotherapy, radiation therapy, and immunotherapy.

[0172] To overcome treatment resistance, combinations of multi-targeted kinase inhibitors and single-targeted kinase inhibitors have been developed.Importantly, the multi-modality combination of targeting agents, including particle-based probes designed with SMI, chemotherapeutic agents, radiotherapy labels, and / or immunotherapy, can enhance the efficacy of treatment and / or improve treatment regimens for malignant brain tumors.Combined with molecular imaging labels, these vehicles can then monitor the delivery, accumulation, and retention of drugs, which can provide optimal therapeutic index.

[0173] One such clinically translated ultrasmall nanoparticle (e.g., nanoparticles with diameters of 20 nm or less, e.g., 15 nm or less, e.g., 10 nm or less) platform, C'dots, is useful for this purpose. This nanoparticle was developed as a tumor-targeted dual-modality (PET-optical) drug delivery vehicle. These favorable motility, internalization, and enhanced tumor retention properties, together with their ability to readily diffuse within the tumor interstitium, suggested that systemic delivery of these particles to the CNS and broader distribution within the extracellular matrix may be suitable to achieve therapeutic concentrations and improve targeted treatment responses. New nanoparticle drug conjugates (NDCs) have been synthesized and characterized for the controlled delivery of prototypic EGFR (gefitinib, gef) and PDGFR (dasatinib, das) SMIs into EGFRmt+ and PDGFB-driven tumor models, respectively. SMIs were attached to the particle surface using several different linker chemistries; loading and release profiles were evaluated in serum-supplemented media.

[0174] In certain embodiments, the nanoparticles have an average diameter of 15 nm or less. In certain embodiments, the nanoparticles have an average diameter of 10 nm or less. In certain embodiments, the nanoparticles have an average diameter of about 5 nm to about 7 nm (e.g., about 6 nm). EXAMPLES

[0175] This example provides a two-pronged approach to demonstrate the feasibility of the nanoparticle platform described herein for treating tumors, particularly malignant brain tumors, in a subject. The first of the two-pronged approaches uses a primary glioma model to understand the behavior and distribution of nanoparticles in the tumor (e.g., when the drug is on the particle, the particle effectively treats the tumor compared to the free drug). The second of the two-pronged approach uses nanoparticle drug conjugates (NDCs) to treat and / or modulate the tumor microenvironment to change the phenotype of macrophages (e.g., in metastatic brain tumors). Xenografts were created and the in vivo efficacy of the provided compositions was established to establish the described compositions for treatment in the brain, as described in detail herein. The examples demonstrate that tumor targeting is achieved with and / or without the attachment of a targeting moiety to the nanoparticle composition. There is evidence that the use of a targeting moiety improves the transport and / or concentration of nanoparticles to / in the tissue / tumor of interest. Example 1 Optimizing distribution, efficacy, and dosing of C'dots in brain tumors

[0176] This example provides (1) the use of a genetically engineered mouse glioma model to determine the intratumoral and intracellular distribution dynamics of C'dots in brain tumors as a function of blood-brain permeability, time, RGD targeting and drug conjugation, and (2) the pharmacological efficacy and dosing optimization of C'dots conjugated to small molecule EGFR inhibitors via cleavable linkers in a metastatic model of EGFR mutant non-small cell lung cancer.

[0177] Following incubation of EGFRmt+ and PDGFB-driven tumor cell lines with gefitinib (or dasatinib)-modified C'dots, cell internalization, inhibition profile, and viability were assessed over a range of particle concentrations and times (i.e., 6, 18 h) compared to native SMI. For EGFRmt+ expressing cell lines, non-small cell lung cancer (NSCLC) lines were tested, including L858R ECLC26, a line containing an activating single-point substitution mutation L858R in exon 21 that confers sensitivity to EGFR tyrosine kinase inhibitors. A less sensitive NSCLC line, H1650, carrying a resistance mutation, was also used. As PDGFR expressing cells, 3T3 cells and primary PDGFB-driven cells were used. In the latter case, cells were derived from a genetically engineered mouse model (GEMM) of high-grade glioma in which the receptor, tv-a, was engineered into mouse lines under the GFAP or nestin promoter (i.e., Gtv-a and Ntv-a, respectively), while RCAS was used for PDGF-B gene transfer. The EGFR and PDGFR phosphorylation status of cells was assayed by Western blot, and findings were used to select lead candidates for in vivo efficacy studies.

[0178] In parallel with the in vitro studies, in vivo baseline studies were performed using the base particle probe (i.e., FDA-IND approved cRGDY-PEG-C'dots) and dasatinib-NDC along with time-dependent vital staining methods that provided an initial assessment of intratumoral penetration and particle distribution kinetics as a function of blood-brain barrier permeability, integrin targeting (versus nonintegrin targeting using cRGDY-PEG-C'dots), and subsequent drug conjugation in RCAS-tva GEMMs of high-grade gliomas.

[0179] Using dose escalation studies with dual-modality particle probes, we will investigate the improvement of delivery, penetration, and maximum treatment response of targeted therapeutics over native drugs for both dasatinib-NDC of PDGFB-driven glioma and gefitinib-NDC in EGFRmt+ preclinical flank / brain xenograft models, and confirm imaging findings histologically. Drug-based escalation studies will be used to assess unanticipated toxicity and evaluate particle dosimetry. Kinetic studies were also performed with these agents. Separate cohorts of mice were injected with the dual-modality particle probes, allowing us to track drug-to-particle delivery and distribution and monitor platform stability. The expected increase in effective drug concentration at the tumor site is based on previously observed preferential tumor retention and the ability to quantitatively estimate therapeutic dosing requirement for tracer NDC doses using PET imaging.

[0180] These SMI-bearing platforms have also been further adapted to targeting peptides, including cRGDY and αMSH, the former for delivering and targeting SMI to integrin and / or melanocortin-1 (MC1) receptors. Integrins are expressed by primary glioma cells and by tumor vascular endothelial cells, while the latter are expressed by tumor-associated macrophages in the microenvironment. The contribution of integrin receptor targeting to the overall intratumoral accumulation of these probes can then be determined for this ultra-small (10 nm or less) particle size. Nonspecific uptake in tumors due to enhanced permeability retention (EPR) effects can also be assessed using scrambled peptide (cRADY)-conjugated C'dots (control) that do not bind to integrin receptors. Without wishing to be bound by theory, it is believed that the ultra-small size of these particles allows for diffusion within the tumor interstitium (see Figures 1-35) to reach a greater number of cellular targets versus larger nanomaterials (i.e., liposomes) that accumulate in large quantities along the vascular walls at sites of vascular leakage (by EPR action). Such theranostic platforms (diagnostic-therapeutic) can be used to treat targets both in the tumor and in the surrounding microenvironment (via macrophages or other immune / inflammatory cell types). For example, dasatinib can be used with cRGDY-conjugated C'dots to target primary glioma cells (and activated endothelium), while inhibitors targeting TAMs (i.e., inhibitors of macrophage CSF-1 receptor (CSF-1R)) or other immune components may be conjugated to αMSH-conjugated C'dots. It should be noted that αMSH is a neuroimmunomodulator and its receptor, MC1-R, is present on macrophages.

[0181] Use the results of preclinical studies to inform clinical trial design. 124 Targeted delivery and penetration of I-cRGDY-conjugated C'dots is currently monitored in pre-operative patients harboring either brain metastases (i.e., NSCLC, breast cancer) or GBM, two tumor types where improved delivery of SMIs to the CNS is likely to be clinically significant.124 Sequential PET-CT imaging will be used to detect, localize, and evaluate particle tracer accumulation within brain tumors over a 24-hour period following intravenous injection of I-cRGDY-conjugated C'dots. Tissue will be analyzed from tumor biopsies targeting areas of tracer uptake within and near the tumor to correlate imaging with molecular abnormalities and tissue particle distribution. The experimental protocol includes: (1) co-registered routine preoperative MRI and PET-CT imaging for confirmation of potential biopsy target(s); 124 (1) I-cRGDY-PEG-C'dots; (2) surgical resection to acquire routine targeted tissue with integrated frameless stereotactic tracking used to annotate biopsy sites and updated by intraoperative MRI (iMRI, 1.5T Siemens magnet). Tissue samples derived from several regions are collected within and near the tumor. Tumor tissue areas that show uptake of the particle tracer and other tissues that show little or no uptake are analyzed for integrin expression. Assays include immunohistochemistry using commercially available antibodies.

[0182] Furthermore, it is contemplated that the conjugates described herein can be used to manipulate (e.g., regulate, control) the behavior of certain cells (e.g., macrophages, tumor-associated macrophages and / or microglia (TAMs), dendritic cells, and / or T cells) in the tumor microenvironment (e.g., in vivo, e.g., in the treatment of cancer, e.g., brain cancer, e.g., malignant cancer, e.g., malignant brain cancer). For example, conjugates of inhibitors of CSF-1 receptor (CSF-1R) and ultra-small nanoparticles can be used to target tumor-associated macrophages in the tumor microenvironment to regulate / control them in the treatment of tumors. For example, the nanoparticle conjugates described can include a modulator moiety (e.g., an inhibitor of colony-stimulating factor-1 (CSF-1R)) for targeting TAMs.

[0183] A chart illustrating the use of the RCAS-PDGFB mouse glioma model to study C'dot distribution with simultaneous vital staining is shown in Figure 10. To further evaluate how this particle could be used therapeutically, particle distribution was further investigated, both within the tumor and at the cellular level. Using the RCAS brain tumor model, a methodology was developed to administer fluorescent labels prior to sacrifice. Hoechst was used to stain cell nuclei as a marker of cellular localization, and green fluorescent FITC-70kDa dextran was used to roughly approximate particle size as a marker of disrupted blood-brain barrier, estimating EPR effects only in the small dextran. Particle distribution over time was also investigated, focusing on the shorter time point of 3 hours post-treatment compared to the 96 hour time point.

[0184] In RCAS tumor-bearing mice 124 Ex of I-RGD-Cys5-C-dot distribution Images from the in vivo study are also provided in FIG. 12. RGD-targeted nanoparticles are strongly retained in the tumor at 96 hours post-injection (pi) and diffuse across 70 kDa dextran given 3 hours prior to sacrifice. RCAS-tva tumor-bearing mice are treated in vivo with RGD-targeted C-dots at 96 hours prior to sacrifice (pts), FITC-dextran at pts3 hours, followed by Hoechst at pts10 minutes. Compared to the Cy5 and FICT signals being very close together when co-administered at pts3 hours, the Cy5 signal at 96 hours post-treatment appears to be more diffuse within the tumor than the FITC signal in the concentrated areas of disrupted BBB and retains high signal intensity. The Cy5 signal is very close to the areas of tumor identified by H&E. RGD-targeted C-dots are retained at 96 hours and appear to diffuse alone through the tumor across the areas of BBB disruption. I-124 autoradiography demonstrated illumination of areas that closely matched the Cy5 signal, suggesting that I-124 remained bound to the Cy5 containing C-dots in vivo.

[0185] Triple fluorescent labeling images of FITC-dextran as a fiducial tracer of similar size to the nanoparticle conjugates of Figures 13A, 13B, and 14 are shown in Figures 15A-15F. As explained above, the data suggest intracellular localization of Cy5-C' dots as early as at least 3 hours after treatment. In addition to tumor distribution, high magnification imaging of tumor sections was obtained to visualize particle distribution at the cellular level. In these images, strong blue nuclear staining can be seen closely surrounded by red nanoparticles. Without wishing to be bound by any theory, the data suggest intracellular localization, possibly in lysosomes.

[0186] Figures 27 and 28 show images and data showing distribution analysis by pixel correlation. High power imaging of focal areas of tumors treated with targeted and non-targeted nanoparticles. RCAS-tva tumor-bearing mice were treated with either radiolabeled RGD-targeted nanoparticles or RAD-nanoparticles, injected with FITC-dextran 3 hours prior to sacrifice, and sacrificed 96 hours after treatment. Frozen sections were analyzed for fluorescent signal using high power imaging of representative areas. The data show areas of BBB disruption marked by FITC and closely overlapping areas of RAD nanoparticle signal compared to a more diffuse pattern of Cy5 signal beyond FITC hotspots in tumors treated with RGD nanoparticles.

[0187] Figure 29 shows images of Western blots showing that Dasatinib-NDC achieves PDGFR inhibition in a dose-dependent manner at levels similar to free drug. TS543 (neurosphere cells) were treated with the indicated drugs for 4 hours, followed by PDGF-BB 20ng / ml for 10 minutes. Prior to treatment, cells were starved for 18 hours with stem cell medium without growth factors. Modified / linker Dasatinib-NDC showed p-PDGFRα inhibition in a dose-dependent manner at doses comparable to those shown by free Dasatinib to inhibit p-PDGFRα.

[0188] Figure 30 shows images of Dasatinib-NDC distribution in tumors 3 and 96 hours after treatment. RCAS-tva tumor-bearing mice were treated with non-targeted Dasatinib-nanoparticle conjugates (Das-NDC) and sacrificed 3 and 96 hours after treatment, followed by injection of FITC-dextran 3 hours before sacrifice. Frozen sections were analyzed for fluorescent signal using high-power imaging of representative areas. A high degree of overlap between Cy5 and FITC was observed at 3 and 96 hours, reproducing similar findings with the corresponding non-targeted non-conjugated nanoparticles (RAD-NP).

[0189] Figure 31 shows H&E and fluorescence images of comparable distribution of fluorescent signal in targeted and non-targeted particle nanoparticle-drug conjugates compared to targeted and non-targeted particles alone. RCAS-tva tumor-bearing mice were treated with non-targeted Dasatinib-nanoparticle conjugates (Das-NDC) and targeted Dasatinib-nanoparticle conjugates (RGD-DAS-NDC) and sacrificed 96 hours after treatment by injection of FITC-dextran 3 hours and Hoechst 10 minutes prior to sacrifice. Frozen sections were analyzed for fluorescent signal using high-power imaging of representative areas. Representative samples showing similar distribution in non-targeted Das-NDC tumors compared to RAD-NP, and RGD-NDC compared to RGD-NP, suggesting retention of nanoparticle uptake and diffusion properties with introduction of drug conjugates.

[0190] SMI was used as a model system to study the kinetics of drug conjugates. Gefitinib drug model, which has efficacy in primary NSCLC but not in the treatment of brain metastases, was used, and its characteristics of being highly protein-bound and cleared in the liver are shown in Figures 17 and 18. If the kinetics of nanoparticles can be improved in this regard with enhanced clearance in the kidney, a higher therapeutic index can be realized. Therefore, gefitinib was attached to C'-dots using optimization of drug-linker combinations. It was then demonstrated that the modified drug-NP conjugates retained efficacy as measured by pEGFR inhibition despite the modification of the drug. Optimization of the delivery and release of SMI from NDC can be investigated.

[0191] Figure 33 shows data from multiple dose treatment of ECLC26 flank tumor-bearing mice resulting in robust tumor control. Growth analysis was examined on days 8 and 9. ECLC26 tumor-bearing mice were treated with either gefitinib-NDC, daily gefitinib PO (150 mg / kg), or daily oral saline vehicle at two time points in a multiple dose model with dose administration indicated by blue arrows. Tumor volume was measured daily using caliper measurements of the largest tumor dimension. The graph shows the natural growth of vehicle-treated tumors over time compared to the steady decrease in tumor volume in the Gef-NDC and gefitinib treatment groups. Of note, there was a recovery of tumor growth in the Gef-NDC treatment group approximately 8 days after treatment, suggesting a possible attenuation of efficacy at this time point.

[0192] Figure 34 shows ECLC26 growth after treatment. Nude mice were implanted with 2 million ECLC26 cells. Tumor-bearing mice were treated with 15 μM Gef-NDC at 10 μL / g for 10 days, either as 200 μL iv saline or 2 doses of gavage with 15 mg / ml gefitinib. Example 2 Modulation of the tumor microenvironment using targeted ultrasmall silica nanoparticle imaging probes (C'dots) for delivery and imaging of small molecule inhibitors

[0193] Therapeutic approaches targeting high-grade gliomas have largely failed. An alternative strategy is to modulate cells such as tumor-associated macrophages and microglia (TAMs) in the tumor microenvironment (TME). TAMs account for as much as 30% of the tumor mass in mouse models of high-grade gliomas and in brain tumor patients. Accumulation of TAMs is associated with more aggressive gliomas and poorer patient prognosis. Colony-stimulating factor-1 (CSF-1) is known to affect macrophage differentiation and survival, as well as their activation or maldistribution state. In a PDGF-driven mouse glioma model, inhibition of CSF-1R has been shown to suppress the M2 phenotype, reduce tumor growth, and improve survival.

[0194] In this example, a small molecule inhibitor, e.g., BLZ945, a CSF-1R drug, is selectively delivered to TAMs by conjugating a synthetic drug and a targeting peptide, e.g., alpha melanocyte stimulating hormone (αMSH), to ultra-small fluorescent silica nanoparticles (C'dots). Such compositions are referred to herein as "nanoparticle drug conjugates (NDCs)". By using a PDGF-driven mouse glioma model with established sensitivity to TAM modulation, the targeted delivery and efficacy of this NDC is evaluated and compared to the established efficacy of BLZ945 as a free drug. Additionally, combination treatment with an integrin-targeted NDC incorporating the PDGF inhibitor dasatinib was evaluated.

[0195] TAMs are the most predominant inflammatory cells in the TME where they comprise a heterogeneous community of distinct functional subtypes. Although the scope of TAM phenotypes is not fully understood, activated TAMs expressing M2 class markers have been shown to contribute to tumor initiation and maintenance, as well as to influence antitumor autoimmunity through cytokine release and inflammatory recruitment in the TME. In turn, tumors may promote the polarization of monocytes into M2 TAMs by releasing factors such as TGF-beta and M-CSF. Therapeutic modulation of TAM subtypes through intact physiological mechanisms is a potentially effective means of influencing the TME in a wide range of cancers.

[0196] As described herein, targeting TAMs in cancer may be most effective when combined with other therapies directed at tumor cells. Indeed, initial attempts at CSF-1R inhibition as a monotherapy for gliomas found little efficacy.

[0197] As described herein, ultrasmall nanoparticles (e.g., C'dots) were used to selectively deliver receptor tyrosine kinase (RTK) inhibitors (e.g., BLZ945) to melanocortin-1 receptor (MC1R)-expressing TAMs by conjugating its ligand, the neuroimmunomodulator alpha melanocyte stimulating hormone (αMSH). BLZ945, a specific CSF-1R inhibitor that regulates macrophage polarization and function, was synthesized and modified for conjugation to C'dots as described in International Application No. PCT / US2015 / 032565 (published as WO2015 / 183882 on December 3, 2015), the contents of which are incorporated herein by reference in their entirety.

[0198] Utilizing the RCAS PDGFB-driven genetically engineered mouse model (GEMM) of high-grade glioma, we leveraged the intense brightness of the resulting NDCs to evaluate uptake of αMSH-targeted particles in macrophages in vitro and in tumors. This model was chosen due to its sensitivity to TME modulation by CSF-1R inhibition, as well as disruption of tumor cell signaling by the PDGF and Src inhibitor dasatinib (das). Thus, we tested the efficacy of particle-based delivery of these drugs alone and potentially in combination. The development of das-RGDY-PEG-C'dots provides a methodology for mapping the delivery and diffusion of das-RGDY-PEG-C'dots and BLZ947-αMSH-PEG-C'dots as a function of blood-brain barrier permeability. Synthesis and characterization of targeted NDCs as combination agents to independently target tumor cells and TAMs in high-grade gliomas

[0199] Two RTK inhibitors, BLZ945 and dasatinib (das), were conjugated to C'dots by using cleavable chemical linkers. BLZ945, a CSF-1R specific RTK inhibitor developed at MSKCC, was adapted to a dipeptide-based chemical linker. This drug-linker construct was conjugated to αMSHPEG-C'dots to form NDC BLZ945-αMSH-PEG-C'dots for targeting TAMs, while dasatinib was conjugated to cRGDY-PEG-C'dots to target integrin-expressing glioma cells. If modification of BLZ945 impairs CSF-1R inhibition, an alternative strategy is to conjugate PLX3397, a CSF-1R multikinase inhibitor.

[0200] Also provided is the synthesis of das-cRGDY-PEG-C'dots. For example, modified dasatinib analogues are conjugated to C'dots via cleavable linkers. Das analogues are conjugated to cRGDY-functionalized particles to form NDC das-cRGDY-PEG-C'dots. In addition, BLZ945-αMSH-PEG-C'dots and das-cRGDY-PEG-C'dots are characterized by HPLC method to evaluate drug loading.

[0201] Drug release in the presence of serine and cysteine ​​proteases (eg, trypsin and cathepsin) was assessed by liquid chromatography-mass spectrometry. Evaluation of C'dots adapted with one or more targeting moieties (BLZ945; αMSH) to activate TAMs expressing CSF-1R and MC1R by assessing cytokine secretion and gene signatures

[0202] RAW264.7 mouse macrophages and primary mouse bone marrow-derived macrophages (BMDMs) were cultured in U-251 glioma-conditioned medium (GCM), which protects macrophages from BLZ945-induced cell death. BMDMs were prepared and cultured. Furthermore, a chelator-free radiolabeling strategy was compared with traditional chelator-based radiolabeling methods for particle radiolabeling in terms of stability, radiochemical yield, specific activity, uptake by tumor targets, and tumor-to-background ratio. The chelator-free approach utilizes the unique C'-dot deprotonated silanol groups (-Si-O-) to selectively cleave the cytoplasmic C'-dots. 89 Relying on Zr labeling, chelator-based methods 89 This involves conjugation of glutathione and desferrioxamine B to C' dot surface-bound PEG chains prior to Zr labeling.

[0203] Competitive binding studies were performed against native "cold" TKIs using MC1-R expressing macrophages and gamma counting detection to determine binding affinity and potency. 89Zr-αMSH-conjugated NDCs were performed. To test the binding specificity, MC1-R blocking experiments were performed using anti-MC1R antibodies prior to particle exposure and flow cytometry. Intracellular trafficking of particles through the endocytic pathway and lysosomal uptake were also examined. To investigate trafficking of C'dots through the endocytic pathway, a fluorescent reporter of endocytic trafficking was expressed and colocalization of ingested targeted NDCs with particle controls was examined by time-lapse microscopy.

[0204] Macrophages were incubated with BLZ945-conjugated αMSH-PEG-C'dots to inhibit CSF-1R signaling and target macrophages via αMSH ligands that bind to MC1R expressed in these cells. Dose- and time-dependent particle uptake into macrophages cultured in control or U-251 glioma-conditioned medium was quantified by flow cytometry and fluorescence microscopy. Cell viability was assayed by standard MTT assay after particle exposure (BLZ945-αMSH-PEG-C'dots, BLZ945-PEG-C'dots, αMSH-PEG-C'dots, PEG-C'dots). If particle treatments are well tolerated under these conditions, their effects on macrophage function can be examined.

[0205] Because treatment with BLZ945 was shown to affect the activation or polarization state of TAMs (e.g., reduced expression of M2 polarization markers), the effect of BLZ945-conjugated αMSH-PEG-C' dots on macrophage polarization and function was examined. The positive results from these initial studies suggesting that BLZ945-conjugated αMSH-PEG-C' dots affect macrophage function in a manner similar to soluble BLZ945 were used to guide further testing of αMSH-PEG-C' dots lacking a CSF-1R inhibitor or PEG-C' dots lacking an αMSH targeting ligand to determine whether the base particles could also contribute to modulating macrophage properties.

[0206] RAW264.7 macrophages and BMDMs cultured in GCM were exposed to increasing doses of BLZ945-αMSH-PEG-C'dots or soluble BLZ945 (at 670 nM) and were shown to upregulate a four-gene signature (Adrenomedulin, Arginase 1, Expression of cytokines (e.g., clotting factor F13a1, mannose receptor) was examined. Cytokines associated with M1 (e.g., TNFα, IL-12P70, IL-1β, IFN-γ) or M2 polarization (e.g., IL-10, TGFβ) were assessed by ELISA-based detection from the culture medium. Target gene expression of early growth receptor 2 (Egr2), a downstream transcription factor of CSF-1R, was quantified in control and treated cells by QRT-PCR to determine the degree of inhibition of CSF-1R activation by particle treatment. Modulation of phagocytic activity of cultured macrophages, a hallmark of M1 polarization shown to be upregulated by BLZ945 treatment, was examined by incubating RAW264.7 or BMDM with apoptotic cells and quantifying the phagocytic index. Evaluation of the binding / uptake properties and specificity of das-cRGDY-PEG-C' dots

[0207] 89 To evaluate the binding affinity and potency of Zr-das-cRGDY-PEG-C'dots, competitive binding studies were performed against a native "cold" TKI (das) using the methods described except using primary cells derived from PDGFB-driven gliomas. Prior to particle exposure, integrin receptor blocking studies were performed using an anti-αv integrin antibody. Viability studies following particle exposure (das-cRGDYPEG-C'dots, das-PEG-C'dots, cRGDY-PEG-C'dots, PEG-C'dots) were performed using the methods described herein. Quantitative assessment of PK profile and tumor-selective accumulation

[0208] In PDGFB-driven high-grade gliomas with histological correlation 89 Zr-NDC (e.g. 89 Zr-BLZ945-, 89 Zr-das-PEG-C'dots) and 89 Zr-labeled particle control (αMSH-,cRGDY-PEG-C'dots) 89 We describe the PK profiles and quantitative evaluation of tumor-selective accumulation of Zr-labeled peptide-conjugated NDCs (e.g., BLZ945-αMSH-PEG-C'dots, das-cRGDY-PEG-C'dots).

[0209] Gliomas were generated by RCAS-mediated transfer of the oncogenic driver PDGFB into nestin+ progenitor cells in the brains of Nestin-tva mice. 89 Zr-αMSH (or 89 Zr-cRGDY)NDC, 89 Zr-NDC, or 89 Following intravenous (iv) injection of Zr-labeled particle controls (~20 μCi per mouse), glioma mice (n=5 per particle) were sacrificed at five specific time points (4 h to 168 h) and blood, urine, tumors, and organs were collected, weighed, and gamma counted to determine %ID / g correlated to decay versus time of injection. Results were compared to those of the respective particle controls. RadioTLC of blood and urine was also performed to assess particle stability over this interval.

[0210] Separate cohorts of mice were used to inject 200 μCi of 89 After iv injection of Zr-labeled peptide-conjugated NDC, non-peptide-conjugated NDC, and control probe, consecutive 15-min still images were acquired on an Inveon PET / CT scanner over a 96-h interval.

[0211] Histological assays of resected tumor tissue specimens, digital autoradiography, and multichannel fluorescence microscopy were performed to evaluate and compare the subcellular localization and distribution of particles among the imaging particle probes. Determine whether improved therapeutic efficacy was achieved for targeted NDC versus particle control

[0212] Glioma studies using CSF-1R inhibitors demonstrated robust responses approximately 1 week after treatment. Gradient-echo MR imaging of brain tumors was obtained on a 4.7 Tesla MRI scanner 4-9 weeks after intracranial inoculation. Region-of-interest analysis was performed to assess tumor volume, and volume-matched pairs of mice were assigned to either treatment or control groups for survival studies. Tumor volumes (mm) were measured on consecutive MRI slices. 3 ) was calculated. Mice (n=15 overall) were injected iv with a single dose of BLZ945-αMSH-PEG-C'dots or BLZ945 against saline vehicle (200 μL) for 10 consecutive days and body weights were recorded daily. At the end of treatment, mice underwent repeated MR imaging to assess changes in tumor volume. Percent tumor volume was calculated for individual mice by dividing post-treatment (day 10) values ​​by pre-treatment (day 0) values, as well as for cohort averages. Efficacy (non-inferiority) was established over a short interval (1-2 weeks). This data was compared to multiple dosing and toxicology of NDC to the free drug to determine if the PK of NDC would improve the therapeutic index versus the free drug. Gliomas were isolated and dissociated, resulting in single cell suspensions that could be stained with dye-labeled antibodies for flow cytometric analysis and sorting. Co-localization of particles in specific TME cell types was achieved by applying a multi-fluorochrome antibody panel (e.g., CD45, CD11b, CD11c) to identify myeloid and lymphoid cell types.

Claims

[Claim 1] The invention as depicted in the drawings.

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