Targeted drug delivery with self-agglomerating nanohydrogels (SANGS)

Abstract

Described herein are compositions comprising a population of nanohydrogels covalently conjugated to drug molecules and methods of treating cancer using said compositions.

Description

[0001] GTZ-01525

[0002] TARGETED DRUG DELIVERY WITH SELF-AGGLOMERRATING NANOHYDROGELS (SANGs)

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 749,315, filed January 24, 2025, which is incorporated by reference herein in its entirety.

[0004] BACKGROUND

[0005] Cancer remains a leading cause of death worldwide, with metastases responsible for over 90% of cancer-related mortality. Despite advances in cancer treatment, the development of effective and safe drug delivery systems for targeting tumors remains a significant challenge. Conventional chemotherapy often suffers from poor pharmacokinetics, limited tumor penetration, and severe side effects due to non-specific distribution throughout the body. Nanoparticle-based drug delivery systems have emerged as a promising approach to improve the efficacy and safety of cancer therapies. These systems aim to overcome biological barriers, enhance drug accumulation in tumors, and reduce systemic toxicity. However, many existing nanoparticle formulations face limitations such as rapid clearance from circulation, inadequate tumor targeting, and insufficient drug release at the target site. There is a continued need for innovative nanoparticle designs that can address these challenges and provide more effective cancer treatment options.

[0006] SUMMARY OF THE INVENTION

[0007] This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the detailed description. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

[0008] According to some aspects of the present disclosure, a drug delivery system is provided. The drug delivery system includes a self-agglomerating nanohydrogel (SANG) particle and a drug molecule conjugated to the SANG particle via a linker. The SANG particle is capable of targeting cancer cells.

[0009] According to other aspects of the present disclosure, the drug delivery system may include one or more of the following features. The linker may be cleavable. The cleavable linker may be pH- sensitive. The cleavable linker may be enzyme- sensitive. The linker may be non-cleavable. The drug molecule may be a chemotherapeutic agent. The chemotherapeutic agent may be selected from the group consisting of mertansine, oxaliplatin, cisplatin, and combinations thereof. The drug molecule may be an immunomodulatory agent. The drug molecule may be a biologic drug. The SANG particle may comprise a polymer selected from 1

[0010] FH13249380.11GTZ-01525

[0011] the group consisting of polyethylene glycol, polyacrylamide, and combinations thereof. The SANG particle may have a size between 10 nm and 200 nm. The SANG particle may comprise surface modifications to enhance tumor targeting. The surface modifications may comprise tumor-targeting ligands. The drug delivery system may further comprise an imaging agent conjugated to the SANG particle. The SANG particle may be capable of penetrating the tumor microenvironment. The SANG particle may exhibit prolonged circulation time in vivo. The drug molecule may be conjugated to the SANG particle at a drug-to-particle ratio of 5:1 to 50:1. The SANG particle may be capable of releasing the drug molecule in a controlled manner. The controlled release may be triggered by changes in pH or presence of specific enzymes in the tumor microenvironment. The SANG particle may be capable of targeting both primary tumors and metastatic lesions.

[0012] According to still other aspects of the present disclosure, a method of treating cancer is provided. The method includes administering to a subject in need thereof a therapeutically effective amount of a drug delivery system comprising a self-agglomerating nanohydrogel (SANG) particle and a drug molecule conjugated to the SANG particle via a linker. The SANG particle is capable of targeting cancer cells.

[0013] According to even further aspects of the present disclosure, the method of treating cancer may include one or more of the following features. The linker may be cleavable. The cleavable linker may be pH- sensitive. The cleavable linker may be enzyme- sensitive. The linker may be non-cleavable. The drug molecule may be a chemotherapeutic agent. The chemotherapeutic agent may be selected from the group consisting of mertansine, oxaliplatin, cisplatin, and combinations thereof. The drug molecule may be an immunomodulatory agent. The SANG particle may comprise a polymer selected from the group consisting of polyethylene glycol, polyacrylamide, and combinations thereof. The SANG particle may have a size between 10 nm and 200 nm. The SANG particle may comprise surface modifications to enhance tumor targeting. The surface modifications may comprise tumortargeting ligands. The method may further comprise administering an imaging agent conjugated to the SANG particle. The SANG particle may be capable of penetrating the tumor microenvironment. The SANG particle may exhibit prolonged circulation time in vivo. The drug molecule may be conjugated to the SANG particle at a drug-to-particle ratio of 5:1 to 50:1. The SANG particle may be capable of releasing the drug molecule in a controlled manner. The controlled release may be triggered by changes in pH or presence of specific enzymes in the tumor microenvironment. The SANG particle may be capable of targeting both primary tumors and metastatic lesions. The cancer may be selected from the group consisting of breast cancer, lung cancer, colorectal cancer, melanoma, and lymphoma.

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[0015] FH13249380.11GTZ-01525

[0016] According to other aspects of the present disclosure, a method of making a drug delivery system is provided. The method includes providing a self-agglomerating nanohydrogel (SANG) particle and conjugating a drug molecule to the SANG particle via a linker. The SANG particle is capable of targeting cancer cells.

[0017] According to other aspects of the present disclosure, the method of making a drug delivery system may include one or more of the following features. The linker may be cleavable. The cleavable linker may be pH- sensitive. The cleavable linker may be enzyme- sensitive. The linker may be non-cleavable. The drug molecule may be a chemotherapeutic agent. The chemotherapeutic agent may be selected from the group consisting of mertansine, oxaliplatin, cisplatin, and combinations thereof. The drug molecule may be an immunomodulatory agent. The SANG particle may comprise a polymer selected from the group consisting of polyethylene glycol, polyacrylamide, and combinations thereof. The SANG particle may have a size between 10 nm and 200 nm. The method may further comprise modifying the surface of the SANG particle to enhance tumor targeting. Modifying the surface may comprise conjugating tumor-targeting ligands to the SANG particle. The method may further comprise conjugating an imaging agent to the SANG particle. Conjugating the drug molecule to the SANG particle may comprise using a linker selected from the group consisting of oxanorbornadiene (OND) linkers and maleimide linkers. The OND linker may be a 9-hour half-life releasing OND-NHS linker. The OND linker may be a non-releasing epoxide OND-NHS linker. Conjugating the drug molecule to the SANG particle may comprise using EDC coupling. The method may further comprise purifying the drug-conjugated SANG particle using size exclusion chromatography. The drug molecule may be conjugated to the SANG particle at a drug-to-particle ratio of 5:1 to 50:1. The method may further comprise characterizing the drug-conjugated SANG particle using a fluorescence assay or inductively coupled plasma mass spectrometry.

[0018] In certain aspects, the present disclosure provides a composition, comprising:

[0019] a population of nanohydrogels; and

[0020] one or more drug molecules or imaging agents,

[0021] wherein each of the population of nanohydrogels comprises a cross-linkable core / shell polymer;

[0022] wherein each of the one or more drug molecules or imaging agents are bound to the population of nanohydrogels via one or more covalent linkages;

[0023] wherein, optionally, the nanohydrogel is configured to self-agglomerate in aberrant vasculature environments;

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[0026] wherein, optionally, the population of nanohydrogels is inducible via tortuous flow of an aberrant microvasculature of a tumor microenvironment (TME) to self-aggregate to form an agglomerated cluster; and

[0027] wherein the agglomerated cluster has an average hydrodynamic size larger than an average hydrodynamic size prior to self-agglomeration.

[0028] In certain embodiments, the composition comprises one or more imaging agents. In certain embodiments, each of the one or more imaging agents is bound to a core of the nanohydrogel. In certain embodiments, the composition releases the one or more imaging agents in a controlled manner.

[0029] In certain embodiments, the composition comprises one or more drug molecules. In certain embodiments, each of the one or more drug molecules is bound to a core of the nanohydrogel.

[0030] In certain embodiments, each of the cross-linkable core / shell polymer comprises a gel-forming polymer. In certain embodiments, each of the gel-forming polymer comprises a biocompatible polymer.

[0031] In certain embodiments, each of the population of nanohydrogels comprises a polymer selected from polyethylene glycol, polyacrylamide, and combinations thereof. In certain embodiments, each of the cross-linkable core / shell polymers comprises polyacrylamide.

[0032] In certain embodiments, each of the cross-linkable core / shell polymers comprises a copolymer of N-isopropylmethacrylamide and N,N'-methylenebis(acrylamide).

[0033] In certain embodiments, a shell of the nanohydrogel comprises a cationic polymer ligand (e.g., poly(aminopropyl methacrylate)).

[0034] In certain embodiments, the nanohydrogel has a hydrodynamic size of from about 10 nm to about 250 nm prior to self-agglomeration. In certain embodiments, the nanohydrogel has a hydrodynamic size of from about 10 nm to about 200 nm prior to self-agglomeration. In certain embodiments, the nanohydrogel has a hydrodynamic size of from about 50 nm to about 250 nm prior to self-agglomeration.

[0035] In certain embodiments, the population of nanohydrogels further comprises surface modifications.

[0036] In certain embodiments, the population of nanohydrogels is inducible via tortuous flow of an aberrant microvasculature of a tumor microenvironment (TME) to selfagglomerate to form an agglomerated cluster, wherein the agglomerated cluster has an

[0037] 4

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[0039] average hydrodynamic size larger than an average hydrodynamic size prior to selfagglomeration.

[0040] In certain embodiments, self-agglomeration of the population of nanohydrogels is concentration-dependent. In certain embodiments, a concentration of nanohydrogels causing self-agglomeration is 10 nM or greater (e.g., 20 nM or greater, 30 nM or greater, 40 nM or greater, 50 nM or greater, 60 nM or greater, 70 nM or greater, 80 nM or greater, 90 nM or greater, 100 nM or greater, 110 nM or greater, 120 nM or greater, 130 nM or greater, 140 nM or greater, 150 nM or greater, 156 nM or greater, 200 nM or greater, 250 nM or greater, 500 nM or greater, 1 pM or greater). In certain embodiments, a concentration of nanohydrogels causing self-agglomeration is from about 10 nM to about 1 pM (e.g., from about 10 nM to about 500 nM, from about 30 nM to about 500 nM, from about 60 nM to about 500 nM, from about 60 nM to about 250 nM, from about 100 nM to about 250 nM, from about 100 nM to about 200 nM, or about 156 nM).

[0041] In certain embodiments, the one or more covalent linkages are cleavable. In certain embodiments, the one or more cleavable covalent linkages are pH-sensitive. In certain embodiments, the one or more cleavable covalent linkages are enzyme- sensitive.

[0042] In certain embodiments, the one or more covalent linkages are non-cleavable.

[0043] In certain embodiments, the one or more drug molecules is one drug molecule.

[0044] In certain embodiments, each of the one or more drug molecules is independently a chemotherapeutic agent. In certain embodiments, each chemotherapeutic agent is independently selected from mertansine, oxaliplatin, cisplatin, and combinations thereof. In certain embodiments, the chemotherapeutic agent is mertansine. In certain embodiments, the chemotherapeutic agent is oxoplatin.

[0045] In certain embodiments, each of the one or more drug molecules is a biologic drug. In certain embodiments, each of the one or more drug molecules is selected from a cytotoxic agent, immunomodulatory agent, complement-activating agent, and receptor blocking agent. In certain embodiments, each of the one or more drug molecules is a cytotoxic agent. In certain embodiments, each of the one or more drug molecules is an immunomodulatory agent. In certain embodiments, each of the one or more drug molecules is a complement-activating agent. In certain embodiments, each of the one or more drug molecules is a receptor-blocking agent.

[0046] In certain embodiments, each of the one or more drug molecules is independently a therapeutic agent selected from monoclonal antibodies, chimeric antibodies, humanized

[0047] 5

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[0049] antibodies, nanobodies, antibody fragments, cholesterol, hormones, peptides, proteins, chemotherapeutics, antineoplastic agents, low molecular weight drugs, vitamins, co-factors, nucleosides, nucleotides, oligonucleotides, polynucleotides, enzymatic nucleic acids, antisense nucleic acids, triplex forming oligonucleotides, antisense DNA or RNA compositions, chimeric DNA:RNA compositions, allozymes, aptamers, ribozyme, decoys, analogs, plasmids, expression vectors, small nucleic acid molecules, mRNA, RNAi agents, short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA (shRNA), peptide nucleic acid (PNA), locked nucleic acid ribonucleotides (LNA), morpholino nucleotides, threose nucleic acid (TNA), glycol nucleic acid (GNA), sisiRNA (small internally segmented interfering RNA), aiRNA (assymetrical interfering RNA), and siRNA with 1, 2, or more mismatches between the sense and anti-sense strand to relevant cells or tissues, or a combination thereof. In certain embodiments, each therapeutic agent independently comprises an RNAi agent. In certain embodiments, each therapeutic agent independently comprises an anticancer agent. In certain embodiments, each therapeutic agent independently comprises a nucleic acid. In certain embodiments, each therapeutic agent independently comprises small interacting RNA (siRNA) and / or microRNA (miRNA). In certain embodiments, each therapeutic agent independently comprises an inhibitor of epidermal growth factor receptor (EGFR), Kirsten rat sarcoma virus oncogene homolog (KRAS), Glul, and / or Zinc Finger E-Box Binding Homeobox 1 (ZEB1). In certain embodiments, each therapeutic agent independently comprises an inhibitor of EGFR. In certain embodiments, each therapeutic agent independently comprises an inhibitor of Glul. In certain embodiments, each therapeutic agent independently comprises an inhibitor of ZEB1. In certain embodiments, each therapeutic agent independently comprises an inhibitor of KRAS. In certain embodiments, the surface modifications comprise tumor-targeting ligands.

[0050] In certain embodiments, the composition further comprises one or more imaging agents conjugated to the population of nanohydrogels. In certain embodiments, each of the one or more imaging agents independently comprises a contrast agent, a heavy metal, or a radioisotope. In certain embodiments, each of the one or more imaging agents independently comprises a fluorophore.

[0051] In certain embodiments, the one or more drug molecules are conjugated to the population of nanohydrogels at a ratio of about 5:1 to about 50:1.

[0052] 6

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[0054] In certain embodiments, the composition releases the one or more drug molecules in a controlled manner. In certain embodiments, the composition releases the one or more imaging agents in a controlled manner.

[0055] In certain embodiments, each covalent linkage independently comprises oxanorbornadiene (OND) or maleimide.

[0056] In certain embodiments, each covalent linkage independently comprises a linker. In certain embodiments, each linker is independently an OND linker. In certain embodiments, each linker is independently a maleimide linker. In certain embodiments, the OND-NHS linker comprises a releasing half-life between about 1 hour to about 10 hours. In certain embodiments, the releasing half-life is about 9 hours. In certain embodiments, the OND linker is a non-releasing epoxide OND-NHS linker.

[0057] In certain aspects, the present disclosure provides a method of treating cancer, comprising:

[0058] administering to a subject in need thereof a therapeutically effective amount of a composition disclosed herein.

[0059] In certain embodiments, the cancer is selected from breast cancer, lung cancer, colorectal cancer, ovarian cancer, pancreatic cancer, melanoma, and lymphoma. In certain embodiments, the cancer is ovarian cancer. In certain embodiments, the cancer is colorectal cancer. In certain embodiments, the cancer is breast cancer. In certain embodiments, the cancer is lung cancer. In certain embodiments, the cancer is pancreatic cancer. In certain embodiments, the cancer is melanoma. In certain embodiments, the cancer is lymphoma.

[0060] In certain embodiments, the cancer is a cancer having an undruggable cancerpromoting gene. In certain embodiments, the undruggable cancer-promoting gene comprises one or more of c-Myc. APC, BRAF, and / or KRAS.

[0061] In certain embodiments, a tumor selectivity of the composition is 1% or greater (e.g., 1.5% or greater, 2% or greater, 2.5% or greater, 3% or greater, 3.5% or greater, 4% or greater, 4.5% or greater, 5% or greater, 5.5% or greater, 6% or greater, 6.5% or greater, 7% or greater, 7.5% or greater, 8% or greater, 9.5% or greater, 10% or greater, 10.5% or greater, 11% or greater, 11.5% or greater, 12% or greater, 12.5% or greater, 13% or greater, 13.5% or greater, 14% or greater, 14.5% or greater, 15% or greater, 20% or greater, or 25% or greater).

[0062] In certain embodiments, a ratio of relative delivery of the composition between the tumor and a liver of the subject is 1:1 or more (e.g., 1.5:1 or more, 2:1 or more, 2.5:1 or more, 3:1 or more, 3.5:1 or more, 4:1 or more, 4.5:1 or more, 5:1 or more, 5.5:1 or more, 6:1 or 7

[0063] FH13249380.11GTZ-01525

[0064] more, 6.5:1 or more, 7:1 or more, 7.5:1 or more, 8:1 or more, 8.5:1 or more, 9:1 or more, 9.5:1 or more, 10:1 or more, 10.5:1 or more, 15:1 or more, 20:1 or more, or 25:1 or more).

[0065] In certain embodiments, 2.5% or more of the composition is retained after 24 hours from an initial dose (e.g., 3% or more, 3.5% or more, 4% or more, 4.5% or more, 5% or more, 5.5% or more, 6% or more, 10% or more).

[0066] In certain embodiments, the administration of the composition comprises administering a first composition comprising a first drug molecule and a second composition comprising a second drug molecule. In certain embodiments, the first composition and second composition are administered concurrently. In certain embodiments, the first drug molecule and the second drug molecule are the same. In certain embodiments, the first drug molecule and the second drug molecule are different. In certain embodiments, the first drug molecule comprises an RNAi agent and the second drug molecule comprises a chemotherapeutic agent.

[0067] In certain embodiments, the composition targets primary tumors. In certain embodiments, the composition targets metastatic lesions. In certain embodiments, the composition targets both primary tumors and metastatic lesions.

[0068] In certain aspects, the present disclosure provides a method of making a composition disclosed herein, comprising:

[0069] providing a population of nanohydrogels; and

[0070] conjugating a drug molecule to the population of nanohydrogels via a covalent linkage.

[0071] In certain embodiments, conjugating the drug molecule to the population of nanohydrogels comprises using l-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) coupling.

[0072] In certain embodiments, the method further comprises purifying the composition using chromatography. In certain embodiments, the chromatography is size-exclusion chromatography.

[0073] In certain embodiments, the method further comprises characterizing the composition using a fluorescence assay or inductively coupled plasma mass spectrometry. In certain embodiments, the composition is characterized using a fluorescence assay. In certain embodiments, the composition is characterized using inductively coupled plasma mass spectrometry.

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[0076] The foregoing general description of the illustrative embodiments and the following detailed description thereof are merely exemplary aspects of the teachings of this disclosure and are not restrictive.

[0077] BRIEF DESCRIPTION OF DRAWINGS

[0078] Fig. 1 shows (a) Whole-body imaging of ovarian cancer-bearing mice 72 hours after iv SANGs or saline control. Ex vivo imaging of tumors and major organs after in vivo imaging was completed, (b) Quantification of SANG delivery and retention to primary tumors and (c) organs shown as fold change (FC) normalized against background tissue fluorescence across 4, 24, 48, 72hr and Iw cohorts (n>4 / time). free siRNA used as quantitative control.

[0079] Fig. 2 shows (a) Representative ex vivo images 24 h after i.v. injection of SANGs to mice with metastatic ovarian cancer (21 days), (b) Quantification of SANG delivery and retention to metastatic tumors, normalized against SANG fluorescence signal in heart. I.V. delivery of free siRNA used as quantitative control, (c) Colocalization analysis of SANG fluorescence and tumor bioluminescence. Dotted line of identity indicates colocalization within ±15% bounding conditions (light blue, >95% on target dose). Upper left (dark blue) = off-target SANGs distribution (3.5% of dose); lower right (orange, the few pixels are difficult to see) = untargeted cancer (<0.01% dose).

[0080] Fig. 3 shows (a) Ex vivo images of lungs from tumor bearing rats relative to non-tumor bearing rats, 24 hours after i.v. SANGs (1 mg*kg-l). (b) Ex vivo images of pancreases from tumor bearing mice (KPC) relative to non-tumor bearing rats, 24 hours after i.v. SANGs (1 mg*kg-l). (c) Ex vivo images of colons from tumor bearing rats (APCPirc) relative to non-tumor bearing rats, at indicated times after i.v. SANGs (1 mg*kg-l). (d) Ex vivo images of primary breast tumor from and major organs from orthotopically implanted MDA-MB-231 24 hours after i.v. SANGs (1 mg*kg-l).

[0081] Fig. 4 shows Bioconjugation Reactions of Mertansine (DM1) onto SANGs via 9-hour Releasing OND-NHS Linker, as described in Example 1.

[0082] Fig. 5 shows Bioconjugation Reactions of Mertansine (DM1) onto SANGs via NonReleasing Epoxide OND-NHS Linker, as described in Example 1.

[0083] Fig. 6 shows bioconjugation reactions of Mertansine (DM1) onto SANGs via non-releasing NHS-maleimide linker, as described in Example 1.

[0084] Fig. 7 shows a diagram of the competition fluorescence assay used to quantify amount of DM1 drug loading onto SANG nanoparticles, as described in Example 2.

[0085] Fig. 8 shows injection and monitoring scheme for in Vivo 4T1 tumor studies treated with SANG-NHS-mal-DMl, as described in Example 4.

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[0088] Fig. 9 shows (a) an exemplary reaction scheme for oxoplatin synthesis from cisplatin, as described in Example 5; (b) oxoplatin conjugation to SANG particles via EDC coupling reaction, as described in Example 5.

[0089] Fig. 10 shows results for Quantification of Mertansine (DM1) on SANGs by Fluorescence Assay, as described in Example 7.

[0090] Fig. 11 shows results for In Vitro Cytotoxicity of DM1 Loaded SANGs on B16F10 and 4T1 Cells by PrestoBlue Assay, as described in Example 8.

[0091] Fig. 12a shows tumor volumes over time in SANG-NHS-mal-DMl treated 4t 1 tumor bearing mice, as described in Example 9.

[0092] Fig. 12b shows survival over time in SANG-NHS-mal-DMl treated 4tl tumor bearing mice, as described in Example 9.

[0093] Fig. 13 shows tumor volumes by individual mouse for each dose of SANG-NHS-mal-DMl condition, as described in Example 9.

[0094] DETAILED DESCRIPTION

[0095] Reference will now be made in detail to the embodiments of the disclosure, examples of which are illustrated in the drawings and the examples. This disclosure may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

[0096] Definitions

[0097] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. The term “comprising” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of’ and “consisting of’ can be used in place of “comprising” and “including” to provide for more specific embodiments and are also disclosed. As used in this disclosure and in the appended claims, the singular forms “a”, “an”, “the”, include plural referents unless the context clearly dictates otherwise.

[0098] Throughout the description and claims of this specification, the word “comprise” and other forms of the word, such as “comprising” and “comprises,” means including but not limited to, and are not intended to exclude, for example, other additives, segments, integers, or steps. Furthermore, it is to be understood that the terms comprise, comprising, and comprises as they

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[0101] relate to various aspects, elements, and features of the disclosed compositions and methods also include the more limited aspects of “consisting essentially of’ and “consisting of.”

[0102] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a “polymer” includes aspects having two or more such polymers unless the context clearly indicates otherwise.

[0103] Ranges can be expressed herein as from “about” one particular value and / or to “about” another particular value. When such a range is expressed, another aspect includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It should be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

[0104] As used herein, the terms “optional” or “optionally” mean that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0105] For the terms "for example" and "such as," and grammatical equivalences thereof, the phrase "and without limitation" is understood to follow unless explicitly stated otherwise.

[0106] The term “about” as used herein when referring to a measurable value such as an amount, a percentage, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, or ±1% from the measurable value.

[0107] “Administration” to a subject or “administering” includes any route of introducing or delivering to a subject an agent. Administration can be carried out by any suitable route, including oral, intravenous, intraperitoneal, intranasal, inhalation and the like. Administration includes self-administration and the administration by another.

[0108] The term “biocompatible" generally refers to a material and any metabolites or degradation products thereof that are generally non-toxic to the recipient and do not cause significant adverse effects to the subject.

[0109] The term “cancer” refers to a malignant tumor (Stedman’s Medical Dictionary, 25th ed.; Hensyl ed.; Williams & Wilkins: Philadelphia, 1990). Exemplary cancers include, but are not limited to, acoustic neuroma; adenocarcinoma; adrenal gland cancer; anal cancer; angiosarcoma (e.g., lymphangiosarcoma, lymphangioendothelio sarcoma, hemangiosarcoma); appendix cancer; benign monoclonal gammopathy; biliary cancer (e.g., cholangiocarcinoma); bladder cancer; breast cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast, mammary cancer, medullary carcinoma of the breast); brain cancer (e.g., meningioma, glioblastomas,

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[0112] glioma (e.g., astrocytoma, oligodendroglioma), medulloblastoma); bronchus cancer; carcinoid tumor; cervical cancer (e.g., cervical adenocarcinoma); choriocarcinoma; chordoma; craniopharyngioma; colorectal cancer (e.g., colon cancer, rectal cancer, colorectal adenocarcinoma); connective tissue cancer; epithelial carcinoma; ependymoma; endotheliosarcoma (e.g., Kaposi's sarcoma, multiple idiopathic hemorrhagic sarcoma); endometrial cancer (e.g., uterine cancer, uterine sarcoma); esophageal cancer (e.g., adenocarcinoma of the esophagus, Barrett's adenocarcinoma); Ewing's sarcoma; ocular cancer (e.g., intraocular melanoma, retinoblastoma); familiar hypereosinophilia; gall bladder cancer; gastric cancer (e.g., stomach adenocarcinoma); gastrointestinal stromal tumor (GIST); germ cell cancer; head and neck cancer (e.g., head and neck squamous cell carcinoma, oral cancer (e.g., oral squamous cell carcinoma), throat cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer)); hematopoietic cancers (e.g., leukemia such as acute lymphocytic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acute myelocytic leukemia (AML) (e.g., B-cell AML, T-cell AML), chronic myelocytic leukemia (CML) (e.g., B-cell CML, T-cell CML), and chronic lymphocytic leukemia (CLL) (e.g., B-cell CLL, T-cell CLL)); lymphoma such as Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) and non-Hodgkin lymphoma (NHL) (e.g., B-cell NHL such as diffuse large cell lymphoma (DLCL) (e.g., diffuse large B-cell lymphoma), follicular lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphomas (e.g., mucosa-associated lymphoid tissue (MALT) lymphomas, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma), primary mediastinal B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma (i.e., Waldenstrom's macroglobulinemia), hairy cell leukemia (HCL), immunoblastic large cell lymphoma, precursor B -lymphoblastic lymphoma and primary central nervous system (CNS) lymphoma; and T-cell NHL such as precursor T-lymphoblastic lymphoma / leukemia, peripheral T-cell lymphoma (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (e.g., mycosis fungoides, Sezary syndrome), angioimmunoblastic T-cell lymphoma, extranodal natural killer T-cell lymphoma, enteropathy type T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, and anaplastic large cell lymphoma); a mixture of one or more leukemia / lymphoma as described above; and multiple myeloma (MM)), heavy chain disease (e.g., alpha chain disease, gamma chain disease, mu chain disease); hemangioblastoma; hypopharynx cancer; inflammatory myofibroblastic tumors; immunocytic amyloidosis; kidney cancer (e.g., nephroblastoma a.k.a. Wilms' tumor, renal cell carcinoma); liver cancer (e.g., hepatocellular cancer (HCC), malignant hepatoma); lung cancer 12

[0113] FH13249380.11GTZ-01525

[0114] (e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung); leiomyosarcoma (LMS); mastocytosis (e.g., systemic mastocytosis); muscle cancer; myelodysplastic syndrome (MDS); mesothelioma; myeloproliferative disorder (MPD) (e.g., polycythemia vera (PV), essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM) a.k.a. myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myelocytic leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES)); neuroblastoma; neurofibroma (e.g., neurofibromatosis (NF) type 1 or type 2, schwannomata sis); neuroendocrine cancer (e.g., gastroenteropancreatic neuroendocrinetumor (GEP-NET), carcinoid tumor); osteosarcoma (e.g., bone cancer); ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma); papillary adenocarcinoma; pancreatic cancer (e.g., pancreatic andenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), Islet cell tumors); penile cancer (e.g., Paget's disease of the penis and scrotum); pinealoma; primitive neuroectodermal tumor (PNT); plasma cell neoplasia; paraneoplastic syndromes; intraepithelial neoplasms; prostate cancer (e.g., prostate adenocarcinoma); rectal cancer; rhabdomyosarcoma; salivary gland cancer; skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cell carcinoma (BCC)); small bowel cancer (e.g., appendix cancer); soft tissue sarcoma (e.g., malignant fibrous histiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma); sebaceous gland carcinoma; small intestine cancer; sweat gland carcinoma; synovioma; testicular cancer (e.g., seminoma, testicular embryonal carcinoma); thyroid cancer (e.g., papillary carcinoma of the thyroid, papillary thyroid carcinoma (PTC), medullary thyroid cancer); urethral cancer; vaginal cancer; and vulvar cancer (e.g., Paget's disease of the vulva).

[0115] A “control” is an alternative subject or sample used in an experiment for comparison purposes. A control can be "positive" or "negative."

[0116] As used herein, the term “crosslinkable” in the context of crosslinkable macromolecules or polymers refers to the ability of macromolecules or polymers to form at least one or more covalent bonds (crosslinks) with one another to form a polymeric network (nanohydrogel) under a conditions effective.

[0117] “Effective amount” of an agent refers to a sufficient amount of an agent to provide a desired effect. The amount of agent that is “effective” will vary from subject to subject, depending on many factors such as the age and general condition of the subject, the particular agent or agents, and the like. Thus, it is not always possible to specify a quantified “effective 13

[0118] FH13249380.11GTZ-01525

[0119] amount.” However, an appropriate “effective amount” in any subject case may be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, and unless specifically stated otherwise, an “effective amount” of an agent can also refer to an amount covering therapeutically effective amounts. An “effective amount” of an agent necessary to achieve a therapeutic effect may vary according to factors such as the age, sex, and weight of the subject. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

[0120] The term “increased” or “increase” as used herein generally means an increase by a statically significant amount; for example, “increased” means an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.

[0121] The term “reduced”, “reduce”, “reduction”, or “decrease” as used herein generally means a decrease by a statistically significant amount. However, for avoidance of doubt, “reduced” means a decrease by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (i.e. absent level as compared to a reference sample), or any decrease between 10-100% as compared to a reference level.

[0122] As used herein, the terms “may,” “optionally,” and “may optionally” are used interchangeably and are meant to include cases in which the condition occurs as well as cases in which the condition does not occur. Thus, for example, the statement that a formulation “may include an excipient” is meant to include cases in which the formulation includes an excipient as well as cases in which the formulation does not include an excipient.

[0123] "Pharmaceutically acceptable" component can refer to a component that is not biologically or otherwise undesirable, i.e., the component may be incorporated into a pharmaceutical formulation and administered to a subject as described herein without causing significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the formulation in which it is contained. When used in reference to

[0124] 14

[0125] FH13249380.11GTZ-01525

[0126] administration to a human, the term generally implies the component has met the required standards of toxicological and manufacturing testing or that it is included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration.

[0127] "Pharmaceutically acceptable carrier" (sometimes referred to as a “carrier”) means a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic, and includes a carrier that is acceptable for veterinary and / or human pharmaceutical or therapeutic use. The terms "carrier" or "pharmaceutically acceptable carrier" can include, but are not limited to, phosphate buffered saline solution, water, emulsions (such as an oil / water or water / oil emulsion) and / or various types of wetting agents.

[0128] As used herein, the term “carrier” encompasses any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations. The choice of a carrier for use in a composition will depend upon the intended route of administration for the composition. The preparation of pharmaceutically acceptable carriers and formulations containing these materials is described in, e.g., Remington’s Pharmaceutical Sciences, 21st Edition, ed. University of the Sciences in Philadelphia, Lippincott, Williams & Wilkins, Philadelphia, PA, 2005. Examples of physiologically acceptable carriers include saline, glycerol, DMSO, buffers such as phosphate buffers, citrate buffer, and buffers with other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN™ (ICI, Inc.; Bridgewater, New Jersey), polyethylene glycol (PEG), and PLURONICS™ (BASF; Florham Park, NJ).

[0129] As used herein, the term “subject” or “host” can refer to living organisms such as mammals, including, but not limited to humans, livestock, dogs, cats, and other mammals.

[0130] Administration of the therapeutic agents can be carried out at dosages and for periods of time effective for treatment of a subject. In some embodiments, the subject is a human.

[0131] The term “selectivity” in the context of therapeutic delivery refers to the relative portion of an administered dose that reaches the targeted location. Thus, the term “tumor selectively” or the like refers to the amount of the administered dose of agent localized within a particular tumor microenvironment.

[0132] 15

[0133] FH13249380.11GTZ-01525

[0134] The terms “treat,” “treating,” “treatment,” and grammatical variations thereof as used herein, include partially or completely delaying, alleviating, mitigating or reducing the intensity of one or more attendant symptoms of cancer or condition and / or alleviating, mitigating or impeding one or more symptoms of cancer. Treatments may be applied palliatively or remedially. Treatments are administered to a subject, for example, during early onset (e.g., upon initial signs and symptoms of cancer), or after an established development of cancer.

[0135] “Therapeutic agent” refers to any composition that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition. The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the terms “therapeutic agent” is used, then, or when a particular agent is specifically identified, it is to be understood that the term includes the agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc.

[0136] “Therapeutically effective amount” or “therapeutically effective dose” of a composition (e.g. a composition comprising an agent) refers to an amount that is effective to achieve a desired therapeutic result. Therapeutically effective amounts of a given therapeutic agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated and the age, gender, and weight of the subject. The term can also refer to an amount of a therapeutic agent, or a rate of delivery of a therapeutic agent (e.g., amount over time), effective to facilitate a desired therapeutic effect. The precise desired therapeutic effect will vary according to the condition to be treated, the tolerance of the subject, the agent and / or agent formulation to be administered (e.g., the potency of the therapeutic agent, the concentration of agent in the formulation, and the like), and a variety of other factors that are appreciated by those of ordinary skill in the art. In some instances, a desired biological or medical response is achieved following administration of multiple dosages of the composition to the subject over a period of days, weeks, or years.

[0137] The term “tumor” refers to an abnormal mass of tissue wherein the growth of the mass surpasses and is not coordinated with the growth of a normal tissue. A tumor may be “benign” or “malignant,” depending on the following characteristics: degree of cellular differentiation (including morphology and functionality), rate of growth, local invasion, and metastasis. A “benign tumor” is generally well differentiated, has characteristically slower growth than a 16

[0138] FH13249380.11GTZ-01525

[0139] malignant tumor, and remains localized to the site of origin. In addition, a benign tumor does not have the capacity to infiltrate, invade, or metastasize to distant sites. Exemplary benign tumors include, but are not limited to, lipoma, chondroma, adenomas, acrochordon, senile angiomas, seborrheic keratoses, lentigos, and sebaceous hyperplasias. In some cases, certain “benign” tumors may later give rise to malignant tumor, which may result from additional genetic changes in a subpopulation of the tumor's neoplastic cells, and these tumors are referred to as “pre-malignant tumor.” An exemplary pre-malignant tumor is a teratoma. In contrast, a “malignant tumor” is generally poorly differentiated (anaplasia) and has characteristically rapid growth accompanied by progressive infiltration, invasion, and destruction of the surrounding tissue. Furthermore, a malignant tumor generally has the capacity to metastasize to distant sites. The term “metastasis,” “metastatic,” or “metastasize” refers to the spread or migration of cancerous cells from a primary or original tumor to another organ or tissue and is typically identifiable by the presence of a “secondary tumor” or “secondary cell mass” of the tissue type of the primary or original tumor and not of that of the organ or tissue in which the secondary (metastatic) tumor is located. For example, a prostate cancer that has migrated to bone is said to be metastasized prostate cancer and includes cancerous prostate cancer cells growing in bone tissue. Reference is also made to the term “tumor microenvironment,” which refers to any and all elements of the tumor milieu including elements that create a structural and or functional environment for the malignant process to survive and / or expand and / or spread.

[0140] Self-Agglomerating Nanohydrogels (SANGs)

[0141] In some embodiments, the crosslinkable core / shell polymer comprises a gel-forming polymer. In some embodiments, the gel-forming polymer comprises a biocompatible polymer.

[0142] Various gel-forming polymers are known in the art and include both natural and synthetic materials or combinations and copolymers thereof. In some aspects, the gel-forming polymer comprises a synthetic material. Examples of synthetic gel-forming polymers include, but are not limited to, polyacrylamide, polylactic acid (PEA), poly(lactic-co-glycolic acid (PEGA), polyethylene glycol (PEG), polyethylene glycol-co-propylene glycol (PEO-PPO), and polyacrylates. In some aspects, the gel-forming polymer comprises a natural gel-forming material. Examples of natural gel-forming polymers include but are not limited to polysaccharides, such as starch, alginate, agarose, cellulosic derivatives, such as, for example, but not limited to, hydroxypropyl methyl cellulose (HPMC), carboxymethyl cellulose (CMC) and n-ethyl cellulose, hyaluronic acid, chitosan, xanthan gum, dextran, alginate, and agar.

[0143] 17

[0144] FH13249380.11GTZ-01525

[0145] In some embodiments, the crosslinkable core / shell polymer comprises polyacrylamide. In some aspects, the crosslinkable core / shell polymer comprises a copolymer of N-isopropylmethacrylamide and N,N’-methylenebis(acrylamide). In some embodiments, a shell of the nanohydrogel comprises a cationic polymer ligand (e.g., poly(aminopropyl methacrylate)). In some embodiments, the shell of the nanohydrogel comprises an amine-doped polymer. In some embodiments, the shell of the nanohydrogel comprises aminomethyl propionic acid (AMPA).

[0146] Methods of synthesizing nanohydrogels are generally known in the art. As an example, core / shell nanohydrogels can be synthesized via precipitation polymerization, such as that described by Lyon et al. (2009) [10.1021 / bc800547c; 10.1007 / s00396-007-1805-7], which is incorporated by reference in its entirety. Briefly, precipitation polymerization involves the use of solvents or solvent mixtures in which the monomers to be polymerized are soluble, but not the polymer which is formed. In some aspects, the nanohydrogels are formed using free-radical precipitation in which monomers are dissolved / dispersed in a polar solvent or solvent mixtures before initiating the polymerization using a free radical-forming compound.

[0147] In some aspects, the crosslinkable core / shell polymer is unfunctionalized. Prior published uses of hydrogels invariably included the attachment of molecules (termed “targeting ligands” here) known to bind to cell surface markers characteristic of cancer cells or other target cells. It was shown that SANGs lacking any targeting ligand (designated as “unfunctionalized SANGs”) were able to effectively and selectively distribute to tumor tissue upon intravenous administration and to operate as a therapeutic delivery agent. This is entirely unexpected, as evidenced by the lack of testing of such unfunctionalized SANGs in any reported account of related materials: those skilled in the art would assume that functionalization would be necessary for selective delivery to tumor tissue. Remarkably, unfunctionalized SANGs can thereby function as a drug delivery platform that exhibits comparable performance among a variety of different cancer models, all of which share common properties of tortuous tumor vasculature but do not necessarily share common cell-surface markers. The term “unfunctionalized” generally refers to a material that is not augmented with a functional group to impart a specific property (e.g., tumor targeting) to the nanohydrogel. For example, the polymer shell can be prepared such that it does not have a targeting moiety selective for a specific tumor. In this regard, the resulting nanohydrogel can respond to a variety of different disorders. In some aspects, the nanohydrogel has a hydrodynamic size of from about 75 nm to about 200 nm prior to self-agglomeration.

[0148] 18

[0149] FH13249380.11GTZ-01525

[0150] In some aspects, the population of nanohydrogels is inducible via tortuous flow of an aberrant microvasculature of a tumor microenvironment (TME) to self-agglomerate to form an agglomerated cluster, wherein the agglomerated cluster has an average hydrodynamic size larger than an average hydrodynamic size prior to self-agglomeration.

[0151] In some aspects, self-agglomeration of the population of nanohydrogels is concentrationdependent. In some aspects, a concentration of nanohydrogels causing self-agglomeration is 10 nM or greater (e.g., 20 nM or greater, 30 nM or greater, 40 nM or greater, 50 nM or greater, 60 nM or greater, 70 nM or greater, 80 nM or greater, 90 nM or greater, 100 nM or greater, 110 nM or greater, 120 nM or greater, 130 nM or greater, 140 nM or greater, 150 nM or greater, 156 nM or greater, 200 nM or greater, 250 nM or greater, 500 nM or greater, 1 pM or greater). In some aspects, a concentration of nanohydrogels causing self-agglomeration is from about 10 nM to about 1 pM (e.g., from about 10 nM to about 500 nM, from about 30 nM to about 500 nM, from about 60 nM to about 500 nM, from about 60 nM to about 250 nM, from about 100 nM to about 250 nM, from about 100 nM to about 200 nM, or about 156 nM).

[0152] Linkers

[0153] In certain aspects, in the composition of the present disclosure, the one or more drug molecules or imaging agents are bound to the population of nanohydrogels via a covalent linkage. In some such aspects, the covalent linkage comprises a linker. In some such aspects, the linker comprises a coupler.

[0154] The term “linker” refers to a molecule that links two or more other molecules. A linker can be a stable linker (also referred to as a non-cleavable linker, or non-enzymatically cleavable linker), a cleavable linker (as defined hereafter), or a combination thereof. The linker can be a cleavable linker or it can be a non-cleavable linker. A non-cleavable linker keeps the drug attached to the SANG under normal metabolic conditions. For purposes of the present disclosure, the linker may, e.g., couple, conjugate, join, connect, tether, etc. the SANG to one or more drug molecules or imaging agents.

[0155] In accordance with some aspects of the disclosure, a linker is covalently bonded to the SANG. Examples of such linkers are known in the art and preferably include, for example, cleavable and non-cleavable linkers. Examples of non-cleavable linkers may include linkers that contain polyethylene glycol chains or polyethylene chains that are not acid or base sensitive (such as hydrazone containing linkers), are not sensitive to reducing or oxidizing agents (such as those containing disulfide linkages), and are not sensitive to enzymes that may be found in cells 19

[0156] FH13249380.11GTZ-01525

[0157] or in the circulatory system. See e.g., U.S. Pat. No. 8,470,980 and U.S. Patent Application 20090202536. Examples of linkers include, without limitation, those selected from the structures below. In these embodiments, the linkers are illustrated in the context of SANGs in accordance with the disclosure. The chemical moiety indicated as “SANG” stands for self-agglomerating nanhohydrogel that each linker is bonded to and at that position of the linker. Likewise, the term “drug” stands for the compound that each linker is bonded to and at that position of the linker (e.g., a drag molecule or imaging agent).

[0158]

[0159] Other examples of linkers include, without limitation, those set forth below:

[0160]

[0161] FH13249380.11GTZ-01525

[0162]

[0163] In the above embodiments, the SANG and drug molecule (or imaging agent) are each attached to the linker via various conjugations (e.g., cysteine and lysine). Other suitable conjugations may be used.

[0164] The term “cleavable linker,” “c-linker,” or a grammatical variation thereof, refers to a linker that can be cleaved under proper conditions. As used herein, a cleavable linker can comprise enzymatically-cleavable linkers, acid cleavable linkers that can be cleaved under low pH conditions, acid sensitive hydrazone linker, Glutathione (GSH) cleavable linkers including disulfide linkers, such as N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB) glutathione cleavable linker that can be cleaved by GSH such as in cytoplasm, Fe(II) cleavable linkers that can be cleaved by elevating levels of ferrous iron, Cathepsin cleavable linkers that can be cleaved by cathepsin such as in lysosomes, Glycosidase cleavable linkers that can be cleaved by p- glucuronidase such as in lysosomes, Phosphatase cleavable linkers that can be cleaved by phosphatase and pyrophosphates such as in lysosomes, Sulfatase cleavable linkers that can be cleaved by sulfatase such as in lysosomes, Photo-responsive cleavable linkers that can be cleaved by irradiation with NIR light (such as A = 650-900 nm), Bioorthogonal cleavable linkers that can be cleaved by the bioorthogonal cleavage pair such as Cu(I)-BTTAA / dsProc, a tumor microenvironment activable linker (TMALIN®), an Ortho Hydroxy-Protected Aryl Sulfate (OHPAS) linker, an enzymatically-cleavable peptide linker, a dipeptide linker, a valine — citrulline linker (VC linker), a valine-alanine linker, a VAPAB linker (Valine-alanine-p-aminobenzyl), L-Val-L-Ala-para-aminobenzyl alcohol (VAP AB-OH) linker (“VAPAB linker” and (VAP AB-OH) linker are herein collectively referred to as “VAPAB linker”), a VCPAB (Valine-citrulline-p-aminobenzyl) linker, L-Val-L-Cit-para-aminobenzyl alcohol

[0165] 21

[0166] FH13249380.11GTZ-01525

[0167] (VCPAB-OH) linker (VCPAB linker and VCPAB-OH are collectively referred to as “VCPAB linker”), a polypeptide linker, Gly-Gly- L-Phe-N-[(carboxymethoxy)methyl]Glycinamid (GGFG-NCM) linker, a GGFG (Glycyl glycyl-L-phenylalanyl glycyl) linker, a glutamic acid-glycine-citrulline (EGCit, or Glu-Gly- Cit) linkers, a tripeptide linker, a glutamic acid — valine — citrulline (Glu-Val-Cit) linker, p- aminobenzyloxycarbonyl (PABC) linker, p-aminobenzyloxycarbonyl (PABC) linker, or a combination thereof. In some cases, a cleavable linker comprises an enzymatically- cleavable peptide linker, such as Gly-Gly-L-Phe-N-[(carboxymethoxy)methyl]Glycinamid (GGFG-NCM) linker, L-Val-L-Ala-para-aminobenzyl alcohol (VAP AB-OH) linker (herein referred to as “VAPAB linker”), L-Val-L-Cit-para-aminobenzyl alcohol (VCPAB-OH) linker, glutamic acid-glycine-citrulline (EGCit) linkers, acid sensitive hydrazone linker, a linker comprising disulfides, a thioether linker, a glutathionesensitive disulfide linker, a linker containing a sugar molecule or moiety, a glucuronide containing linker, a glycosidase cleavable linker, a phosphatase cleavable linker, an esterase cleavable linker, a hydrolysis cleavable linker, a modified monodispersed PEG linker, a modified PEO linker, a modified poly-PEG linker, a dipeptide linker, a tripeptide linker, a valine — citrulline linker, a glutamic acid — valine — citrulline (Glu-Val-Cit) linker, a valinealanine linker, a VAPAB linker (Valine-alanine-p-aminobenzyl), a VCPAB (Valine-citrulline-p-aminobenzyl) linker, a glutamic acid-glycine-citrulline (EGCit, or Glu-Gly -Cit) linker, a polypeptide linker, a GGFG (Glycyl glycyl-L-phenylalanyl glycyl) linker, p-aminobenzyloxycarbonyl (PABC) linker, or a combination thereof. Various linkers comprising glycine-glycine- phenylalanine-glycine, such as Gly-Gly-L-Phe-N-[(carboxymethoxy)methyl]Glycinamid (GGFG-NCM) linker or other forms glycine-glycine-phenylalanine-glycine (GGFG) linkers, are collectively referred to as a “GGFG linker”. Various linkers comprising valine and alanine, such as the aforementioned L-Val-L-Ala-para-aminobenzyl alcohol (VAP AB-OH) linker or other forms of valine and alanine linkers, are collectively referred to as a “VAPAB linker” or “VAPAB”. In some cases, a PEOX polymer comprising disulfide bonds can also be suitable as a cleavable linker. The terms “modified monodispersed PEG linker,” “modified PEO linker,” or “modified poly-PEG linker” refer to a corresponding polymer linker that has been modified to comprise one or more cleavable bonds, such as one or more -S-S- bonds or other cleavable bonds disclosed herein. The terms “linker,” “cleavable linker,” “GGFG Linker,” “GGFG,” “VAPAB linker,” or “VAPAB” can also be used to describe a corresponding linker in a reacted form, i.e. , a linker residue that is reacted and incorporated into a SANG, in text or in a drawing. The term “polymer,” “PEG,” “PEOX,” or a 22

[0168] FH13249380.11GTZ-01525

[0169] variation thereof, can also be used to describe a corresponding polymer in a reacted form, i.e., a polymer that is reacted and incorporated into a SANG, in text or in a drawing.

[0170] In some embodiments, the coupler can be selected from succinimidyl-4-(N-maleimidomethyl)cyclohexane-l -carboxylate (SMCC), maleimide (Mai), maleimidocaproyl (MC), maleimidomethyl cyclohexane- 1 -carboxylate (MCC), dibromomaleimide (DBM), glycan-based coupler, N-acetylglucosamine (GlcNAc)-based coupler, and a combination thereof.

[0171] In some embodiments, the linker can comprise a cleavable linker that can comprise an enzymatically-cleavable linker, acid cleavable linkers, acid sensitive hydrazone linker, a glutathione (GSH) cleavable linker, a linker comprising disulfides, a SPDB glutathione cleavable linker, a thioether linker, a glutathione-sensitive disulfide linker, a Fe(ll) cleavable linker, a Cathepsin cleavable linker, a linker containing a sugar molecule or moiety, a glucuronide containing linker (such as a P-Glucuronide Linker), a glycosidase cleavable linker, a phosphatase cleavable linker, a sulfatase cleavable linker, an esterase cleavable linker, a hydrolysis cleavable linker, a Photo-responsive cleavable linker, a Bioorthogonal cleavable linker, a tumor microenvironment activable linker (TMALIN®), an Ortho Hydroxy-Protected Aryl Sulfate (OHPAS) linker, a modified monodispersed PEG linker, a modified PEG linker, a modified poly-PEG linker, an enzymatically-cleavable peptide linker, a dipeptide linker, a valine — citrulline linker (VC linker), a valine-alanine linker, a VAPAB linker (Valine-alanine-p-aminobenzyl), L-Val-L-Ala-para-aminobenzyl alcohol (VAP AB-OH) linker, a VCPAB (Valine-citrulline-p-aminobenzyl) linker, L-Val-L- Cit-para-aminobenzyl alcohol (VCP AB-OH) linker, a polypeptide linker, Gly-Gly-L-Phe-N- [(carboxymethoxy)methyl]Glycinamid (GGFG-NCM) linker, a GGFG (Glycyl glycyl-L- phenylalanyl glycyl) linker, a glutamic acid-glycine-citrulline (EGCit, or Glu-Gly-Cit) linkers, a tripeptide linker, a glutamic acid — valine — citrulline (Glu-Val-Cit) linker, p- aminobenzyloxycarbonyl (PABC) linker, or a combination thereof.

[0172] In some embodiments, the linker can comprise a cleavable linker selected from the Formula ID 101-141 , or a combination thereof:

[0173] 23

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[0190]

[0191] In some embodiments, the linker comprises cysteine-based coupler, succinimidyl-4- (N-maleimidomethyl)cyclohexane-l -carboxylate (SMCC), maleimide (Mai), dibromomaleimide (DBM), maleimidocaproyl (MC), maleimidomethyl cyclohexane- 1- carboxylate (MCC), Mal-PEG, a CL2A linker that is a cleavable complicated PEG8- and triazole-containing PABC-peptide-MC linker that is cleavable through pH sensitivity, giving rise to bystander effect, and binds the SANG at a cysteine residue via a disulfide bond, glycan-based coupler, N-acetylglucosamine (GlcNAc)-based coupler, or a combination thereof.

[0192] In some embodiments, the linker is selected from Formula ID 201-209, or a combination thereof:

[0193]

[0194] Targeted Delivery with Self- Agglomerating Nanohydrogels (SANGs)

[0195] Cancer is a systemic disease, with more than 90% of deaths caused by metastases. As a result, it requires systemic therapy (Mehlen, P. & Puisieux, A. Nat. Rev. Cancer 6, 449-458 (2006)). Developing an efficient and safe platform to deliver therapeutic agents to tumors is crucial for cancer treatment, but formidable barriers, such as the navigation of vascular, transvascular, and interstitial compartments (Boucher, Y. & Jain, R. K. Cancer Res. 52, 5110-5114. PMID: 1516068 (1992); Gazit, Y„ Berk, D. A., Leunig, M„ Baxter, L. T. & Jain, R. K. Phys. Rev. Lett. 75, 2428 (1995); Griffon-Etienne, G., Boucher, Y., Brekken, C., Suit, H.

[0196] 26

[0197] FH13249380.11GTZ-01525

[0198] D. & Jain, R. K. Cancer Res. 59, 3776-3782. PMID: 10446995 (1999); Helmlinger, G., Netti, P. A., Lichtenbeld, H. C., Melder, R. J. & Jain, R. K. Nat. Biotechnol. 15, 778-783 (1997); Leunig, M. et al. Cancer Res. 52, 6553-6560. PMID: 1384965 (1992); Netti, P. A., Baxter, L. T., Boucher, Y., Skalak, R. & Jain, R. K. Cancer Res. 55, 5451-5458. PMID: 7585615 (1995); Padera, T. P. et al. Nature 427, 695 (2004); Sevick, E. M. & Jain, R. K. Cancer Res. 49, 3506-3512. PMID: 2731172 (1989); Yuan, F. et al. Cancer Res. 54, 4564-4568. PMID: 8062241 (1994)), stand in the way of this goal. Any antitumor drug or delivery vehicle must overcome abnormal characteristics of the tumor microenvironment (TME), target the cell type(s) of interest, and release sufficient therapeutic payloads, all while minimizing toxicity. The possibility of engineering nanostructures to overcome these barriers and selectively destroy cancer cells in vivo has been an area of intense efforts. However, results have been muted and clinical translation of nanostructures has been limited.

[0199] Substantial efforts to develop tumor-targeting nanostructures have been dominated by cationic polymers and lipid nanoparticles (LNPs) (Urban-Klein, B., Werth, S., Abuharbeid, S., Czubayko, F. & Aigner, A. Gene Ther. 12, 461-466 (2005); Wittrup, A. et al. Nat.

[0200] Biotechnol. 33, 870-876 (2015); Sahay, G. et al. Nat. Biotechnol. 31, 653-658 (2013);

[0201] Rietwyk, S. & Peer, D. ACS Nano 11, 7572-7586 (2017); Semple, S. C. et al. Nat.

[0202] Biotechnol. 28, 172-176 (2010)). While these strategies have improved delivery, resulting in several formulations in clinical applications, liposomal trafficking is poorly understood, and these particles have drawbacks (Lokugamage, M. P. et al. Adv. Mater. 32, 1904905 (2020); Paunovska, K. et al. ACS Nano 12, 8341-8349 (2018); Paunovska, K., Loughrey, D. & Dahlman, J. E. Nature Reviews Genetics 23, 265-280 (2022); Paunovska, K. et al. Nano Lett.

[0203] 18, 2148-2157 (2018)). Most obviously, while some success has been observed in diverting LNPs away from liver (Nakamura, T. et al. Mol. Pharm 17, 944-953 (2020); Sago, C. D. et al. J. Am. Chem. Soc. 140, 17095-17105 (2018); Dahlman, J. E. et al. Nat. Nanotechnol. 9, 648-655 (2014), the majority of most injected LNP formulations are trapped in the liver and are not taken up by cancerous cells after systemic injection, even after functionalization (Zuckerman, J. E. & Davis, M. E. Nat. Rev. Drug Discov. 14, 843-856 (2015); Hattab, D., Gazzali, A. M. & Bakhtiar, A. Pharmaceutics 13, 1009 (2021); Gustafson, H. H., Holt-Casper, D., Grainger, D. W. & Ghandehari, H. Nano Today 10, 487-510 (2015). As a result, indications for LNP formulations have been largely limited to hepatic cancers or those receptive to intra-tumoral injection. This leaves the majority of cancers, especially metastatic lesions, untreated because

[0204] 27

[0205] FH13249380.11GTZ-01525

[0206] they require systemic rather than local administration (Zuckerman, J. E. & Davis, M. E. Nat. Rev. Drug Discov. 14, 843-856 (2015)).

[0207] Cationic polymers have a different set of challenges: they bind nonspecifically to many cells types (Yeung, T. et al. Science 319, 210-213 (2008)) and interact with serum components, resulting in short circulation times and toxic side effects. Surface shielding by PEGylation can improve stability, reduce nonspecific binding and clearance, but it also impairs tumor entrainment and cellular uptake by target cells (Fang, Y. et al. Drug Deliv. 24, 22-32 (2017); Miteva, M. et al. Biomaterials 38, 97-107 (2015)). Targeting specificity can be improved through functionalization with ligand conjugation which facilitates ligand-receptor interactions on target cells. This approach introduces increased manufacturing complexity and often a reduction in stability; the last was the principal reason for a recent clinical trial failure (Zuckerman, J. E. & Davis, M. E. Nat. Rev. Drug Discov. 14, 843-856 (2015); Hattab, D., Gazzali, A. M. & Bakhtiar, A. Pharmaceutics 13, 1009 (2021)). Ligand-based targeting requires a priori knowledge of the cell type of interest and necessitates vehicle redesign for each new target. Moreover, cancer cells respond to treatment-based and immunological selection pressure by undergoing rapid clonal evolution, often changing the nature or expression level of target receptor(s), thereby challenging peptide / antibody targeting strategies and contributing to drug resistance (Vasan, N., Baselga, J. & Hyman, D. M. Nature 575, 299-309 (2019)).

[0208] Like LNPs, polymeric nanoparticles face challenges with initial trafficking to tumors even before cell targeting can take place. Altered vasculature in solid tumors has long been suggested to afford preferential drug delivery by the enhanced permeability and retention (EPR) effect, but prior successes have been limited for passively and actively targeted nanoparticles. Tumor / liver delivery ratios provide a measure of efficiency of tumor entrainment that can be compared across different methods and platforms. While not all studies describe organ level biodistribution data to compute such ratios (Dahlman, J. E. et al. Nat. Nanotechnol. 9, 648-655 (2014); Wang, Q. et al. Nature Nanotechnology, 19, 95-105 (2024)) , reported values range from 0.5-1.5 (Ren, Y. et al. Sci. Transl. Med. 4, 147rall2-147rall2 (2012); Benezra, M. et al. J. Clin. Invest. 121, 2768-2780 (2011); Chen, Y. et al. Nat. Nanotechnol. 18, 193-204 (2023); Wen, Y. et al. Sci. Adv. 6, eabc2148 (2020); Gu, L„ Deng, Z. J., Roy, S. & Hammond, P. T. Clin. Cancer Res. 23, 7312-7323 (2017); Xue, W. et al. Proc. Natl. Acad. Sci. USA 111, E3553-E3561 (2014)), showing that most platforms fail to demonstrate preferential delivery or result in only nominally higher delivery to tumors.

[0209] 28

[0210] FH13249380.11GTZ-01525

[0211] Quantitative biodistribution studies reveal the stark reality of the above-mentioned limitations. A recent meta-analysis of nanoparticles demonstrate median tumor accumulation of less than 0.7% of the injected dose whereas up to 90% is rapidly sequestered in the liver and spleen. Two additional challenges face nanostructures: penetration and retention. First, while tumor entrainment is necessary, penetrating the mechanical barriers to reach cancerous cells is also required and is challenging and often requires additional interventions, e.g. local hyperthermic treatment for PLGA (Wang, Q. et al. Nature Nanotechnology, 19, 95-105 (2024)), a complication that diminishes clinical application. Second, delivery typically occurs over short timescales, peaking at 24 hours before rapidly declining (Chen, Y. et al. Nat. Nanotechnol. 18, 193-204 (2023); Wen, Y. et al. Sci. Adv. 6, eabc2148 (2020)). Short retention windows may lead to suboptimal drug delivery and may necessitate repeated or exaggerated doses.

[0212] Two additional challenges face nanostructures: penetration and retention. First, while tumor entrainment is necessary, penetrating the mechanical barriers to reach cancerous cells is also required and is challenging. A recent example showed that extravasation of PLGA nanoparticles through the vascular endothelial and basement membrane requires local hyperthermic treatment (Wang, Q. et al. Nature Nanotechnology, 19, 95-105 (2024)), a complication that diminishes clinical application. Second, delivery typically occurs over short timescales, peaking at 24 hours before rapidly declining (Ren, Y. et al. Targeted tumorpenetrating siRNA nanocomplexes for credentialing the ovarian cancer oncogene ID4. Sci. Transl. Med. 4, 147rall2-147rall2 (2012); Benezra, M. et al. J. Clin. Invest. 121, 2768-2780 (2011); Chen, Y. et al. Nat. Nanotechnol. 18, 193-204 (2023); Wen, Y. et al. Sci. Adv. 6, eabc2148 (2020); Gu, L., Deng, Z. J., Roy, S. & Hammond, P. T. Clin. Cancer Res. 23, 7312-7323 (2017). Short retention windows may lead to suboptimal drug delivery and may necessitate repeated or exaggerated doses.

[0213] All told, there is an urgent need to improve cancer drug delivery to improve the outcomes for the nearly 20 million individuals diagnosed in 2020 and reduce global economic impact of cancer that is projected to reach $25.2 trillion between 2020 and 2050.

[0214] To meet these challenges, a new nanostructure (‘SANGs’) was developed that achieves preferential tumor delivery, penetration, and retention in primary (Fig. 1) and advanced metastatic OC (Fig. 2) after i.v. injection. Extensive preliminary data indicate that SANGs successfully deliver non-covalently incorporated oligonucleotides, which knock down oncogene expression in drug-resistant tumors. SANGs also accumulate in other cancers (colorectal, breast, lung, pancreas: Fig. 3) in multiple species and in both sexes. Altogether, SANGs demonstrate 29

[0215] FH13249380.11GTZ-01525

[0216] favorable biodistribution to both primary tumors and metastasis, high retention rates, while being nearly absent from noncancerous organs. SANGs are also safe and simple to manufacture and work without additional interventions (Wang, Q. et al. Nature Nanotechnology, 19, 95-105 (2024); Housley, Stephen N., et al. Nature Communications VIA (2026): 184).

[0217] In certain embodiments, this disclosure involves a nanostructure platform that does not depend on direct functionalization for tumor targeting. By directly conjugating or through the use of dynamic drug linkers, drug cargos are released in a controlled manner by these environmentally responsive, self-agglomerating nanohydrogels (SANGs). In certain embodiments, the SANG-drug platform involves the successful conjugation of chemically distinct drug classes. Collectively, these data support SANGs potential as a drug- and tumoragnostic delivery platform.

[0218] As described herein, a library of chemically unique SANG-drug derivatives has been successfully developed. Without wishing to be bound by any particular theory, by directly conjugating or through the use linkers, controlled release of drug cargos by these environmentally responsive, self-agglomerating nanohydrogels (SANGs) is shown. In certain embodiments, the disclosure relates to the development and validation of chemically distinct SANG-drug particles with drug molecules for the treatment of solid tumors and metastases in a wide variety of cancers. In certain embodiments, the disclosure relates to:

[0219] a) The attachment of drug molecules to SANGs polymers by non-cleavable covalent linkage(s), and the use of such materials as anticancer agents.

[0220] b) The attachment of drug molecules to SANGs polymers by cleavable covalent linkage(s), wherein the drag is released from the polymer before or after cellular uptake, and the use of such materials as anticancer agents.

[0221] c) The noncovalent interaction or entrainment of drag molecules with SANGs polymers, and the use of such materials as anticancer agents.

[0222] d) In the embodiments above, the disclosure relies on the cancer-targeting properties of SANGs materials.

[0223] e) The disclosure applies to the use of both small and biologic drag molecules. f) The disclosure applies to all mechanisms of action of the drag molecules used, including cytotoxic agents (by any mechanism), immunomodulatory agents, complementactivating agents, receptor-blocking agents, etc.

[0224] g) The disclosure applies to compositional variations in SANGs polymers, covering the use of different monomers.

[0225] 30

[0226] FH13249380.11GTZ-01525

[0227] EXEMPLIFICATION

[0228] I. Methods

[0229] Example 1: Mertansine (DM1) Bioconjugation Reactions

[0230] The tubulin-inhibiting drug mertansine (DM1) is currently used as a chemotherapeutic agent in many different antibody-drug conjugates, including Trastuzumab emtansine or Kadcyla for HER2+ breast cancer, Lorvotuzumab mertansine for CD56+ small cell lung cancer, ovarian cancer, and others, and Bivatuzumab mertansine for CD44+ squamous cell carcinomas.

[0231] Mertansine (DM1) was conjugated to SANG nanoparticles via both releasable and non-releasable linkers. Oxanorbomadiene (OND) linkers modified with different functional groups provide different release rates, and can be added to the nanoparticles via amine-reactive n-hydroxysuccinimide (NHS) ester chemistry to allow for programmable release of small molecule drug cargo. A 9-hour half-life releasing OND-NHS linker and a non-releasing epoxide OND-NHS linker were used (structures shown in Figs. 4 and 5), as well as a non-releasing maleimide-PEG2-NHS linker (Sigma 746223, structure shown in Fig. 6). The NHS-OND or NHS-maleimide linkers were conjugated to the free amines on the surface of the SANGs via NHS-ester reaction. For each linker, a lOmM solution was made by weighing out the amount of linker and dissolving it in the respective volume of DMSO. To ImL of Img / mL SANGs resuspended in 0.1M potassium phosphate buffer at a pH of 7.8 (which contains approximately 0.025umol free amines), multiple reactions were ran with OND linkers added in varying amounts, ranging from a 4x molar excess of NHS-linker to free amines on the SANGs (lOuL of lOmM or O.lumol OND-NHS or maleimide-NHS), 16x excess (42uL of lOmM or 0.42umol OND-NHS or maleimide-NHS), 28x excess (70uL of lOmM or 0.7umol OND-NHS or maleimide-NHS), and 80x excess (200uL of lOmM or 2umol OND-NHS or maleimide-NHS). These were reacted overnight at 4°C. The next day, the reactions were purified by 7K MWCO ZebaTM Desalting Spin Columns following the manufacture’s protocol.

[0232] Next, the thiol-containing DM1 drag cargo was reacted to the linkers via thiol-ene reaction. The drag was added in a 1:1 molar equivalent DM1 linker (so it followed the same 4x, 16x, 28x, and 80x excess of drag to free amines on SANGs). A lOmM solution of DM1 was made by weighing out the amount of drag and dissolving it in the respective volume of DMSO. So, to the cleaned up SANG-linker reactions from above, lOuL of lOmM or O.lumol DM1 was added to the 4x reaction, 42uL of lOmM or 0.42umol DM1 was added to the 16x reaction, 70uL of lOmM or 0.7umol DM1 was added to the 28x reaction, and 200uL of lOmM or 2umol DM1

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[0234] FH13249380.11GTZ-01525

[0235] was added to the 8 Ox reaction. This was reacted for 15 minutes at room temperature before being purified by 7K MWCO ZebaTM Desalting Spin Columns following the manufacture’s protocol.

[0236] The drug cargo then releases from the 9-hour OND linker via retro-Diels-Alder (rDA) reaction into the products shown below, and the drug cargo should remain attached to the particles with the epoxide OND linker and NHS-maleimide linker. All the structures for all the reactions are shown in Figs. 4-6.

[0237] Example 2: Quantification and Characterization of Mertansine (DM1)

[0238] Due to the inability to get monodisperse, clean readings of polymer nanoparticles by most mass-based characterization methods, a competition fluorescence assay was performed to quantify the amount of drag loading on SANGs. A 5-FAM cysteamine (AAT Bioquest, 5210) pictured below was purchased to have a thiol-containing fluorophore to compete in the OND-DM1 or maleimide-DMl thiol-ene reactions. The structure of 5-FAM Cysteamine is shown below.

[0239]

[0240] After the reaction and purification of NHS-OND or NHS-maleimide to the SANGs (Step #1 in Fig. 7), an aliquot of these reactions was reacted with an excess of the 5-FAM cysteamine to quantify how many ONDs or maleimides there were available for a thiol to react to (Step #2 in Fig. 7). To 300uE of the SANG-NHS-OND or SANG-NHS-maleimide reactions, 12uE of lOmM 5-FAM cysteamine was added and reacted for 30 minutes before purification by 7K MWCO ZebaTM Desalting Spin Columns following the manufacture’s protocol. Then, the rest of the SANG-NHS-OND or SANG-NHS-maleimide reactions were reacted with the thiol-containing drag DM1 (Step #3 in Fig. 7). Following purification by 7K MWCO ZebaTM Desalting Spin Columns of this reaction, an aliquot was reacted with an excess of the 5-FAM cysteamine (Step #4 in Fig. 7). To 300uE of the SANG-DM1 reactions, 12uE of lOmM 5-FAM cysteamine was added and reacted for 30 minutes before purification by 7K MWCO ZebaTM Desalting Spin Columns following the manufacture’s protocol. All of these steps of reactions were read on a fluorescence plate reader at excitation / emission of 493 / 517 nm. The amount of

[0241] FH13249380.11GTZ-01525

[0242] DM1 that successfully reacted to the particles was then able to be quantified by the difference or reduction in the fluorescence signal from Step #2 to Step #4.

[0243] A standard curve was made for this assay by starting with a solution of 2.5mM (or 1.1 mg / mL) 5-FAM cysteamine and cutting it in half 24 times to range down to a concentration of 3.0e-7mM (or 1.3e-7mg / mL) 5-FAM cysteamine.

[0244] Example 3: In Vitro Toxicity of SANG-Mertansine (DM1)

[0245] One day before treatment, B16F10 cells and 4T1 cells were plated at 10,000 cells per well in lOOuL per well in 96 well plates (using DMEM or RPMI media with 10% FBS respectively). The following day, cells were treated with 50uL of the respective conditions and concentrations. Two days after treatment, the PrestoBlue™ Cell Viability Assay was used to measure toxicity according to the manufacturer’s protocol. Briefly, 15uL of PrestoBlue™ Cell Viability Reagent was added to each well, the plates were placed protected from light in the cell incubator for 3 hours, and then the plates were read on a fluorescence plate reader at excitation / emission 560 / 590 nm.

[0246] Example 4: In Vivo Toxicity of SANG-NHS-mal-Mertansine (DM1) in Breast Tumor Model Six days before treatment, Balb / c mice were injected with 350k 4T1 cells in 50uL saline into the right mammary fat pad. After six days of tumor growth, the mice were randomized and injected intravenously in the jugular vein with lOOuL of non-releasing SANG-NHS-mal-DMl treatment (scheme in Fig. 8) at doses varying from 25mg / mL down to 0.5mg / mL of SANGs, including controls of saline and free DM1 at the maximum dose. Every 2 days, the tumor sizes were measured with calipers and the mice were weighed. Mice were euthanized according to IACUC protocols or when tumor volume exceeded 1000mm3, and the overall survival of the mice was monitored. The in vivo protocol scheme is pictured in Fig. 8.

[0247] Example 5: Oxoplatin Bioconjugation Reactions

[0248] Cisplatin is a platinum-based DNA-interacting chemotherapeutic agent currently used as an intravenous chemotherapy for a wide variety of cancers including lymphomas, breast cancer, testicular cancer, ovarian cancer, head and neck cancer, cervical cancer, and sarcomas. A derivative of cisplatin can be made into a platinum(IV) prodrug called oxoplatin that becomes very active under acidic conditions.

[0249] 33

[0250] FH13249380.11GTZ-01525

[0251] The oxoplatin platinum(IV) prodrug was made as previously described (Fig. 9, panel a). Briefly, lOOmg cisplatin was mixed with 170uL 30% hydrogen peroxide and 1.34mL of DI water, and placed in a glass vial with a magnetic stir bar at 70°C for 5 hours in the dark. After 5 hours, this reaction was moved off of heat and let cool down while being stirred overnight. The reaction was placed under vacuum and then placed at 4°C for 2 hours so that the product precipitated. The product was collected by vacuum filtration, washing 3 times with cold DI water and 3 times with ethanol. Then, this product was mixed with 60mg succinic anhydride and 200uL DMF in a glass vial with a magnetic stir bar at 70°C for 24 hours in the dark. This final oxoplatin product was recrystallized from acetone at -20°C and collected via vacuum filtration.

[0252] Oxoplatin was conjugated to SANG particles via l-Ethyl-3-(3-dimethylaminopropyljcarbodiimide (EDC) coupling reaction of the carboxylic acid on the oxoplatin to the free amines on the surface of SANGs (Fig. 9, panel b). A solution of oxoplatin was made by dissolving the weighed out material in DMSO to a concentration of 50mM, and a solution of 50mM EDC was made by dissolving the EDC in water. To 980uL of Img / mL SANGs, lOuL of 50mM EDC and lOuL of 50mM oxoplatin were added and reacted for 3 hours at room temperature before purification by 7K MWCO Zeba™ Desalting Spin Columns following the manufacture’s protocol.

[0253] Example 6: Quantification and Characterization of Oxoplatin

[0254] Due to the single platinum ion contained in each oxoplatin molecule, inductively coupled plasma mass spectrometry (ICP-MS) was used to quantify amount of oxoplatin in a solution or sample by quantifying the number of platinum ions. A standard curve was made using platinum standard solutions ranging from concentrations of 10 parts per million down to IO’7parts per million of platinum. For the same reason and in a similar way, atomic absorption spectrophotometry can also be used to quantify the amount of oxoplatin.

[0255] II. Results

[0256] Example 7: Quantification and Characterization of Mertansine (DM1)

[0257] Using the fluorescence assay described above, the amount of mertansine (DM1) drug that loaded onto SANGs with the respective linkers was quantified. Ranging from 4x excess of NHS-OND or NHS-maleimide to 80x excess, the difference in fluorescence from the SANG-linker reacted with the 5-FAM cysteamine (labeled “4x fluor”) to the fluorescence from the SANG-linker-DMl reacted with the 5-FAM cysteamine (labeled “4x DM1”) was measured.

[0258] 34

[0259] FH13249380.11GTZ-01525

[0260] Using the standard curve of 5-FAM cysteamine, this could be converted to an approximate mass of drug that was loaded onto the SANGs. Since the mass of SANGs in each well was known and the mass of drags loaded was calculated by the assay, the number of drags per particle could also be calculated. As seen in Fig. 10, the amount of drag loading increased, almost tripling, from 4x to 16x excess of linker to free amines on SANGs. However, from 16x excess to 80x excess of linker to free amines on SANGs there was no significant increase in drag loading and the number of drags per particle plateaued, indicating the maximum drag loading was achieved at about 15 DMl / particle for the 9hrOND and at about 30 DMl / particle for the non-releasing NHS-maleimide. This indicates that by the 16x excess reaction conditions, linkers added to all possible reactive amines on SANGs and then DM1 added to all available linkers. This was true and was recapitulated for both releasing (NHS-9hrOND) and non-releasing (NHS-maleimide and NHS-epoxideOND) linkers. All conditions were ran in triplicate.

[0261] Controls were ran for this assay as well to make sure there was no background signal. These controls included just SANG particles in buffer, SANG particles mixed with the 5-FAM cysteamine and cleaned up in the same way as all the other reactions, and SANG particles mixed with DM1 and cleaned up in the same way as all the other reactions, and these all produced almost zero fluorescence signal.

[0262] Example 8: In Vitro Toxicity of SANG-Mertansine (DM1) in B16F10 and 4T1 Cell Culture To test the cytotoxicity of DM1 drag loaded SANGs in vitro, murine melanoma cells (B16F10) and murine breast cancer cells (4T1) were dosed with a variety of conditions and controls in concentrations ranging from ~3uM down to ~0.5pM in PBS. The non-releasing condition explained above of SANG-NHS-maleimide-DMl was tested. For this condition, a control using beta-mercaptoethanol instead of DM1 was made, as the beta-mercaptoethanol was a sacrificial thiol (not very toxic at these low concentrations) to react with the NHS-maleimide linker, to test the cytotoxicity of the SANG-linker condition. Unmodified SANGs, free DM1 drag, and addition of only saline were also controls that were tested for cytotoxicity.

[0263] The results in Fig. 11 indicate that DM1 attached to SANG particles via the nonreleasing NHS-maleimide linker is approximately as toxic as free DM1 itself, and that SANG particles alone (or SANG particles functionalized with maleimide linker and the non-toxic molecule b-mercaptoethanol) show little to no toxicity. This is encouraging data to support the cytotoxicity of the DM1 loaded SANGs to move in vivo.

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[0265] FH13249380.11GTZ-01525

[0266] Example 9: In Vivo Toxicity of SANG-NHS-mal-Mertansine (DM1) in 4T1 Breast Tumor Model

[0267] The effect of non-releasing DM1 loaded SANGs (SANG-NHS-mal-DMl) on tumors in vivo was tested in a 4T1 triple negative breast tumor model in Balb / c mice. SANGs were loaded with approximately 23 DM1 drugs per particle via the non-releasing NHS-maleimide linker, and this was quantified by the fluorescent assay. They were then intravenously injected into 4T1 tumor bearing mice 6 days after tumor cell injection. The different conditions received different doses of treatment, from a maximum concentration of SANGs of 25mg / mL, decreasing doses of 12.5mg / mL, 5mg / mL, 2mg / mL, 0.5mg / mL, and a control of no treatment just saline. The calculated maximum dose of DM1 that was delivered in the 25mg / mL condition with 23 DM1 per particle was also injected intravenously as a free drug control.

[0268] The results in Figs. 12a, 12b, and 13 show significant tumor volume decrease with the maximum 25mg / mL and 12.5mg / mL SANG-NHS-mal-DMl doses compared to the control saline and free drug mice. They also show a significant increase in survival with the single dose of either 25mg / mL and 12.5mg / mL SANG-NHS-mal-DMl. The free DM1 dose did not provide any tumor protection or survival benefit, showing that the systemic dose of DM1 is not enough to treat the tumor but that same dose of DM1 on SANGs did provide tumor protection due to the targeted drag delivery.

[0269] INCORPORATION BY REFERENCE

[0270] All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0271] EQUIVALENTS

[0272] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the compounds and methods of use thereof described herein. Such equivalents are considered to be within the scope of this disclosure and are covered by the following claims. Those skilled in the art will also recognize that all combinations of embodiments described herein are within the scope of the disclosure.

[0273] 36

[0274] FH13249380.11

Claims

GTZ-01525CLAIMS1. A composition, comprising:a population of nanohydrogels; andone or more drug molecules or imaging agents,wherein each of the population of nanohydrogels comprises a cross-linkable core / shell polymer;wherein each of the one or more drug molecules or imaging agents are bound to the population of nanohydrogels via one or more covalent linkages;wherein, optionally, the nanohydrogel is configured to self-agglomerate in aberrant vasculature environments;wherein, optionally, the population of nanohydrogels is inducible via tortuous flow of an aberrant microvasculature of a tumor microenvironment (TME) to self-aggregate to form an agglomerated cluster; andwherein the agglomerated cluster has an average hydrodynamic size larger than an average hydrodynamic size prior to self-agglomeration.

2. The composition of claim 1, wherein the composition comprises one or more imaging agents.

3. The composition of claim 1 or 2, wherein each of the one or more imaging agents is bound to a core of the nanohydrogel.

4. The composition of any one of claims 1-3, wherein the composition releases the one or more imaging agents in a controlled manner.

5. The composition of claim 1, wherein the composition comprises one or more drug molecules.

6. The composition of claim 1 or 5, wherein each of the one or more drug molecules is bound to a core of the nanohydrogel.

7. The composition of any one of claims 1-6, wherein each of the cross-linkable core / shell polymer comprises a gel-forming polymer.37FH13249380.11GTZ-015258. The composition of claim 7, wherein each of the gel-forming polymer comprises a biocompatible polymer.

9. The composition of any one of claims 1-8, wherein each of the population of nanohydrogels comprises a polymer selected from polyethylene glycol, polyacrylamide, and combinations thereof.

10. The composition of any one of claims 1-9, wherein each of the cross-linkable core / shell polymers comprises polyacrylamide.

11. The composition of any one of claims 1-10, wherein each of the cross-linkable core / shell polymers comprises a copolymer of N-isopropylmethacrylamide and N,N'-methylenebis (acrylamide) .

12. The composition of any one of claims 1-11, wherein a shell of the nanohydrogel comprises a cationic polymer ligand (e.g., poly(aminopropyl methacrylate)).

13. The composition of any one of claims 1-12, wherein the nanohydrogel has a hydrodynamic size of from about 10 nm to about 250 nm prior to self-agglomeration.

14. The composition of claim 13, wherein the nanohydrogel has a hydrodynamic size of from about 10 nm to about 200 nm prior to self-agglomeration.

15. The composition of claim 13, wherein the nanohydrogel has a hydrodynamic size of from about 50 nm to about 250 nm prior to self-agglomeration.

16. The composition of any one of claims 1-15, wherein the population of nanohydrogels further comprises surface modifications.

17. The composition of any one of claims 1-16, wherein the population of nanohydrogels is inducible via tortuous flow of an aberrant microvasculature of a tumor microenvironment (TME) to self- agglomerate to form an agglomerated cluster, wherein the agglomerated cluster 38FH13249380.11GTZ-01525has an average hydrodynamic size larger than an average hydrodynamic size prior to selfagglomeration.

18. The composition of claim 17, wherein self-agglomeration of the population of nanohydrogels is concentration-dependent.

19. The composition of claim 17 or 18, wherein a concentration of nanohydrogels causing self-agglomeration is 10 nM or greater (e.g., 20 nM or greater, 30 nM or greater, 40 nM or greater, 50 nM or greater, 60 nM or greater, 70 nM or greater, 80 nM or greater, 90 nM or greater, 100 nM or greater, 110 nM or greater, 120 nM or greater, 130 nM or greater, 140 nM or greater, 150 nM or greater, 156 nM or greater, 200 nM or greater, 250 nM or greater, 500 nM or greater, 1 pM or greater).

20. The composition of any one of claims 17-19, wherein a concentration of nanohydrogels causing self-agglomeration is from about 10 nM to about 1 pM (e.g., from about 10 nM to about 500 nM, from about 30 nM to about 500 nM, from about 60 nM to about 500 nM, from about 60 nM to about 250 nM, from about 100 nM to about 250 nM, from about 100 nM to about 200 nM, or about 156 nM).

21. The composition of any one of clams 1-20, wherein the one or more covalent linkages are cleavable.

22. The composition of claim 21, wherein the one or more cleavable covalent linkages are pH-sensitive.

23. The composition of claim 21, wherein the one or more cleavable covalent linkages is enzyme-sensitive.

24. The composition of any one of claims 1-20, wherein the one or more covalent linkages are non-cleavable.39FH13249380.11GTZ-0152525. The composition of any one of claims 5-24, wherein the one or more drug molecules is one drug molecule.

26. The composition of any one of claims 5-25, wherein each of the one or more drug molecules is independently a chemotherapeutic agent.

27. The composition of claim 26, wherein each chemotherapeutic agent is independently selected from mertansine, oxaliplatin, cisplatin, and combinations thereof.

28. The composition of claim 27, wherein the chemotherapeutic agent is mertansine.

29. The composition of claim 27, wherein the chemotherapeutic agent is oxoplatin.

30. The composition of any one of claims 5-25, wherein each of the one or more drug molecules is a biologic drug.

31. The composition of any one of claims 5-25, wherein each of the one or more drug molecules is selected from a cytotoxic agent, immunomodulatory agent, complementactivating agent, and receptor blocking agent.

32. The composition of claim 31, wherein each of the one or more drug molecules is a cytotoxic agent.

33. The composition of claim 31, wherein each of the one or more drug molecules is an immunomodulatory agent.

34. The composition of claim 31, wherein each of the one or more drug molecules is a complement-activating agent.

35. The composition of claim 31, wherein each of the one or more drug molecules is a receptor-blocking agent.40FH13249380.11GTZ-0152536. The composition of any one of claims 5-24, wherein each of the one or more drug molecules is independently a therapeutic agent selected from monoclonal antibodies, chimeric antibodies, humanized antibodies, nanobodies, antibody fragments, cholesterol, hormones, peptides, proteins, chemotherapeutics, antineoplastic agents, low molecular weight drugs, vitamins, co-factors, nucleosides, nucleotides, oligonucleotides, polynucleotides, enzymatic nucleic acids, antisense nucleic acids, triplex forming oligonucleotides, antisense DNA or RNA compositions, chimeric DNA:RNA compositions, allozymes, aptamers, ribozyme, decoys, analogs, plasmids, expression vectors, small nucleic acid molecules, mRNA, RNAi agents, short interfering nucleic acid (siNA), short interfering RNA (siRNA), double- stranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA (shRNA), peptide nucleic acid (PNA), locked nucleic acid ribonucleotides (LNA), morpholino nucleotides, threose nucleic acid (TNA), glycol nucleic acid (GNA), sisiRNA (small internally segmented interfering RNA), aiRNA (assymetrical interfering RNA), and siRNA with 1, 2, or more mismatches between the sense and anti-sense strand to relevant cells or tissues, or a combination thereof.

37. The composition of claim 36, wherein each therapeutic agent independently comprises an RNAi agent.

38. The composition of claim 36, wherein each therapeutic agent independently comprises an anticancer agent.

39. The composition of claim 36, wherein each therapeutic agent independently comprises a nucleic acid.

40. The composition of claim 36, wherein each therapeutic agent independently comprises small interacting RNA (siRNA) and / or microRNA (miRNA).

41. The composition of claim 36, wherein each therapeutic agent independently comprises an inhibitor of epidermal growth factor receptor (EGFR), Kirsten rat sarcoma virus oncogene homolog (KRAS), Glul, and / or Zinc Finger E-Box Binding Homeobox 1 (ZEB1).41FH13249380.11GTZ-0152542. The composition of claim 36, wherein each therapeutic agent independently comprises an inhibitor of EGFR.

43. The composition of claim 36, wherein each therapeutic agent independently comprises an inhibitor of Glul.

44. The composition of claim 36, wherein each therapeutic agent independently comprises an inhibitor of ZEB1.

45. The composition of claim 36, wherein each therapeutic agent independently comprises an inhibitor of KRAS.

46. The composition of any one of claims 16-45, wherein the surface modifications comprise tumor-targeting ligands.

47. The composition of any one of claims 5-46, further comprising one or more imaging agents conjugated to the population of nanohydrogels.

48. The composition of any one of claims 1-4 and 47, wherein each of the one or more imaging agents independently comprises a contrast agent, a heavy metal, or a radioisotope.

49. The composition of any one of claims 1-4 and 47, wherein each of the one or more imaging agents independently comprises a fluorophore.

50. The composition of any one of claims 5-49, wherein the one or more drug molecules are conjugated to the population of nanohydrogels at a ratio of about 5:1 to about 50:1.

51. The composition of any one of claims 5-50, wherein the composition releases the one or more drug molecules in a controlled manner.

52. The composition of any one of claims 47-51, wherein the composition releases the one or more imaging agents in a controlled manner.42FH13249380.11GTZ-0152553. The composition of any one of claims 1-52, wherein each covalent linkage independently comprises oxanorbornadiene (OND) or maleimide.

54. The composition of any one of claims 1-53, wherein each covalent linkage independently comprises a linker.

55. The composition of claim 54, wherein each linker is independently an OND linker.

56. The composition of claim 54, wherein each linker is independently a maleimide linker.

57. The composition of claim 54, wherein the OND-NHS linker comprises a releasing half-life between about 1 hour to about 10 hours.

58. The composition of claim 57, wherein the releasing half-life is about 9 hours.

59. The composition of claim 54, wherein the OND linker is a non-releasing epoxide OND-NHS linker.

60. A method of treating cancer, comprising:administering to a subject in need thereof a therapeutically effective amount of the composition of any one of claims 1-59.

61. The method of claim 60, wherein the cancer is selected from breast cancer, lung cancer, colorectal cancer, ovarian cancer, pancreatic cancer, melanoma, and lymphoma.

62. The method of claim 61, wherein the cancer is ovarian cancer.

63. The method of claim 61, wherein the cancer is colorectal cancer.

64. The method of claim 61, wherein the cancer is breast cancer.

65. The method of claim 61, wherein the cancer is lung cancer.43FH13249380.11GTZ-0152566. The method of claim 61, wherein the cancer is pancreatic cancer.

67. The method of claim 61, wherein the cancer is melanoma.

68. The method of claim 61, wherein the cancer is lymphoma.

69. The method of any one of claims 60-68, wherein the cancer is a cancer having an undruggable cancer-promoting gene.

70. The method of claim 69, wherein the undruggable cancer-promoting gene comprises one or more of c-Myc. APC, BRAF, and / or KRAS.

71. The method of any one of claims 60-70, wherein a tumor selectivity of the composition is 1% or greater (e.g., 1.5% or greater, 2% or greater, 2.5% or greater, 3% or greater, 3.5% or greater, 4% or greater, 4.5% or greater, 5% or greater, 5.5% or greater, 6% or greater, 6.5% or greater, 7% or greater, 7.5% or greater, 8% or greater, 9.5% or greater, 10% or greater, 10.5% or greater, 11% or greater, 11.5% or greater, 12% or greater, 12.5% or greater, 13% or greater, 13.5% or greater, 14% or greater, 14.5% or greater, 15% or greater, 20% or greater, or 25% or greater).

72. The method of any one of claims 60-71, wherein a ratio of relative delivery of the composition between the tumor and a liver of the subject is 1:1 or more (e.g., 1.5:1 or more, 2:1 or more, 2.5:1 or more, 3:1 or more, 3.5:1 or more, 4:1 or more, 4.5:1 or more, 5:1 or more, 5.5:1 or more, 6:1 or more, 6.5:1 or more, 7:1 or more, 7.5:1 or more, 8:1 or more, 8.5:1 or more, 9:1 or more, 9.5:1 or more, 10:1 or more, 10.5:1 or more, 15:1 or more, 20:1 or more, or 25:1 or more).

73. The method of any one of claims 60-72, wherein 2.5% or more of the composition is retained after 24 hours from an initial dose (e.g., 3% or more, 3.5% or more, 4% or more, 4.5% or more, 5% or more, 5.5% or more, 6% or more, 10% or more).44FH13249380.11GTZ-0152574. The method of any one of claims 60-73, wherein the administration of the composition comprises administering a first composition comprising a first drug molecule and a second composition comprising a second drug molecule.

75. The method of claim 74, wherein the first composition and second composition are administered concurrently.

76. The method of claim 74 or 75, wherein the first drug molecule and the second drug molecule are the same.

77. The method of claim 74 or 75, wherein the first drug molecule and the second drug molecule are different.

78. The method of claim 74 or 75, wherein the first drug molecule comprises an RNAi agent and the second drug molecule comprises a chemotherapeutic agent.

79. The composition of any one of claims 60-78, wherein the composition targets primary tumors.

80. The composition of any one of claims 60-78, wherein the composition targets metastatic lesions.

81. The composition of any one of claims 60-78, wherein the composition targets both primary tumors and metastatic lesions.

82. A method of making the composition of any one of claims 1-59, comprising: providing a population of nanohydrogels; andconjugating a drug molecule to the population of nanohydrogels via a covalent linkage.

83. The method of claim 82, wherein conjugating the drug molecule to the population of nanohydrogels comprises using l-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) coupling.45FH13249380.11GTZ-0152584. The method of claim 82 or 83, further comprising purifying the composition using chromatography.

85. The method of claim 84, wherein the chromatography is size-exclusion chromatography.

86. The method of any one of claims 83-85, further comprising characterizing the composition using a fluorescence assay or inductively coupled plasma mass spectrometry.

87. The method of claim 86, wherein the composition is characterized using a fluorescence assay.

88. The method of claim 86, wherein the composition is characterized using inductively coupled plasma mass spectrometry.FH13249380.11

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