Antibody-drug conjugates and methods of use

Abstract

Antibody-drug conjugates and pharmaceutical compositions are provided herein. Methods of treating cancers using the antibody-drug conjugates and pharmaceutical compositions as well as methods of using the same are further provided herein.

Description

ANTIBODY-DRUG CONJUGATES AND METHODS OF USE GOVERNMENT INTERESTS

[0001] This invention was made with government support under CA097098, CA262991, and CA233084 awarded by the National Institutes of Health. The government has certain rights in the invention. RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 603,059 filed on November 27, 2023 and U.S. Provisional Application No.63 / 604,528 filed on November 30, 2023, which are incorporated by reference in their entirety herein. SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing that has been submitted electronically in XML format and is incorporated by reference in its entirety. Said XML copy, created on November 25, 2024, is named SeqListing_91016-417349.xml and is 69,000 bytes in size. FIELD

[0004] The present disclosure relates to antibody-drug conjugates and pharmaceutical compositions as well as methods of treatment and use thereof. BACKGROUND

[0005] Cancer remains the second most common cause of death worldwide. MUC1 is involved in multiple biochemical pathways that are dysregulated in a variety of different cancers. MUC1 is overexpressed in various cancers and can indicate more aggressive, metastatic cancers that are less likely to respond to conventional therapies. The MUC1 structure is known and is formed by a non- covalent heterodimer of the MUC1 C-terminal (MUC1-C) and N-terminal (MUC1-N) subunits. Although MUC1 is an oncogene with both subunits overexpressed in various cancers, no drugs or biologics targeting MUC1 have been approved for clinical use. MUC1-C can drive lineage plasticity and self-renewal in cancer stem cells (CSCs) derived from epithelial cells in the prostate, breast, respiratory tract, and other organs with protective barrier functions; for example, castration-resistantprostate cancer (CRPC), neuroendocrine prostate cancer (NEPC), triple negative breast cancer (TNBC), small cell lung cancer (SCLC), and Merkel cell carcinoma (MCC) cells depend on MUC1- C for self-renewal capacity and tumorigenicity. To date, there has been limited success in developing small molecules effective in suppressing MUC1-C expression and / or function.

[0006] Ferroptosis is iron-dependent cell death, which is involved in tumor suppression and dysregulated in various cancer cell types, which require more iron than non-cancerous cells to survive. Further, multiple cancers have suppressed ferroptosis, and inducing ferroptosis in cancer cells can reverse therapeutic resistance. However, no drugs or biologics inducing or restoring ferroptosis in cancer patients have been approved for clinical use. Thus, effective therapies targeting MUC1 and ferroptosis are desirable. SUMMARY

[0007] Provided herein are antibody-drug conjugates of the formula: Formula I. A –S, wherein: A is an antibody or fragment thereof that binds MUC1-C; and S is SAL, a derivative thereof, or a pharmaceutically acceptable salt thereof.

[0008] Provided herein are antibody-drug conjugates of the formula: Formula II. A – B – S, wherein: A is an antibody or fragment thereof that binds MUC1-C; B is a drug delivery nanoparticle; S is SAL, a derivative thereof, or a pharmaceutically acceptable salt thereof; and the drug delivery nanoparticle encapsulates the SAL, derivative thereof, or pharmaceutically acceptable salt thereof.

[0009] Provided herein are antibody-drug conjugates of the formula: Formula III. A –S, wherein: A is an antibody means for binding MUC1-C; and S is SAL, a derivative thereof, or a pharmaceutically acceptable salt thereof.

[0010] Provided herein are antibody-drug conjugates of the formula:Formula IV. A – B – S, wherein: A is an antibody means for binding MUC1-C; B is a drug delivery nanoparticle; S is SAL, a derivative thereof, or a pharmaceutically acceptable salt thereof; and the drug delivery nanoparticle encapsulates the SAL, derivative thereof, or pharmaceutically acceptable salt thereof.

[0011] Provided herein are pharmaceutical compositions comprising antibody-drug conjugates of Formulas I-IV, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0012] Provided herein are methods of treating cancer in a patient in need thereof, comprising administering to the patient (a) an antibody-drug conjugate comprising any one of Formulas I-IV, or (b) pharmaceutical compositions comprising Formulas I-IV, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0013] Provided herein are methods of inducing ferroptosis in a subject, comprising administering to the subject (a) an antibody-drug conjugate comprising any one of Formulas I-IV, or (b) pharmaceutical composition comprising Formulas I-IV, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0014] Provided herein are uses of (a) an antibody-drug conjugate comprising any one of Formulas I-IV, or (b) pharmaceutical composition comprising Formulas I-IV, and one or more pharmaceutically acceptable carriers, diluents, or excipients, for treating cancer.

[0015] Provided herein are antibody-drug conjugates of any one of Formulas I-IV, or the pharmaceutical compositions comprising Formulas I-IV, and one or more pharmaceutically acceptable carriers, diluents, or excipients, for use in treating cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIGS.1A-1J show embodiments in which salinomycin (SAL) suppresses NF-^B / MUC1-C auto-inductive circuit. In FIGS. 1A and 1B, DU-145 (FIG. 1A) and H660 (FIG.1B) cells were treated with vehicle (DMSO) or the indicated concentrations of SAL for 72 hours. Viability was assessed by Alamar Blue staining. The results (mean±SD of 4 determinations) are expressed as relative viability compared to untreated cells (assigned a value of 100%). In FIGS. 1C and 1D, DU- 145 and H660 cells treated with vehicle or 1 ^M SAL for 24 hours were analyzed for MUC1-Ctranscripts (FIG. 1C). The results (mean±SD of 4 determinations) are expressed as relative MUC1-C mRNA levels compared to vehicle-treated cells (assigned a value of 1). Lysates were immunoblotted with antibodies against the indicated proteins (FIG. 1D). In FIGS. 1E and 1F, DU-145 (FIG.1E) and H660 (FIG. 1F) cells expressing a control CshRNA or NF-^BshRNA were analyzed for MUC1- C transcripts (left). The results (mean±SD of 4 determinations) are expressed as relative MUC1-C mRNA levels compared to CshRNA cells (assigned a value of 1). Lysates were immunoblotted with antibodies against the indicated proteins (right). In FIGS.1G and 1H, DU-145 (FIG. 1G) and H660 (FIG. 1H) cells untreated or treated with 10 ^M BAY11-7082 for 24 hours were analyzed for MUC1-C transcripts (left). The results (mean±SD of 4 determinations) are expressed as relative MUC1-C mRNA levels compared to untreated cells (assigned a value of 1). Lysates were immunoblotted with antibodies against the indicated proteins (right). FIGS. 1I and 1J show SAL downregulates MUC1-C expression in TNBC cells. Lysates from BT-549 (A) and MDA-MB-468 (B) cells treated with vehicle or 1 ^M SAL for 48 hours were immunoblotted with antibodies against the indicated proteins.

[0017] FIGS.2A-2L show embodiments in which silencing MUC1-C induces lipid peroxidation and enhances the effects of SAL treatment. In FIG.2A, DU-145 cells treated with vehicle or 1 ^M SAL for 24 hours were analyzed for lipid peroxidation. Shown are histograms (left) and quantitation (mean±SD of three determinations) (right) of the PE / FITC ratios. In FIG. 2B, DU-145 cells treated with vehicle or 1 ^M SAL for 24 hours were analyzed for TfR1 expression by flow cytometry. Listed are the gMFI values. In FIG. 2C, DU-145 / tet-MUC1shRNA cells treated with vehicle or DOX for 7 days were analyzed for lipid peroxidation. Shown are histograms (left) and quantitation (mean±SD of three determinations) (right) of the PE / FITC ratios. FIG.2D. DU-145 / tet- MUC1shRNA cells treated with vehicle or DOX for 7 days and then incubated with 1 ^M SAL for 24 hours were analyzed for lipid peroxidation. Shown are histograms (left) and quantitation (mean±SD of three determinations) (right) of the PE / FITC ratios. FIG.2E. DU-145 / tet- MUC1shRNA cells treated with vehicle or DOX for 7 days and then incubated with 1 ^M SAL for 24 hours were analyzed for TfR1 expression by flow cytometry. Listed are the gMFI values. FIG. 2F. Lysates from DU-145 cells expressing tet-MUC1shRNA and / or tet-MUC1-C / CD vectors treated with vehicle or DOX for 7 days were immunoblotted with antibodies against the indicated proteins. FIG. 2G. DU-145 cells expressing tet- MUC1shRNA and / or tet-MUC1-C / CD vectors treated with vehicle or DOX for 7 days and then incubated with 1 ^M SAL for 24 hours were analyzed for TfR1expression by flow cytometry. Listed are the gMFI values. FIGS 2H-2L show embodiments of the effects of silencing MUC1-C on induction of ferroptosis. FIGS 2H-2I show DU-145 / tet- MUC1shRNA (FIG.2H) and H660 / tet- MUC1shRNA (FIG. 2I) cells treated with vehicle or DOX for 7 days were analyzed for MUC1-C mRNA levels by qRT-PCR. The results (mean±SD of four determinations) are expressed as relative levels compared to that obtained for vehicle- treated cells (assigned a value of 1) (left). Lysates were immunoblotted with antibodies against the indicated proteins (right). FIG.2J shows H660 / tet-MUC1shRNA cells that were treated with vehicle or DOX for 7 days and analyzed for lipid peroxidation. Shown are histograms (left) and quantitation (mean±SD of three determinations) (right) of the PE / FITC ratios. FIG.2K shows H660 / tet- MUC1shRNA cells that were treated with vehicle or DOX for 7 days and then incubated with 1 uM SAL for 24 hours were analyzed for lipid peroxidation. Shown are histograms (left) and quantitation (mean±SD of three determinations) (right) of the PE / FITC ratios. FIG.2L shows DU-145 cells expressing tet-MUC1shRNA and tet-MUC1-C / CD vectors that were treated with vehicle or DOX for 5 days, then 1 ^M SAL for an additional 2 days, and thereafter analyzed for cell death by PI staining. The results (mean±SD of four determinations) are expressed as relative cell death compared to vehicle-treated cells (assigned a value of 1).

[0018] FIGS.3A-3J show embodiments in which MUC1-C activates GSR expression by a MYC- dependent mechanism. In FIGS. 3A and 3B, DU-145 cells treated with vehicle or 1 ^M SAL for 24 hours (FIG. 3A) and DU-145 / tet-MUC1shRNA cells treated with vehicle or DOX for 7 days (FIG. 3B) were analyzed for GSR transcripts (left). The results (mean±SD of 4 determinations) are expressed as relative GSR mRNA levels compared to vehicle-treated cells (assigned a value of 1). Lysates were immunoblotted with antibodies against the indicated proteins (right). FIG.3C shows Genome browser snapshots of ATAC-seq data from the GSR gene in DU-145 / tet-MUC1shRNA cells treated with vehicle or DOX for 7 days (left). Chromatin was analyzed for accessibility by nuclease digestion (right). The results (mean±SD of 3 determinations) are expressed as % untreated chromatin. FIG. 3D shows DU-145 / tet-MUC1shRNA cells treated with vehicle or DOX for 7 days were analyzed for GSR gene transcription. The results (mean±SD of 3 determinations) are expressed as relative GSR transcription compared to that obtained in vehicle-treated cells (assigned a value of 1). FIG. 3E shows DU-145 / tet-MYCshRNA cells treated with vehicle or DOX for 7 days were analyzed for GSR transcripts (left). The results (mean±SD of 4 determinations) are expressed as relative GSR mRNA levels compared to vehicle-treated cells (assigned a value of 1). Lysates wereimmunoblotted with antibodies against the indicated proteins (right). FIG.3F shows DU-145 / tet- MUC1shRNA cells treated with vehicle or DOX for 7 days were analyzed for GSH levels. The results (mean±SD of 3 determinations) are expressed as relative GSH levels compared to that obtained in vehicle-treated cells (assigned a value of 1). FIG.3G shows DU-145 cells expressing tet-MUC1shRNA and / or tet-Flag- MUC1-C / CD treated with vehicle or DOX for 7 days were analyzed for GSR transcripts (left). The results (mean±SD of 4 determinations) are expressed as relative GSR mRNA levels compared to that obtained in vehicle-treated cells (assigned a value of 1). Lysates were immunoblotted with antibodies against the indicated proteins (right). FIGS. 3H-3J show embodiments in which silencing MUC1-C with MUC1shRNA#2 suppresses GSR expression. FIG. 3H shows DU-145 / CshRNA and DU-145 / MUC1shRNA#2 that were analyzed for GSR transcripts (left). The results (mean±SD of 4 determinations) are expressed as relative GSR mRNA levels compared to CshRNA cells (assigned a value of 1). Lysates were immunoblotted with antibodies against the indicated proteins (right). FIG. 3I shows H660 / tet-MUC1shRNA cells treated with vehicle or DOX for 7 days that were analyzed for GSR transcripts (left). The results (mean±SD of 4 determinations) are expressed as relative GSR mRNA levels compared to vehicle-treated cells (assigned a value of 1). Lysates were immunoblotted with antibodies against the indicated proteins (right). FIG. 3J shows H660 / tet-MUC1shRNA cells treated with vehicle or DOX for 7 days that were analyzed for GSH levels. The results (mean±SD of 3 determinations) are expressed as relative GSH levels compared to that obtained in vehicle-treated cells (assigned a value of 1).

[0019] FIGS.4A-4H show embodiments in which MUC1-C induces LRP8 and GPX4 expression. FIGS. 4A and B show DU-145 cells treated with vehicle or 1 ^M SAL for 24 hours (FIG.4A) and DU-145 / tet- MUC1shRNA cells treated with vehicle or DOX for 7 days (FIG. 4B) were analyzed for LRP8 transcripts (left). The results (mean±SD of 4 determinations) are expressed as relative LRP8 mRNA levels compared to that obtained in vehicle-treated cells (assigned a value of 1). Lysates were immunoblotted with antibodies against the indicated proteins (right). FIG.4C shows Genome browser snapshots of ATAC-seq data from the LRP8 gene in DU- 145 / tet-MUC1shRNA cells treated with vehicle or DOX for 7 days (left). Chromatin was analyzed for accessibility by nuclease digestion (right). The results (mean±SD of 3 determinations) are expressed as % untreated chromatin. FIG. 4D shows DU-145 / tet-MUC1shRNA cells treated with vehicle or DOX for 7 days were analyzed for LRP8 gene transcription. The results (mean±SD of 3 determinations) are expressed as relative LRP8 transcription compared to that obtained in vehicle-treated cells (assigneda value of 1). FIG. 4E shows DU-145 / tet-MYCshRNA cells treated with vehicle or DOX for 7 days were analyzed for LRP8 transcripts (left). The results (mean±SD of 4 determinations) are expressed as relative LRP8 mRNA levels compared to that obtained in vehicle-treated cells (assigned a value of 1). Lysates were immunoblotted with antibodies against the indicated proteins (right). FIGS. 4F and 4G show lysates from DU-145 / tet-MUC1shRNA cells treated with vehicle or DOX for 7 days were immunoblotted with antibodies against the indicated proteins (FIG. 4F) and analyzed for GPX activity (FIG.4G). The results (mean±SD of 3 determinations) are expressed as relative GPX activity compared to that obtained in vehicle-treated cells (assigned a value of 1). FIG.4H shows lysates from DU-145 cells expressing tet-MUC1shRNA and / or tet-Flag- MUC1-C / CD treated with vehicle or DOX for 7 days were immunoblotted with antibodies against the indicated proteins. FIGS. 4I-4N show embodiments of the effects of MUC1-C on LRP8 and GPX4 expression. FIG.4I shows H660 cells treated with vehicle or 1 ^M SAL for 24 hours were analyzed for LRP8 transcripts (left). The results (mean±SD of 4 determinations) are expressed as relative LRP8 mRNA levels compared to that obtained in vehicle-treated cells (assigned a value of 1). Lysates were immunoblotted with antibodies against the indicated proteins (right). FIG. 4J shows DU- 145 / CshRNA and DU-145 / MUC1shRNA#2 were analyzed for LRP8 transcripts (left). The results (mean±SD of 4 determinations) are expressed as relative GSR mRNA levels compared to that obtained in CshRNA cells (assigned a value of 1). Lysates were immunoblotted with antibodies against the indicated proteins (right). FIG. 4K shows H660 / tet-MUC1shRNA cells treated with vehicle or DOX for 7 days were analyzed for LRP8 transcripts (left). The results (mean±SD of 4 determinations) are expressed as relative LRP8 mRNA levels compared to that obtained in vehicle- treated cells (assigned a value of 1). Lysates were immunoblotted with antibodies against the indicated proteins (right). FIG.4L shows DU-145 / tet- MUC1shRNA cells treated with vehicle or DOX for 7 days were analyzed for GPX4 transcripts. The results (mean±SD of 4 determinations) are expressed as relative levels compared to that obtained in vehicle-treated cells (assigned a value of 1). FIG. 4M and 4N shows lysates from H660 / tet-MUC1shRNA cells treated with vehicle or DOX for 7 days were immunoblotted with antibodies against the indicated proteins (FIG. 4M) and analyzed for GPX activity (FIG. 4N). The results (mean±SD of 3 determinations) are expressed as relative GPX activity compared to vehicle-treated cells (assigned a value of 1).

[0020] FIGS.5A-5G show embodiments in which targeting MUC1-C with GO-203 induces ferroptosis in association with suppression of GSR, LRP8 and GPX4 expression. FIG. 5A showsDU-145 cells were treated with PBS as a control vehicle or the indicated concentrations of GO-203 for 72 hours. Viability was assessed by Alamar Blue staining. The results (mean±SD of 4 determinations) are expressed as relative viability compared to untreated cells (assigned a value of 100%). FIG. 5B shows DU-145 cells left untreated or treated with 2 ^M GO-203 for 24 hours were analyzed for lipid peroxidation. Shown are histograms (left) and quantitation (mean±SD of three determinations) (right) of the PE / FITC ratios. FIG.5C shows DU-145 cells left untreated or treated with 2 ^M GO-203 for 12 hours were analyzed for cell surface TfR1 expression by flow cytometry. Listed are gMFI values. FIG. 5D shows DU-145 cells left untreated or treated with 2 ^M GO-203 for 24 hours were analyzed for GSR transcripts (left). The results (mean±SD of 4 determinations) are expressed as relative GSR mRNA levels compared to untreated cells (assigned a value of 1). Lysates were immunoblotted with antibodies against the indicated proteins (right). FIG. 5E shows lysates from DU-145 cells left untreated or treated with 2 ^M GO-203 for 24 hours were analyzed for GSH levels. The results (mean±SD of 3 determinations) are expressed as relative GSH levels compared to that obtained in untreated cells (assigned a value of 1). FIG. 5F shows DU-145 cells left untreated or treated with 2 ^M GO-203 for 8 hours were analyzed for LRP8 transcripts (left). The results (mean±SD of 4 determinations) are expressed as relative LRP8 mRNA levels compared to untreated cells (assigned a value of 1). Lysates were immunoblotted with antibodies against the indicated proteins (right). FIG.5G shows lysates from DU-145 cells left untreated or treated with 2 ^M GO- 203 for 24 hours were analyzed for GPX activity. The results (mean±SD of 3 determinations) are expressed as relative GPX activity compared to untreated cells (assigned a value of 1). FIGS. 5H- 5M show embodiments of the effects of targeting MUC1-C with GO-203 in H660 cells. FIG.5H shows H660 cells that were treated with vehicle or the indicated concentrations of GO-203 for 72 hours. Viability was assessed by Alamar Blue staining. The results (mean±SD of 3 determinations) are expressed as relative viability compared to untreated cells (assigned a value of 100%). FIG. 5I shows H660 cells left untreated or treated with 2 ^M GO-203 for 24 hours were analyzed for lipid peroxidation. Shown are histograms (left) and quantitation (mean±SD of three determinations) (right) of the PE / FITC ratios. FIG. 5J shows H660 cells left untreated or treated with 2 ^M GO-203 for 24 hours were analyzed for GSR transcripts (left). The results (mean±SD of 4 determinations) are expressed as relative GSR mRNA levels compared to that obtained in untreated cells (assigned a value of 1). Lysates were immunoblotted with antibodies against the indicated proteins (right). FIG. 5K shows lysates from H660 cells left untreated or treated with 2 ^M GO-203 for 24 hours wereanalyzed for GSH levels. The results (mean±SD of 3 determinations) are expressed as relative GSH levels compared to that obtained in untreated cells (assigned a value of 1). FIG. 5L shows H660 cells left untreated or treated with 2 ^M GO-203 for 24 hours were analyzed for LRP8 transcripts (left). The results (mean±SD of 4 determinations) are expressed as relative LRP8 mRNA levels compared to that obtained in untreated cells (assigned a value of 1). Lysates were immunoblotted with antibodies against the indicated proteins (right). FIG. 5M shows lysates from H660 cells left untreated or treated with 2 ^M GO-203 for 24 hours were analyzed for GPX activity. The results (mean±SD of 3 determinations) are expressed as relative GPX4 activity compared to untreated cells (assigned a value of 1).

[0021] FIGS.6A-6F show embodiments in which purified CSCs are dependent on MUC1-C for self-renewal capacity and resistance to ferroptosis. FIG. 6A shows DU-145 cells growing as monolayers were seeded in tumorsphere culture medium. After 10 days, the sphere 1 (S1) cells were isolated and reseeded for selection of S2 cells. Photomicrographs are shown for the serially passaged tumorspheres up to S13 (left). Scale bar: 100 µm. The sphere forming efficiency (SFE) was determined by the percentage of cells that formed tumorspheres as a function of the number of seeded cells. The results (mean±SD of three determinations) are expressed as % SFE (right). FIG. 6B shows DU-145 / tet-MUC1shRNA CSCs treated with vehicle or DOX for 7 days were analyzed for tumorsphere formation. The results (mean±SD of three determinations) are expressed as relative SFE compared to vehicle treated cells (assigned a value of 1). FIG.6C shows DU-145 / tet- MUC1shRNA CSCs treated with vehicle or DOX in the absence and presence of 10 ^M Fer-1 for 24 hours were analyzed for tumorsphere formation. Photomicrographs are shown for the treated tumorspheres (left). The results (mean±SD of three determinations) are expressed as relative SFE compared to DOX alone treated cells (assigned a value of 1) (right). FIG.6D shows DU-145 / tet- MUC1shRNA CSCs without and with transfection of tet-Flag-MUC1-C / CD were treated with vehicle or DOX for 7 days. Photomicrographs are shown for the treated tumorspheres (left). The results (mean±SD of three determinations) are expressed as relative SFE compared to vehicle treated cells (assigned a value of 1) (right). FIG.6E shows DU-145 CSCs treated with vehicle or 2 ^M GO- 203 for 24 hours were analyzed for tumorsphere formation. Photomicrographs are shown for the treated tumorspheres (left). The results (mean±SD of three determinations) are expressed as relative SFE compared to that obtained in vehicle treated cells (assigned a value of 1)(right). The results (mean±SD of three determinations) are expressed as SFE. FIG. 6F shows DU-145 CSCs treatedwith vehicle or 2 ^M GO-203 in the absence and presence of 10 ^M Fer-1 for 24 hours were analyzed for tumorsphere formation. Photomicrographs are shown for the treated tumorspheres (left). The results (mean±SD of three determinations) are expressed as relative SFE compared to GO-203 alone treated cells (assigned a value of 1)(right). FIGS.6G-6K show embodiments of the effects of SAL and targeting MUC1-C in CSCs. FIG.6G shows enriched S8 CSCs treated with vehicle or 1 ^M SAL for 24 hours that were analyzed for the indicated mRNA levels by qRT-PCR. The results (mean±SD of 4 determinations) are expressed as relative mRNA levels compared to that obtained in vehicle-treated cells (assigned a value of 1)(left). Lysates were immunoblotted with antibodies against the indicated proteins (right). FIG. 6H shows DU-145 CSCs treated with vehicle or 1 ^M SAL for 24 hours were analyzed for tumorsphere formation. Photomicrographs are shown for the treated tumorspheres (left). The results (mean±SD of three determinations) are expressed as relative SFE compared to control cells (assigned a value of 1)(right). FIG.6I shows DU-145 CSCs treated with 1 ^M SAL in the absence and presence of 10 ^M Fer-1 for 7 days were analyzed for tumorsphere formation. Photomicrographs are shown for the treated tumorspheres (left). The results (mean±SD of three determinations) are expressed as relative SFE compared to that obtained in SAL alone treated cells (assigned a value of 1)(right). FIG.6J shows lysates from DU-145 cells left untreated or treated with 2 ^M GO-203 for 24 hours that were immunoblotted with antibodies against the indicated proteins. FIG. 6K shows DU-145 CSCs treated with 10 ^M BAY11-7082 for 7 days that were analyzed for tumorsphere formation. Photomicrographs are shown for the treated tumorspheres (left). The results (mean±SD of three determinations) are expressed as relative SFE compared to BAY11-7082-treated cells (assigned a value of 1)(right).

[0022] FIGS.7A-7F show embodiments in which MUC1-C is a target of SAL-nanoparticles (SAL / NPs) for inhibition of tumorigenicity. FIG. 7A shows DU-145 cells that were treated with SAL-nanoparticles at the indicated concentrations of SAL for 24-96 hours. Viability was assessed by Alamar Blue staining. The results (mean±SD of 4 determinations) are expressed as relative viability compared to untreated cells (assigned a value of 100%). FIG. 7B shows lysates from DU-145 cells treated with SAL-nanoparticles or an equivalent amount of empty nanoparticles for 24 hours that were immunoblotted with antibodies against the indicated proteins. FIG. 7C shows DU-145 cells that were treated with SAL-nanoparticles or an equivalent amount of empty nanoparticles for 24 hours and analyzed for lipid peroxidation. Shown are histograms (left) and quantitation (mean±SD of three determinations) (right) of the PE / FITC ratios. FIG. 7D shows DU-145 cells that weretreated with SAL-nanoparticles or an equivalent amount of empty nanoparticles for 24 hours and analyzed for TfR1 expression by flow cytometry. Listed are the gMFI values. FIG. 7E shows six- week old nude mice that were injected subcutaneously in the flank with 1 × 107 DU-145 cells. Mice pair-matched into two groups of 6 mice each when tumors reached 150–200 mm3 were treated with SAL-nanoparticles or empty nanoparticles each week x 5 weeks. Tumor volumes are expressed as the mean±SEM for six mice. FIG.7F shows lysates of tumors harvested on day 35 that were immunoblotted with antibodies against the indicated proteins. FIG.7G shows an example schema depicting the effects of SAL on inhibiting MUC1- C-driven ferroptosis resistance. Without being bound by theory, MUC1-C interacts directly with NF-^B and contributes to the activation of NF-^B target genes, including MUC1 in an auto-inductive loop. Salinomycin inhibits this inflammatory circuit by suppressing NF-^B and MUC1-C expression. The MUC1-C cytoplasmic domain also binds directly to MYC and activates MYC target genes. The present disclosure demonstrates that this MUC1-C-MYC pathway increases chromatin accessibility of the GSR and LRP8 promoter regions and activation of these genes. The dependency on MUC1-C for inducing GSR and LRP8 expression was further shown in the present disclosure by rescuing MUC1-C silencing with expression of the MUC1-C cytoplasmic domain, which includes the CQC motif that binds to MYC. Moreover, this MUC1-C dependency was further shown in the present disclosure by the MUC1-C inhibitor GO-203, which blocks the CQC motif and similarly suppresses GSR and LRP8 expression. GPX4 is a negative regulator of ferroptosis. GSR regulates GSH levels necessary for GPX4 activity. In addition, LRP8 contributes to translation of the GPX4 protein. The present disclosure shows that targeting MUC1-C genetically and pharmacologically with SAL or GO-203 downregulates GPX4 levels. In the present disclosure, rescue of MUC1-C silencing with the MUC1-C cytoplasmic domain further shows that MUC1-C facilitates GPX4 expression and ferroptosis resistance. Without being bound by theory, the examples of the present disclosure show that SAL induces ferroptosis by downregulating of MUC1-C and suppressing GSR, LRP8, and GPX4. FIGS 7H-7I show embodiments of the effects of empty nanoparticles (NPs). FIG. 7H shows DU-145 cells that were treated with empty NPs for 24-96 hours at equivalent amounts used in studies of SAL / NPs shown in FIG. 7A. Viability was assessed by Alamar Blue staining. The results (mean±SD of 4 determinations) are expressed as relative viability compared to untreated cells (assigned a value of 100%). FIG. 7I shows six-week-old nude mice that were injected subcutaneously in the flank with 1 × 107DU-145 cells. Mice pair-matched into two groups of 6 mice each when tumors reached 150–200 mm3were treated with SAL / NPs or empty NPs each week x 5 weeks. Body weights are expressed as the mean±SEM for six mice. DETAILED DESCRIPTION

[0023] Without being bound by theory, provided in the present disclosure, the MUC1-C addiction of CSCs includes resistance to ferroptosis, and salinomycin (SAL) downregulates MUC1-C, suppressing effectors of ferroptosis resistance. Provided in the present disclosure, silencing MUC1-C and targeting MUC1-C with the GO-203 inhibitor induces ferroptosis, showing MUC1-C facilitates ferroptosis resistance. Thus, without being bound by theory, SAL induces ferroptosis by downregulating MUC1-C. Rescue of MUC1-C downregulation with MUC1-C Conjugated Drug (MUC1-C / CD) restored ferroptosis resistance. Without being bound by theory, the present disclosure provides that SAL is an effective small molecule inhibitor of MUC1-C signaling and that CSCs are dependent on MUC1-C for resistance to ferroptosis. Terms

[0024] Used herein, an "antibody" is an immunoglobulin molecule comprising two heavy chains (HCs) and two light chains (LCs) interconnected by disulfide bonds. The amino terminal portion of each LC and HC includes a variable region of about 100-120 amino acids primarily responsible for antigen recognition via the complementary determining region (CDRs) contained therein. The CDRs are interspersed with regions that are well-known and generally conserved among and between species (e.g., mouse and human), which are termed framework regions (FRs).

[0025] In embodiments, the CDRs are interspersed with FRs. Antibodies disclosed herein have four FRs, termed FR1, FR2, FR3, and FR4. In embodiments, the FRs are human FRs (e.g., Antibody Engineering: Methods and Protocols (Damien Nevoltris and Patrick Chames eds., 3d ed. 2018)).

[0026] The three CDRs of the LC are referred to as "LCDR1, LCDR2, and LCDR3," and the three CDRs of the HC are referred to as "HCDR1, HCDR2, and HCDR3." The functional ability of an antibody to bind a particular antigen is largely determined by the six CDRs. Assignment of amino acids to CDR domains within the LCVR and HCVR regions of the antibodies of the present disclosure is based on the well-known Kabat numbering conventions (Andrew Martin, Protein Sequence and Structure Analysis of Antibody Variable Domains in Antibody Engineering (Roland Kontermann and Stefan Dübel eds., 2d ed. 2010)).

[0027] The constant region of the antibody defines the isotype of an antibody. The antibodies of the present disclosure include IgG and IgM. IgG antibodies can be further divided into subclasses, e.g., IgG1, IgG2, IgG3, IgG4. In a particular embodiment, the antibodies of the present disclosure are IgM, IgG, IgG1, IgG2, IgG2a. The carboxy-terminal portion of each HC defines a constant region primarily responsible for effector function. In a particular embodiment, the antibodies of the present disclosure have one or more modifications in the constant region of each HC that reduces effector function.

[0028] The term “antibody means for binding MUC1-C” refers to an antibody that binds MUC1-C, such as at the MUC1-C extracellular domain (ECD). Antibodies within the scope of antibody means are the disclosed antibodies and functional equivalents thereto. Functional equivalent antibodies comprise different specific amino acid residues but bind the MUC1-C at the MUC1-C / ECD. Functional equivalent antibodies that bind the MUC1-C at the MUC1-C / ECD would differ insubstantially to deliver a payload and have a therapeutic effect.

[0029] Herein, “MUC1” refers to Mucin 1, which is a heterodimer transmembrane protein of the mucin family encoded in humans by the MUC1 gene. MUC1 helps form mucin and regulate protein transcription and activation and is involved in immune function and epithelial regulation.

[0030] The phrase “complementary determining means” as used herein describes the six complimentary determining regions (CDRs) that collectively form specific interactions with the MUC1-C / ECD. CDRs within the scope of complementary determining means are the disclosed CDRs and functional equivalents thereto. Functional equivalent CDRs comprise different specific amino acid residues but bind the MUC1-C at the MUC1-C / ECD. Functional equivalent CDRs that bind the MUC1-C at the MUC1-C / ECD would differ insubstantially to deliver a payload and have a therapeutic effect.

[0031] Contemplated herein are conservative variants of the disclosed antibodies and fragments thereof. A protein is a conservative variant where it contains conservative amino acid substitutions that do not substantially affect or decrease the affinity of a protein. For example, an antibody that binds MUC1-C / ECD can include at least 1, 2, 5, 10, or 15 conservative substitutions, for example, in a constant domain, and bind the MUC1-C / ECD. Conservative amino acid substitution tables providing functionally similar amino acids are well-known to one of ordinary skill in the art. The following groups are examples of amino acids that are considered conservative substitutions for one another: 1) serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N),glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) phenylalanine (F), tyrosine (Y), tryptophan (W).

[0032] Herein, a "degenerate variant" refers to a polynucleotide encoding a polypeptide (such as an antibody or fragment thereof) that includes a sequence that is degenerate based on the genetic code (i.e., the 20 natural amino acids can be specified by more than one codon). All degenerate nucleotide sequences encoding the disclosed antibody and fragment polypeptide sequences are included.

[0033] Further contemplated are variants of the disclosed antibodies and fragments thereof. Herein, “sequence identity“ is referred to as the similarity between amino acid or nucleic acid sequences, which is expressed as the similarity between the sequences. Sequence identity is frequently measured as percent identity, in which two sequences are considered more similar the higher the percentage. Homologs or variants of a polypeptide or nucleic acid molecule possess a relatively high degree of sequence identity when aligned using standard methods, which are well-known. Ceslovas Venclovas, Methods for Sequence-Structure Alignment in Homology Modeling: Methods and Protocols, 55-82 (Andrew Orry and Ruben Abagyan, eds., 2012)).

[0034] Herein, “binding” (or “binds”) refers to the well-understood interaction between and antibody or antibody-drug conjugate and a target protein, peptide, or polysaccharide. Binding can be measured in a variety of ways (see, e.g., Antibody Engineering: Methods and Protocols (Damien Nevoltris and Patrick Chames eds., 3d ed. 2018)). A particular antibody, antibody-drug conjugate, or protein binds to a particular target protein, peptide, or polysaccharide and does not bind in a significant amount to other proteins or polysaccharides present in a sample or subject disclosed herein. Binding occurs between the disclosed antibodies, fragments thereof, or antibody-drug conjugates and an epitope of MUC1-C / ECD. Herein, "epitope" refers to discrete sites of an antigen recognized by the disclosed antibodies, fragments thereof, and antibody-drug conjugates. Epitopes may be linear or three-dimensional. In embodiments, the epitope is the alpha helix 3 (SEQ ID NO: 58) or alpha helix 4 (SEQ ID NO: 59) of the MUC1-C / ECD (SEQ ID NO: 57). An antibody, fragment thereof, or antibody-drug conjugate binds to a target protein when the interaction has a KDof less than 10-6molar, such as less than 10-7molar, less than 10-8molar, less than 10-9molar, or less than 10-10molar.

[0035] The antibodies, fragments thereof, or antibody-drug conjugates disclosed herein can be administered to subjects or patients. Herein, “administration” refers to the act of the attending physician or caregiver, prescribing the agent for administration and thereby causing the applicationof an agent to a subject, through ingestion, infusion, injection, or any other means, whether self- administered or administered by a clinician or other qualified care giver. Herein, a “subject” includes both human patient and veterinary subjects, including human and non-human mammals. In embodiments, the subject or patient has or has a risk of cancer, such as cancers that express MUC1- C on their cell surface, renal cancer, leukemia, acute myeloid leukemia, lymphoma, cutaneous T cell lymphoma, multiple myeloma, breast cancer, triple negative breast cancer, Merkel cell carcinoma, small cell lung cancer, non-small cell lung cancer, castration-resistant prostate cancer, neuroendocrine prostate cancer, urothelial carcinoma, or colorectal cancer.

[0036] Herein, an “effective amount” is a quantity sufficient to achieve a desired effect in a subject. For instance, this can be the amount necessary to prevent, treat, or ameliorate a disease, for example, inhibiting or suppressing cancer, such as cancers that express MUC1-C on their cell surface, including but not limited to, renal cancer, leukemia, acute myeloid leukemia, lymphoma, cutaneous T cell lymphoma, multiple myeloma, breast cancer, triple negative breast cancer, Merkel cell carcinoma, small cell lung cancer, non-small cell lung cancer, castration-resistant prostate cancer, neuroendocrine prostate cancer, urothelial carcinoma, or colorectal cancer. In embodiments, an effective amount is the amount necessary to eliminate, reduce the size, or prevent metastasis of cancer or a tumor. Efficacy is first evident in the cellular response, for which a variety of in vitro and cell-based assays are well-known to measure. Kristina V. Kitaeva et al., Cell Culture Based In vitro Test Systems for Anticancer Drug Screening, 8 Front. Bioeng. Biotechnol.322(2020)). In embodiments, an “effective amount” is the amount necessary to significantly inhibit or reduce cancer cell proliferation or migration, invasion, or adhesion. A cellular response manifests as significantly reduced tumor size, reduced or inhibited disease progression, and improvement in survival in a subject. More particularly, an effective amount provides an improvement in important cancer endpoints, Overall Survival (OS), Disease-Free Survival (DFS), Objective Response Rate, Complete Response Rate or Progression Free Survival (PFS). See Dept. of Health and Human Services, Food and Drug Admin, Clinical Trial Endpoints for the Approval of Cancer Drugs and Biologics: Guidance for Industry (2018); E.A. Eisenhauer et al., New Response Evaluation Criteria in Solid Tumours: Revised RECIST Guideline (Version 1.1), 45 Eur. J. Cancer 228 (2009).

[0037] The term “therapeutic” in conjunction with antibody-drug conjugates disclosed herein refers to antibody-drug conjugates suitable for use in human treatment of cancer, such as cancers that express MUC1-C on their cell surface, renal cancer, leukemia, acute myeloid leukemia, lymphoma,cutaneous T cell lymphoma, multiple myeloma, breast cancer, triple negative breast cancer, Merkel cell carcinoma, small cell lung cancer, non-small cell lung cancer, castration-resistant prostate cancer, neuroendocrine prostate cancer, urothelial carcinoma, or colorectal cancer. Such an antibody-drug conjugate has a KDof less than 10-6molar, such as less than 10-7molar, less than 10-8molar, less than 10-9molar, or less than 10-10molar and any toxic or detrimental effects of the antibody-drug conjugate are outweighed by the therapeutic beneficial effects. “Anti-cancer therapy” as disclosed herein refers to clinical therapies that treat CSCs in cancers. CSCs expressing various surface markers, such as CD34+and CD38−, CD133, nestin, and CD44, are found in many non-solid and solid tumors, and CSCs form the bulk of the tumor through self-renewal and differentiation into multiple cellular subtypes. Many anti-cancer therapies are directed to targeting CSCs, including genotoxic drugs, such as tamoxifen, trastuzumab, afatinib, vemurafenib, and Osimertinib.

[0038] Further disclosed herein are antibody fragments and antibody-drug conjugates comprising antibody fragments, such as well-characterized Fabs (e.g., Fab, Fab', F(ab')2), Fvs (the variable region of the light chain and the variable region of the heavy chain expressed as two chains), and single-chain fragments (e.g., single-chain variable region fragments, scFv, and single chain Fabs, scFab), which also bind to the MUC1-C / ECD (SEQ ID NO: 57), such as at the alpha helix 3 (SEQ ID NO: 58) or alpha helix 4 (SEQ ID NO: 59). Methods of making these fragments and antibody- drug conjugates comprising antibody fragments are routine (see, e.g., Antibody Engineering: Methods and Protocols (Damien Nevoltris and Patrick Chames eds., 3d ed. 2018)).

[0039] The antibodies herein are monoclonal antibodies ("mAbs"). mAbs can be produced, for example, by hybridoma technologies, recombinant technologies, phage display technologies, synthetic technologies (e.g., CDR or specificity-determining residue, SDR, grafting), or combinations of such or other technologies known in the art. mAbs are antibodies derived from a single copy or clone including, for example, any eukaryotic, prokaryotic or phage clone. A variety of well-known methods and tools can be used for producing and purifying the mAbs disclosed herein, including vectors, for example, plasmids, virus, or other vehicles for polynucleotide insertion or expression, and hosts, for example, microbial, yeast, insect, and mammalian organisms (see, e.g., Process Scale Purification of Antibodies (Uwe Gottschalk, ed., 2d ed. 2017)).

[0040] Further antibodies having a complementary binding means can be prepared and screened by well-known methods, such as hybridoma, transgenic animals, and phage or yeast display (see, e.g., Monoclonal Antibodies: Methods and Protocols (Vincent Ossipow and Nicolas Fischer, eds., 2d ed.2014)). Antibodies having equivalent complementary binding means differ in their amino acid sequence but perform the same function of binding the target through CDR-target interaction acting as (inhibitor / agonist / antagonist) to achieve the same result (inhibiting tumor growth). Preferably, the complementary binding means functions through the same epitope as the disclosed antibodies.

[0041] The antibodies, fragments thereof, and antibody-drug conjugates disclosed herein can be used in therapy. In embodiments, the antibodies, fragments thereof, and antibody-drug conjugates disclosed herein can be used to treat, prevent (such as through prophylactic treatment), or ameliorate a cancer, such as cancers that express MUC1-C on their cell surface, renal cancer, leukemia, acute myeloid leukemia, lymphoma, cutaneous T cell lymphoma, multiple myeloma, breast cancer, triple negative breast cancer, Merkel cell carcinoma, small cell lung cancer, non-small cell lung cancer, castration-resistant prostate cancer, neuroendocrine prostate cancer, urothelial carcinoma, or colorectal cancer. Herein, "preventing" a disease refers to inhibiting the full development of a disease, such as cancer, such as cancers that express MUC1-C on their cell surface, renal cancer, leukemia, acute myeloid leukemia, lymphoma, cutaneous T cell lymphoma, multiple myeloma, breast cancer, triple negative breast cancer, Merkel cell carcinoma, small cell lung cancer, non-small cell lung cancer, castration-resistant prostate cancer, neuroendocrine prostate cancer, urothelial carcinoma, or colorectal cancer. "Treating" refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop, such as a reduction in tumor burden or a decrease in the number of size of metastases. "Ameliorating" refers to the reduction in the number or severity of signs or symptoms of a disease, such as cancer, such cancers that express MUC1-C on their cell surface, renal cancer, leukemia, acute myeloid leukemia, lymphoma, cutaneous T cell lymphoma, multiple myeloma, breast cancer, triple negative breast cancer, Merkel cell carcinoma, small cell lung cancer, non-small cell lung cancer, castration-resistant prostate cancer, neuroendocrine prostate cancer, urothelial carcinoma, or colorectal cancer. A "prophylactic" treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing a pathology, such as cancer, such as cancers that express MUC1-C on their cell surface, renal cancer, leukemia, acute myeloid leukemia, lymphoma, cutaneous T cell lymphoma, multiple myeloma, breast cancer, triple negative breast cancer, Merkel cell carcinoma, small cell lung cancer, non-small cell lung cancer, castration-resistant prostate cancer, neuroendocrine prostate cancer, urothelial carcinoma, or colorectal cancer.

[0042] The pharmaceutically acceptable carriers of use are conventional (e.g., as described in Remington, The Science and Practice of Pharmacy, 22nd Edition, Loyd V., ed., Pharmaceutical Press, 2012). In general, the nature of the carrier will depend on the mode of administration. For instance, parenteral formulations typically comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids, such as water, physiological saline, balanced salt solutions or the like as a vehicle. Pharmaceutical compositions can additionally include minor amounts of non- toxic auxiliary substances for stability.

[0043] In embodiments, the carrier may be sterile and / or suspended or otherwise contained in a unit dosage form including one or more measured doses of the composition suitable for administration to a subject of an effective amount of the antibodies and fragments thereof disclosed herein. Medications for use in treatment may also be included in embodiments. In embodiments, the unit dosage form may be in a sealed vial that contains sterile contents or a syringe for injection into a subject, lyophilized for subsequent solubilization and administration, or in a solid or controlled release dosage.

[0044] A pharmaceutical composition of the present disclosure contains an "effective" or "therapeutically effective" amount, as used interchangeably herein, of a monoclonal antibody of the present disclosure. The dosages and dosage regimen to achieve the desired therapeutic result depending on the means of administration and may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the monoclonal antibody to elicit a desired response in the individual. An effective amount is also one in which any toxic or detrimental effects of the monoclonal antibody of the present disclosure are outweighed by the therapeutically beneficial effects. Compositions of the Present Disclosure Antibody-Drug Conjugates

[0045] Provided in the present disclosure are antibody-drug conjugates (ADCs). In embodiments, the provided ADCs are of the formulas:

[0046] Formula I. A – S wherein:A is an antibody or fragment thereof that binds MUC1-C; and S is salinomycin, a derivative thereof, or a pharmaceutically acceptable salt thereof.

[0047] Formula II. A – B – S wherein: A is an antibody or fragment thereof that binds MUC1-C; B is a drug delivery nanoparticle; S is salinomycin, a derivative thereof, or a pharmaceutically acceptable salt thereof; and the drug delivery nanoparticle encapsulates the salinomycin, derivative thereof, or pharmaceutically acceptable salt thereof.

[0048] Formula III. A – S wherein: A is an antibody means for binding MUC-1C; and S is salinomycin, a derivative thereof, or a pharmaceutically acceptable salt thereof.

[0049] Formula IV. A – B – S wherein: A is an antibody means for binding MUC-1C; B is a drug delivery nanoparticle; S is salinomycin, a derivative thereof, or a pharmaceutically acceptable salt thereof; and the drug delivery nanoparticle encapsulates the salinomycin, derivative thereof, or pharmaceutically acceptable salt thereof.

[0050] In embodiments, the antibody or fragment thereof that binds MUC1-C (A) is conjugated, or linked, through a linkage to the salinomycin, a derivative thereof, or a pharmaceutically acceptable salt thereof (S) of Formula I. In embodiments, the antibody means for binding MUC1-C (A) is conjugated, or linked, through a linkage to the salinomycin, a derivative thereof, or a pharmaceutically acceptable salt thereof (S) of Formula III.

[0051] Various linkages are possible for embodiments of Formula I or Formula III. Conventionally, ADCs are linked to antibodies or fragments thereof through lysine or cysteine amine or sulfhydrylgroups either by random or site-directed conjugation methods. Common linkages contemplated in the present disclosure include THIOMAB, non-natural amino acid-based, non-cleavable, cleavable (chemical or protease- or ^-glucuronic acid-based), and enzymatically mediated conjugation linkages. Thus, various drug-antibody ratios (DARs) are also contemplated in the present disclosure, such as a DAR of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8; a DAR of 1-2, 1-3, 1-4, 1-5, 1-6, 2-6, 1-7, 2-7, or 1-8; or an average DAR of 1, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or 8.

[0052] Additional linkages that are useful in embodiments of ADCs of the present disclosure can be found in US Patent Publication No. US10617773B2 and Antibody-Mediated Drug Delivery Systems (Pathak and Benita, eds., 2012), both of which are incorporated by reference in their entireties on November 30, 2023.

[0053] In embodiments, the drug delivery nanoparticle (B) of Formula II or Formula IV comprises polymers, a mixture of polymers and lipids, or lipids. Polymers contemplated in the present disclosure include, but are not limited to, polyethylenimine (PEI), poly(lactic acid) (PLA), poly(glutamic acid) (PGLA), poly(cyanoacrylate), chitosan, poly(ethylene glycol) (PEG), poly(propylene glycol) (PPG), and derivatives thereof. Lipids contemplated in the present disclosure include, but are not limited to, cholesterols or saturated or unsaturated phospholipids, which can be functionalized with various polymers, such as PEG and derivatives thereof.

[0054] In embodiments, the drug delivery nanoparticle (B) comprises a PLA-PEG-PPG-PEG tetra- block copolymer. Additional nanoparticles that are useful in embodiments of ADCs of the present disclosure can be found in US Patent Publication No. US20200046648A1, which is incorporated by reference in its entirety on November 30, 2023.

[0055] In embodiments, the antibody or fragment thereof that binds MUC1-C (A) is linked to the drug delivery nanoparticle (B) of Formula II. In embodiments, the antibody means for binding MUC1-C (A) is linked to the drug delivery nanoparticle (B) of Formula IV. Various linkages are possible for embodiments of Formula I or Formula III. Conventionally, nanoparticles are linked to antibodies or fragments thereof through lysine or cysteine amine or sulfhydryl groups or carbohydrates either by random or site-directed conjugation methods. Common linkages contemplated in the present disclosure include THIOMAB, non-natural amino acid-based, non- cleavable, cleavable (chemical or protease- or ^-glucuronic acid-based), and enzymatically mediated conjugation linkages.

[0056] Additional linkages that are useful in embodiments of ADCs of the present disclosure can be found in US Patent Publication No. US10617773B2 and Antibody-Mediated Drug Delivery Systems (Pathak and Benita, eds., 2012), both of which are incorporated by reference in their entireties on November 30, 2023.

[0057] The ADCs of the present disclosure provides a salinomycin, a derivative thereof, or a pharmaceutically acceptable salt thereof (S). In embodiments, the salinomycin, a derivative thereof, or a pharmaceutically acceptable salt thereof (S) is a salinomycin. Salinomycin is an ionophoric monocarboxylic polyether as follows, which, as provided by the present disclosure, decreases MUC1-C expression:Salinomycin (2R)-2-[(2R,5S,6R)-6-[(2S,3S,4S,6R)-6-[(3S,5S,7R,9S,10S,12R,15R)-3-[(2R,5R,6S)-5-ethyl-5- hydroxy-6-methyloxan-2-yl]-15-hydroxy-3,10,12-trimethyl-4,6,8-trioxadispiro[4.1.57.35]pentadec- 13-en-9-yl]-3-hydroxy-4-methyl-5-oxooctan-2-yl]-5-methyloxan-2-yl]butanoic acid

[0058] In embodiments, the salinomycin, a derivative thereof, or a pharmaceutically acceptable salt thereof (S) decreases MUC1-C expression in cancer cells. In embodiments, the salinomycin, a derivative thereof, or a pharmaceutically acceptable salt thereof (S) decreases MUC1-C expression in cancers that express MUC1-C on their cell surface, renal cancer, leukemia, acute myeloid leukemia, lymphoma, cutaneous T cell lymphoma, multiple myeloma, breast cancer, triple negative breast cancer, Merkel cell carcinoma, small cell lung cancer, non-small cell lung cancer, castration- resistant prostate cancer, neuroendocrine prostate cancer, urothelial carcinoma, or colorectal cancer. A variety of salinomycin derivatives, which can also be referred to as salinomycin analogues, are contemplated by the present disclosure. In embodiments, the salinomycin derivative comprises ameans for decreasing MUC1-C expression. In embodiments, the salinomycin derivative decreases MUC1-C expression in cancer cells. In embodiments, the salinomycin derivative decreases MUC1- C expression in cancers that express MUC1-C on their cell surface, renal cancer, leukemia, acute myeloid leukemia, lymphoma, cutaneous T cell lymphoma, multiple myeloma, breast cancer, triple negative breast cancer, Merkel cell carcinoma, small cell lung cancer, non-small cell lung cancer, castration-resistant prostate cancer, neuroendocrine prostate cancer, urothelial carcinoma, or colorectal cancer. In embodiments, the salinomycin, a derivative thereof, or a pharmaceutically acceptable salt thereof (S) is ironomycin, as follows:Ironomycin (2R)-2-[(2R,5S,6R)-6-[(2S,3S,4S,6R)-6-[(3S,5S,7R,9S,10S,12R,15R)-3-[(2R,5R,6S)-5-ethyl-5- hydroxy-6-methyloxan-2-yl]-3,10,12-trimethyl-15-(prop-2-ynylamino)-4,6,8- trioxadispiro[4.1.57.35]pentadec-13-en-9-yl]-3-hydroxy-4-methyl-5-oxooctan-2-yl]-5-methyloxan-2- yl]butanoic acid

[0059] Additional SAL derivatives that are useful in embodiments of ADCs of the present disclosure can be found in US Patent Publication Nos. US10287298B2, US10364253B2, and US20220402932A1 as well as PCT Patent Publication No. WO2022187849A1, each of which is incorporated by reference in its entirety on November 30, 2023.

[0060] Additional combinations with the salinomycin, a derivative thereof, or a pharmaceutically acceptable salt thereof (S) are also contemplated herein. In embodiments, the drug delivery nanoparticle (B) of Formula II or Formula IV comprises salinomycin, a derivative thereof, or a pharmaceutically acceptable salt thereof (S) and a small molecule drug that targets CSCs. In embodiments, the drug delivery nanoparticle (B) of Formula II or Formula IV comprises salinomycin, a derivative thereof, or a pharmaceutically acceptable salt thereof (S) and a genotoxic drug. In embodiments, the drug delivery nanoparticle (B) of Formula II or Formula IV comprises salinomycin, a derivative thereof, or a pharmaceutically acceptable salt thereof (S) and tamoxifen. Inembodiments, the drug delivery nanoparticle (B) of Formula II or Formula IV comprises salinomycin, a derivative thereof, or a pharmaceutically acceptable salt thereof (S) and afatinib. In embodiments, the drug delivery nanoparticle (B) of Formula II or Formula IV comprises salinomycin, a derivative thereof, or a pharmaceutically acceptable salt thereof (S) and vemurafenib. In embodiments, the drug delivery nanoparticle (B) of Formula II or Formula IV comprises salinomycin, a derivative thereof, or a pharmaceutically acceptable salt thereof (S) and osimertinib. Antibodies of Use in the Antibody-Drug Conjugates of the Present Disclosure

[0061] In embodiments, the antibody or fragment thereof binds MUC1-C. In embodiments, the antibody or fragment thereof binds to MUC1-C extracellular domain (ECD; SEQ ID NO: 57): SVVVQLTLAFREGTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGAG (SEQ ID NO: 57, MUC1-C / ECD)

[0062] In embodiments, the antibody-drug conjugate of any one of claim 6, wherein antibody or fragment thereof binds the MUC1-C / ECD at the ^3 helix (VHDVETQFNQY, SEQ ID NO: 58) or ^4 helix (TEAASPYRN, SEQ ID NO: 59).

[0063] In embodiments, the antibody or fragment thereof comprises a variable region and a constant region, wherein the variable region comprises framework regions and a complementary determining means for binding MUC1-C ECD (SEQ ID NO: 57). In embodiments, the complementary determining means for binding comprises binding SEQ ID NO: 57, 58, or 59. In embodiments, the constant region is IgG or IgM. In embodiments, the constant region is IgG. In embodiments, the constant region is IgG1. In embodiments, the constant region is IgG2. In embodiments, the constant region is IgG2a.

[0064] In embodiments, the antibody or fragment thereof comprises heavy chain (HC) variable region (HCVR) and a light chain (LC) variable region (LCVR). In embodiments, the HCVR comprises the complementarity determining regions (CDRs) HCDR1, HCDR2, and HCDR3. In embodiments, the LCVR further comprises CDRs LCDR1, LCDR2, and LCDR3. Table 1 includes example sequences that can be used in the present disclosure:Table 1. Example sequences for use in the ADCs of the present disclosure.^^^^^^

[0065] In embodiments, the amino acid sequence of the HCVR is SEQ ID NO: 1, and the amino acid sequence of the LCVR is SEQ ID NO: 2. In embodiments, the amino acid sequence of the HCVR is SEQ ID NO: 3, and the amino acid sequence of the LCVR is SEQ ID NO: 4. In embodiments, the amino acid sequence of the HCVR is SEQ ID NO: 5, and the amino acid sequence of the LCVR is SEQ ID NO: 6. In embodiments, the amino acid sequence of the HCVR is SEQ ID NO: 7, and the amino acid sequence of the LCVR is SEQ ID NO: 8. In embodiments, the amino acid sequence of the HCVR is SEQ ID NO: 9, and the amino acid sequence of the LCVR is SEQ ID NO: 10. In embodiments, the amino acid sequence of the HCVR is SEQ ID NO: 11, and the amino acid sequence of the LCVR is SEQ ID NO: 12. In embodiments, the amino acid sequence of the HCVR is SEQ ID NO: 13, and the amino acid sequence of the LCVR is SEQ ID NO: 14.

[0066] In embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 15, the amino acid sequence of HCDR2 is SEQ ID NO: 16, the amino acid sequence of HCDR3 is SEQ ID NO: 17, the amino acid sequence of LCDR1 is SEQ ID NO: 18, the amino acid sequence of LCDR2 is SEQ ID NO: 19, and the amino acid sequence of LCDR3 is SEQ ID NO: 20. In embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 21, the amino acid sequence of HCDR2 is SEQ ID NO:22, the amino acid sequence of HCDR3 is SEQ ID NO: 23, the amino acid sequence of LCDR1 is SEQ ID NO: 24, the amino acid sequence of LCDR2 is SEQ ID NO: 25, and the amino acid sequence of LCDR3 is SEQ ID NO: 26. In embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 27, the amino acid sequence of HCDR2 is SEQ ID NO: 28, the amino acid sequence of HCDR3 is SEQ ID NO: 29, the amino acid sequence of LCDR1 is SEQ ID NO: 30, the amino acid sequence of LCDR2 is SEQ ID NO: 31, and the amino acid sequence of LCDR3 is SEQ ID NO: 32. In embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 33, the amino acid sequence of HCDR2 is SEQ ID NO: 34, the amino acid sequence of HCDR3 is SEQ ID NO: 35, the amino acid sequence of LCDR1 is SEQ ID NO: 36, the amino acid sequence of LCDR2 is SEQ ID NO: 37, and the amino acid sequence of LCDR3 is SEQ ID NO: 38. In embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 39, the amino acid sequence of HCDR2 is SEQ ID NO: 40, the amino acidsequence of HCDR3 is SEQ ID NO: 41, the amino acid sequence of LCDR1 is SEQ ID NO: 42, the amino acid sequence of LCDR2 is SEQ ID NO: 43, and the amino acid sequence of LCDR3 is SEQ ID NO: 44. In embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 45, the amino acid sequence of HCDR2 is SEQ ID NO: 46, the amino acid sequence of HCDR3 is SEQ ID NO: 47, the amino acid sequence of LCDR1 is SEQ ID NO: 48, the amino acid sequence of LCDR2 is SEQ ID NO: 49, and the amino acid sequence of LCDR3 is SEQ ID NO: 50. In embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 51, the amino acid sequence of HCDR2 is SEQ ID NO: 52, the amino acid sequence of HCDR3 is SEQ ID NO: 53, the amino acid sequence of LCDR1 is SEQ ID NO: 54, the amino acid sequence of LCDR2 is SEQ ID NO: 55, and the amino acid sequence of LCDR3 is SEQ ID NO: 56.

[0067] In embodiments, the antibody or fragment thereof comprises an antibody fragment. In embodiments, antibody fragment comprises a F(ab). In embodiments, the F(ab) comprises an HCVR and a LCVR. In embodiments, the HCVR comprises the CDRs HCDR1, HCDR2, and HCDR3. In embodiments, the LCVR comprises LCDRs LCDR1, LCDR2, and LCDR3. In embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 15, the amino acid sequence of HCDR2 is SEQ ID NO: 16, the amino acid sequence of HCDR3 is SEQ ID NO: 17, the amino acid sequence of LCDR1 is SEQ ID NO: 18, the amino acid sequence of LCDR2 is SEQ ID NO: 19, and the amino acid sequence of LCDR3 is SEQ ID NO: 20. In embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 21, the amino acid sequence of HCDR2 is SEQ ID NO:22, the amino acid sequence of HCDR3 is SEQ ID NO: 23, the amino acid sequence of LCDR1 is SEQ ID NO: 24, the amino acid sequence of LCDR2 is SEQ ID NO: 25, and the amino acid sequence of LCDR3 is SEQ ID NO: 26. In embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 27, the amino acid sequence of HCDR2 is SEQ ID NO: 28, the amino acid sequence of HCDR3 is SEQ ID NO: 29, the amino acid sequence of LCDR1 is SEQ ID NO: 30, the amino acid sequence of LCDR2 is SEQ ID NO: 31, and the amino acid sequence of LCDR3 is SEQ ID NO: 32. In embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 33, the amino acid sequence of HCDR2 is SEQ ID NO: 34, the amino acid sequence of HCDR3 is SEQ ID NO: 35, the amino acid sequence of LCDR1 is SEQ ID NO: 36, the amino acid sequence of LCDR2 is SEQ ID NO: 37, and the amino acid sequence of LCDR3 is SEQ ID NO: 38. In embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 39, the amino acid sequence of HCDR2 is SEQ ID NO: 40, the amino acid sequence of HCDR3 is SEQ ID NO: 41, the amino acid sequence of LCDR1 is SEQ ID NO: 42, the amino acid sequence of LCDR2 is SEQ IDNO: 43, and the amino acid sequence of LCDR3 is SEQ ID NO: 44. In embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 45, the amino acid sequence of HCDR2 is SEQ ID NO: 46, the amino acid sequence of HCDR3 is SEQ ID NO: 47, the amino acid sequence of LCDR1 is SEQ ID NO: 48, the amino acid sequence of LCDR2 is SEQ ID NO: 49, and the amino acid sequence of LCDR3 is SEQ ID NO: 50. In embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 51, the amino acid sequence of HCDR2 is SEQ ID NO: 52, the amino acid sequence of HCDR3 is SEQ ID NO: 53, the amino acid sequence of LCDR1 is SEQ ID NO: 54, the amino acid sequence of LCDR2 is SEQ ID NO: 55, and the amino acid sequence of LCDR3 is SEQ ID NO: 56.

[0068] In embodiments, the antibody fragment comprises an Fv or scFv. In embodiments, the Fv or scFv comprises an HCVR and a LCVR. In embodiments, the HCVR comprises the CDRs HCDR1, HCDR2, and HCDR3. In embodiments, the LCVR comprises the CDRs LCDR1, LCDR2, and LCDR3. In embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 15, the amino acid sequence of HCDR2 is SEQ ID NO: 16, the amino acid sequence of HCDR3 is SEQ ID NO: 17, the amino acid sequence of LCDR1 is SEQ ID NO: 18, the amino acid sequence of LCDR2 is SEQ ID NO: 19, and the amino acid sequence of LCDR3 is SEQ ID NO: 20. In embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 21, the amino acid sequence of HCDR2 is SEQ ID NO:22, the amino acid sequence of HCDR3 is SEQ ID NO: 23, the amino acid sequence of LCDR1 is SEQ ID NO: 24, the amino acid sequence of LCDR2 is SEQ ID NO: 25, and the amino acid sequence of LCDR3 is SEQ ID NO: 26. In embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 27, the amino acid sequence of HCDR2 is SEQ ID NO: 28, the amino acid sequence of HCDR3 is SEQ ID NO: 29, the amino acid sequence of LCDR1 is SEQ ID NO: 30, the amino acid sequence of LCDR2 is SEQ ID NO: 31, and the amino acid sequence of LCDR3 is SEQ ID NO: 32. In embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 33, the amino acid sequence of HCDR2 is SEQ ID NO: 34, the amino acid sequence of HCDR3 is SEQ ID NO: 35, the amino acid sequence of LCDR1 is SEQ ID NO: 36, the amino acid sequence of LCDR2 is SEQ ID NO: 37, and the amino acid sequence of LCDR3 is SEQ ID NO: 38. In embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 39, the amino acid sequence of HCDR2 is SEQ ID NO: 40, the amino acid sequence of HCDR3 is SEQ ID NO: 41, the amino acid sequence of LCDR1 is SEQ ID NO: 42, the amino acid sequence of LCDR2 is SEQ ID NO: 43, and the amino acid sequence of LCDR3 is SEQ ID NO: 44. In embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 45, the amino acid sequence of HCDR2 is SEQ ID NO: 46, the amino acid sequence of HCDR3 is SEQ ID NO: 47, theamino acid sequence of LCDR1 is SEQ ID NO: 48, the amino acid sequence of LCDR2 is SEQ ID NO: 49, and the amino acid sequence of LCDR3 is SEQ ID NO: 50. In embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 51, the amino acid sequence of HCDR2 is SEQ ID NO: 52, the amino acid sequence of HCDR3 is SEQ ID NO: 53, the amino acid sequence of LCDR1 is SEQ ID NO: 54, the amino acid sequence of LCDR2 is SEQ ID NO: 55, and the amino acid sequence of LCDR3 is SEQ ID NO: 56.

[0069] In embodiments, the antibody fragment comprises an scFv. In embodiments, the antibody fragment comprises a humanized scFv. In embodiments, the humanized scFv comprises SEQ ID NO: 60. In embodiments, the humanized scFv comprises SEQ ID NO: 61. In embodiments, the humanized scFv comprises SEQ ID NO: 62. In embodiments, the humanized scFv comprises SEQ ID NO: 63. In embodiments, the humanized scFv comprises SEQ ID NO: 64. In embodiments, the humanized scFv comprises SEQ ID NO: 65. In embodiments, the humanized scFv comprises SEQ ID NO: 66. In embodiments, the humanized scFv comprises SEQ ID NO: 67. In embodiments, the humanized scFv comprises SEQ ID NO: 68. In embodiments, the humanized scFv comprises SEQ ID NO: 69. In embodiments, the humanized scFv comprises SEQ ID NO: 70. In embodiments, the humanized scFv comprises SEQ ID NO: 71.

[0070] In embodiments, the antibody fragment comprises a VhH. In embodiments, the VhH comprises an HCVR. In embodiments, the HCVR comprises the CDRs HCDR1, HCDR2, and HCDR3. In embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 15, the amino acid sequence of HCDR2 is SEQ ID NO: 16, and the amino acid sequence of HCDR3 is SEQ ID NO: 17. In embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 21, the amino acid sequence of HCDR2 is SEQ ID NO:22, and the amino acid sequence of HCDR3 is SEQ ID NO: 23. In embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 27, the amino acid sequence of HCDR2 is SEQ ID NO: 28, and the amino acid sequence of HCDR3 is SEQ ID NO: 29. In embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 33, the amino acid sequence of HCDR2 is SEQ ID NO: 34, and the amino acid sequence of HCDR3 is SEQ ID NO: 35. In embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 39, the amino acid sequence of HCDR2 is SEQ ID NO: 40, and the amino acid sequence of HCDR3 is SEQ ID NO: 41. In embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 45, the amino acid sequence of HCDR2 is SEQ ID NO: 46, and the amino acid sequence of HCDR3 is SEQ ID NO: 47. In embodiments, the amino acid sequence of HCDR1 is SEQ ID NO: 51, the amino acid sequence ofHCDR2 is SEQ ID NO: 52, and the amino acid sequence of HCDR3 is SEQ ID NO: 53.

[0071] Additional antibodies and fragments thereof that are useful in embodiments of ADCs of the present disclosure can be found in US Patent Publication Nos. US10617773B2, US10059775B2, and US20230265208A1, each of which is incorporated by reference in its entirety on November 30, 2023. Methods of Treating and Using Compositions of the Present Disclosure

[0072] The present disclosure further provides pharmaceutical compositions comprising an ADC of the present disclosure and one or more pharmaceutically acceptable carriers, diluents, or excipients. Further, the present disclosure provides a method of treating cancer, comprising administering to a patient in need thereof a pharmaceutical composition comprising an ADC of the present disclosure. In another embodiment, the present disclosure provides a method of treating cancer, comprising administering an effective amount of an ADC of the present disclosure. Particularly, the present disclosure provides a method of treating a MUC1-positive cancer, selected from cancers that express MUC1-C on their cell surface, renal cancer, leukemia, acute myeloid leukemia, lymphoma, cutaneous T cell lymphoma, multiple myeloma, breast cancer, triple negative breast cancer, Merkel cell carcinoma, small cell lung cancer, non-small cell lung cancer, castration-resistant prostate cancer, neuroendocrine prostate cancer, urothelial carcinoma, or colorectal cancer, comprising administering to said patient an effective amount of an ADC of the present disclosure. Particularly, the present disclosure provides a method of treating a MUC1-positive cancer, selected from cancers that express MUC1-C on their cell surface, renal cancer, leukemia, acute myeloid leukemia, lymphoma, cutaneous T cell lymphoma, multiple myeloma, breast cancer, triple negative breast cancer, Merkel cell carcinoma, small cell lung cancer, non-small cell lung cancer, castration-resistant prostate cancer, neuroendocrine prostate cancer, urothelial carcinoma, or colorectal cancer, comprising administering to said patient an effective amount of a pharmaceutical composition comprising an ADC of the present disclosure.

[0073] In another embodiment, the present disclosure provides a method of inducing ferroptosis in a subject, comprising administering to the subject a pharmaceutical composition comprising an ADC of the present disclosure. In another embodiment, the present disclosure provides a method of inducing ferroptosis in a subject, comprising administering to the subject an effective amount of an ADC of the present disclosure. Particularly, the present disclosure provides a method of inducingferroptosis in a patient with a MUC1-positive cancer, selected from cancers that express MUC1-C on their cell surface, renal cancer, leukemia, acute myeloid leukemia, lymphoma, cutaneous T cell lymphoma, multiple myeloma, breast cancer, triple negative breast cancer, Merkel cell carcinoma, small cell lung cancer, non-small cell lung cancer, castration-resistant prostate cancer, neuroendocrine prostate cancer, urothelial carcinoma, or colorectal cancer, comprising administering to said patient an effective amount of an ADC of the present disclosure. Particularly, the present disclosure provides a method of inducing ferroptosis in a subject with MUC1-positive cancer, selected from cancers that express MUC1-C on their cell surface, renal cancer, leukemia, acute myeloid leukemia, lymphoma, cutaneous T cell lymphoma, multiple myeloma, breast cancer, triple negative breast cancer, Merkel cell carcinoma, small cell lung cancer, non-small cell lung cancer, castration-resistant prostate cancer, neuroendocrine prostate cancer, urothelial carcinoma, or colorectal cancer, comprising administering to said patient an effective amount of a pharmaceutical composition comprising an ADC of the present disclosure.

[0074] In another embodiment, the present disclosure provides a method of inhibiting cancer stem cell self-renewal capacity in a patent, comprising administering to a patient in need thereof a pharmaceutical composition comprising an ADC of the present disclosure. In another embodiment, the present disclosure provides a method of inhibiting cancer stem cell self-renewal capacity in a patent, comprising administering to the patient an effective amount of an ADC of the present disclosure. Particularly, the present disclosure provides a method of inhibiting cancer stem cell self- renewal capacity in a patient with a MUC1-positive cancer, selected from cancers that express MUC1-C on their cell surface, renal cancer, leukemia, acute myeloid leukemia, lymphoma, cutaneous T cell lymphoma, multiple myeloma, breast cancer, triple negative breast cancer, Merkel cell carcinoma, small cell lung cancer, non-small cell lung cancer, castration-resistant prostate cancer, neuroendocrine prostate cancer, urothelial carcinoma, or colorectal cancer, comprising administering to said patient an effective amount of an ADC of the present disclosure. Particularly, the present disclosure provides a method of inhibiting cancer stem cell self-renewal capacity in a patent with a MUC1-positive cancer, selected from cancers that express MUC1-C on their cell surface, renal cancer, leukemia, acute myeloid leukemia, lymphoma, cutaneous T cell lymphoma, multiple myeloma, breast cancer, triple negative breast cancer, Merkel cell carcinoma, small cell lung cancer, non-small cell lung cancer, castration-resistant prostate cancer, neuroendocrine prostate cancer, urothelial carcinoma, or colorectal cancer, comprising administering to said patient aneffective amount of a pharmaceutical composition comprising an ADC of the present disclosure.

[0075] In another embodiment, the present disclosure provides a method of decreasing tumor cell viability in a patient, comprising administering to a patient in need thereof a pharmaceutical composition comprising an ADC of the present disclosure. In another embodiment, the present disclosure provides a method of decreasing tumor cell viability in a patient, comprising administering an effective amount of an ADC of the present disclosure. Particularly, the present disclosure provides a method of decreasing tumor cell viability in a patient with a MUC1-positive cancer, selected from cancers that express MUC1-C on their cell surface, renal cancer, leukemia, acute myeloid leukemia, lymphoma, cutaneous T cell lymphoma, multiple myeloma, breast cancer, triple negative breast cancer, Merkel cell carcinoma, small cell lung cancer, non-small cell lung cancer, castration-resistant prostate cancer, neuroendocrine prostate cancer, urothelial carcinoma, or colorectal cancer, comprising administering to said patient an effective amount of an ADC of the present disclosure. Particularly, the present disclosure provides a method of decreasing tumor cell viability in a patient with a MUC1-positive cancer, selected from cancers that express MUC1-C on their cell surface, renal cancer, leukemia, acute myeloid leukemia, lymphoma, cutaneous T cell lymphoma, multiple myeloma, breast cancer, triple negative breast cancer, Merkel cell carcinoma, small cell lung cancer, non-small cell lung cancer, castration-resistant prostate cancer, neuroendocrine prostate cancer, urothelial carcinoma, or colorectal cancer, comprising administering to said patient an effective amount of a pharmaceutical composition comprising an ADC of the present disclosure.

[0076] In another embodiment, the present disclosure provides a method of reversing resistance to anti-cancer therapy in a patient, comprising administering to a patient in need thereof a pharmaceutical composition comprising an ADC of the present disclosure. In another embodiment, the present disclosure provides a method of reversing resistance to anti-cancer therapy in a patient, comprising administering an effective amount of an ADC of the present disclosure. Particularly, the present disclosure provides a method of decreasing tumor cell viability in a patient with a MUC1- positive cancer, selected from cancers that express MUC1-C on their cell surface, renal cancer, leukemia, acute myeloid leukemia, lymphoma, cutaneous T cell lymphoma, multiple myeloma, breast cancer, triple negative breast cancer, Merkel cell carcinoma, small cell lung cancer, non-small cell lung cancer, castration-resistant prostate cancer, neuroendocrine prostate cancer, urothelial carcinoma, or colorectal cancer, comprising administering to said patient an effective amount of anADC of the present disclosure. Particularly, the present disclosure provides a method of reversing resistance to anti-cancer therapy in a patient with a MUC1-positive cancer, selected from cancers that express MUC1-C on their cell surface, renal cancer, leukemia, acute myeloid leukemia, lymphoma, cutaneous T cell lymphoma, multiple myeloma, breast cancer, triple negative breast cancer, Merkel cell carcinoma, small cell lung cancer, non-small cell lung cancer, castration-resistant prostate cancer, neuroendocrine prostate cancer, urothelial carcinoma, or colorectal cancer, comprising administering to said patient an effective amount of a pharmaceutical composition comprising an ADC of the present disclosure.

[0077] The present disclosure also provides an ADC of the present disclosure for use in therapy. In an embodiment, the present disclosure provides an ADC of the present disclosure for use in the treatment of cancer. ln another embodiment, the present disclosure provides an ADC of the present disclosure for use in treatment of MUC1-positive cancer, selected from cancers that express MUC1- C on their cell surface, renal cancer, leukemia, acute myeloid leukemia, lymphoma, cutaneous T cell lymphoma, multiple myeloma, breast cancer, triple negative breast cancer, Merkel cell carcinoma, small cell lung cancer, non-small cell lung cancer, castration-resistant prostate cancer, neuroendocrine prostate cancer, urothelial carcinoma, or colorectal cancer. The present disclosure also provides an ADC of the present disclosure for use in therapy. In an embodiment, the present disclosure provides a pharmaceutical composition comprising an ADC of the present disclosure for use in the treatment of cancer. ln another embodiment, the present disclosure provides an ADC of the present disclosure for use in treatment of MUC1-positive cancer, selected from cancers that express MUC1-C on their cell surface, renal cancer, leukemia, acute myeloid leukemia, lymphoma, cutaneous T cell lymphoma, multiple myeloma, breast cancer, triple negative breast cancer, Merkel cell carcinoma, small cell lung cancer, non-small cell lung cancer, castration-resistant prostate cancer, neuroendocrine prostate cancer, urothelial carcinoma, or colorectal cancer. In embodiments, the anti-cancer therapy is at least one genotoxic drug. In embodiments, the anti-cancer therapy is at least one of tamoxifen, trastuzumab, afatinib, vemurafenib, or osimertinib.

[0078] The present disclosure also provides an ADC of the present disclosure for use in inducing ferroptosis. In an embodiment, the present disclosure provides an ADC of the present disclosure for use in inducing ferroptosis. ln another embodiment, the present disclosure provides an ADC of the present disclosure for use in inducing ferroptosis in MUC1-positive cancer, selected from cancers that express MUC1-C on their cell surface, renal cancer, leukemia, acute myeloid leukemia,lymphoma, cutaneous T cell lymphoma, multiple myeloma, breast cancer, triple negative breast cancer, Merkel cell carcinoma, small cell lung cancer, non-small cell lung cancer, castration-resistant prostate cancer, neuroendocrine prostate cancer, urothelial carcinoma, or colorectal cancer. The present disclosure also provides an ADC of the present disclosure for use in inducing ferroptosis. In an embodiment, the present disclosure provides a pharmaceutical composition comprising an ADC of the present disclosure for use in inducing ferroptosis. ln another embodiment, the present disclosure provides an ADC of the present disclosure for use in inducing ferroptosis in MUC1- positive cancer, selected from cancers that express MUC1-C on their cell surface, renal cancer, leukemia, acute myeloid leukemia, lymphoma, cutaneous T cell lymphoma, multiple myeloma, breast cancer, triple negative breast cancer, Merkel cell carcinoma, small cell lung cancer, non-small cell lung cancer, castration-resistant prostate cancer, neuroendocrine prostate cancer, urothelial carcinoma, or colorectal cancer.

[0079] The present disclosure also provides an ADC of the present disclosure for use in inhibiting cancer stem cell self-renewal capacity. In an embodiment, the present disclosure provides an ADC of the present disclosure for use in inhibiting cancer stem cell self-renewal capacity. ln another embodiment, the present disclosure provides an ADC of the present disclosure for use in inhibiting cancer stem cell self-renewal capacity in MUC1-positive cancer, selected from cancers that express MUC1-C on their cell surface, renal cancer, leukemia, acute myeloid leukemia, lymphoma, cutaneous T cell lymphoma, multiple myeloma, breast cancer, triple negative breast cancer, Merkel cell carcinoma, small cell lung cancer, non-small cell lung cancer, castration-resistant prostate cancer, neuroendocrine prostate cancer, urothelial carcinoma, or colorectal cancer. The present disclosure also provides an ADC of the present disclosure for use in inhibiting cancer stem cell self- renewal capacity. In an embodiment, the present disclosure provides a pharmaceutical composition comprising an ADC of the present disclosure for use in inhibiting cancer stem cell self-renewal capacity. ln another embodiment, the present disclosure provides an ADC of the present disclosure for use in inhibiting cancer stem cell self-renewal capacity in MUC1-positive cancer, selected from cancers that express MUC1-C on their cell surface, renal cancer, leukemia, acute myeloid leukemia, lymphoma, cutaneous T cell lymphoma, multiple myeloma, breast cancer, triple negative breast cancer, Merkel cell carcinoma, small cell lung cancer, non-small cell lung cancer, castration-resistant prostate cancer, neuroendocrine prostate cancer, urothelial carcinoma, or colorectal cancer.

[0080] The present disclosure also provides an ADC of the present disclosure for use in decreasingtumor cell viability. In an embodiment, the present disclosure provides an ADC of the present disclosure for use in decreasing tumor cell viability. ln another embodiment, the present disclosure provides an ADC of the present disclosure for use in decreasing tumor cell viability in MUC1- positive cancer, selected from cancers that express MUC1-C on their cell surface, renal cancer, leukemia, acute myeloid leukemia, lymphoma, cutaneous T cell lymphoma, multiple myeloma, breast cancer, triple negative breast cancer, Merkel cell carcinoma, small cell lung cancer, non-small cell lung cancer, castration-resistant prostate cancer, neuroendocrine prostate cancer, urothelial carcinoma, or colorectal cancer. The present disclosure also provides an ADC of the present disclosure for use in decreasing tumor cell viability. In an embodiment, the present disclosure provides a pharmaceutical composition comprising an ADC of the present disclosure for use in decreasing tumor cell viability. ln another embodiment, the present disclosure provides an ADC of the present disclosure for use in decreasing tumor cell viability in MUC1-positive cancer, selected from cancers that express MUC1-C on their cell surface, renal cancer, leukemia, acute myeloid leukemia, lymphoma, cutaneous T cell lymphoma, multiple myeloma, breast cancer, triple negative breast cancer, Merkel cell carcinoma, small cell lung cancer, non-small cell lung cancer, castration- resistant prostate cancer, neuroendocrine prostate cancer, urothelial carcinoma, or colorectal cancer.

[0081] The present disclosure also provides an ADC of the present disclosure for use in reversing resistance to anti-cancer therapy. In an embodiment, the present disclosure provides an ADC of the present disclosure for use in reversing resistance to anti-cancer therapy. ln another embodiment, the present disclosure provides an ADC of the present disclosure for use in reversing resistance to anti- cancer therapy in MUC1-positive cancer, selected from cancers that express MUC1-C on their cell surface, renal cancer, leukemia, acute myeloid leukemia, lymphoma, cutaneous T cell lymphoma, multiple myeloma, breast cancer, triple negative breast cancer, Merkel cell carcinoma, small cell lung cancer, non-small cell lung cancer, castration-resistant prostate cancer, neuroendocrine prostate cancer, urothelial carcinoma, or colorectal cancer. The present disclosure also provides an ADC of the present disclosure for use in reversing resistance to anti-cancer therapy. In an embodiment, the present disclosure provides a pharmaceutical composition comprising an ADC of the present disclosure for use in reversing resistance to anti-cancer therapy. ln another embodiment, the present disclosure provides an ADC of the present disclosure for use in reversing resistance to anti-cancer therapy in MUC1-positive cancer, selected from cancers that express MUC1-C on their cell surface, renal cancer, leukemia, acute myeloid leukemia, lymphoma, cutaneous T cell lymphoma, multiplemyeloma, breast cancer, triple negative breast cancer, Merkel cell carcinoma, small cell lung cancer, non-small cell lung cancer, castration-resistant prostate cancer, neuroendocrine prostate cancer, urothelial carcinoma, or colorectal cancer. In embodiments, the anti-cancer therapy is at least one genotoxic drug. In embodiments, the anti-cancer therapy is at least one of tamoxifen, trastuzumab, afatinib, vemurafenib, or osimertinib.

[0082] The methods and uses herein further contemplate combination therapies. The combination therapy may provide synergy and prove synergistic, that is, the effect achieved when the active ingredients used together is greater than the sum of the effects that results from using the compounds separately. A synergistic effect may be attained when the active ingredients are: (1) coformulated and administered or delivered simultaneously in a combined, unit dosage formulation; (2) delivered by alternation or in parallel as separate formulations; or (3) by some other regimen. In embodiments, an ADC of the present disclosure is administered as a combination therapy with a biologic that targets CSCs or small molecule drug that targets CSCs. In embodiments, an ADC of the present disclosure is administered as a combination therapy with a genotoxic biologic or a genotoxic small molecule drug. In embodiments, an ADC of the present disclosure is administered as a combination therapy with tamoxifen. In embodiments, an ADC of the present disclosure is administered as a combination therapy with trastuzumab. In embodiments, an ADC of the present disclosure is administered as a combination therapy with afatinib. In embodiments, an ADC of the present disclosure is administered as a combination therapy with vemurafenib. In embodiments, an ADC of the present disclosure is administered as a combination therapy with osimertinib. ^ EXAMPLES

[0083] Example 1. This example shows embodiments in which SAL downregulates MUC1-C expression in human cancer cells. Treatment of DU-145 CRPC cells with increasing SAL concentrations suppresses their viability (IC50=0.45 ^M) (Fig. 1A). Similar results were observed with H660 NEPC cells; that is, a SAL concentration- dependent loss of viability (IC50=0.15 ^M) (Fig.1B). SAL treatment of DU-145 and H660 cells was associated with decreases in MUC1-C transcripts (Fig. 1C, left and right). SAL suppressed MUC1-C and NF-^B expression in DU-145 and H660 cells (Fig. 1D, left and right). Silencing NF-^B decreased MUC1-C transcripts and protein (Figs. 1E and 1F). Moreover, treatment with the NF-^B inhibitor BAY11-7082 suppressed MUC1-C expression (Figs 1G and 1H), confirming that SAL inhibits the NF-^B / MUC1-C auto-inductivepathway. SAL also downregulated MUC1-C expression in BT-549 and MDA-MB-436 TNBC cells (Figs. 1I and 1J), demonstrating this response in a variety of cancer cell types.

[0084] Example 2. This example shows embodiments in which SAL drives ferroptosis by downregulating MUC1-C expression. Treatment of DU-145 cells with SAL was associated with induction of lipid peroxidation as evidenced by staining with the ratiometric lipid peroxidase sensor (Fig.2A). Consistent with these results, SAL increased cell surface expression of the transferrin receptor 1 (TfR1) marker of ferroptosis 35 (Fig.2B). MUC1-C was silenced in DU-145 and H660 cells (Figs.2H-2I), and increases in lipid peroxidation were detected (Fig. 2C and 2J). In addition, silencing MUC1-C increased SAL-induced lipid peroxidation (Fig.2D and 2K) and TfR1 expression (Fig.2E). MUC1-C downregulation was rescued with expression of the MUC1-C cytoplasmic domain (tet- Flag-MUC1-C / CD), which, unlike the endogenous MUC1 gene, is under control of a tet-promoter (Fig.2F). Rescue of MUC1-C expression suppressed the effects of silencing MUC1-C alone and in combination with SAL on the induction of ferroptosis (Fig. 2G) and cell death (Fig. 2L). Thus, without being bound by theory, SAL induces ferroptosis, at least in large part, by suppressing MUC1-C expression.

[0085] Example 3. This example shows embodiments in which MUC1-C induces GSR expression and GSH production. Glutathione-disulfide reductase (GSR) catalyzes the reduction of glutathione disulfide (GSSG) with NADPH as the electron donor. In this way, GSR generates GSH, which confers resistance to ferroptosis. The present disclosure shows that SAL treatment decreased GSR mRNA and protein levels (Fig.3A). Moreover, silencing MUC1-C suppressed GSR expression (Fig. 3B and 3H-3I). ATAC-seq revealed dependence on MUC1-C for increasing chromatin accessibility of the GSR promoter region (Fig. 3C). In addition, silencing MUC1-C decreased GSR transcription (Fig.3D). MYC binding motifs were identified in the GSR promoter. Silencing MYC also suppressed GSR expression in concert with MUC1-C to MYC to GSR signaling (Fig. 3E) Silencing MUC1-C decreased GSH levels (Fig.3F and 3J). Rescuing MUC1-C silencing with MUC1-C / CD reestablished expression of GSR transcripts and protein (Fig.3G).

[0086] Example 4. This example shows embodiments in which MUC1-C signaling regulates LRP8 and GPX4 expression. The present disclosure shows that SAL treatment of DU-145 and H660 cells decreases LRP8 expression (Fig. 4A and 4I). Silencing MUC1-C in these cells also downregulated LRP8 transcripts and protein (Fig.4B and 4J-4K). Silencing MUC1-C decreased LRP8 gene chromatin accessibility (Fig. 4C) and transcription (Fig. 4D). MYC binding motifs were observed inthe LRP8 promoter region, and silencing MYC downregulated LRP8 expression (Fig. 4E). Silencing MUC1-C had little if any effect on GPX4 mRNA levels (Fig.4L), but decreased expression of the GPX4 protein (Fig. 4F and 4M). Without being bound by theory, these data demonstrate a post- transcriptional effect. Silencing MUC1-C was also associated with decreased GPX activity (Fig. 4G and 4N). Moreover, rescue of MUC1-C silencing with MUC1-C / CD reversed the suppression of LRP8 and GPX4 expression (Fig.4H). Without being bound by theory, these findings demonstrate that MUC1-C drives (i) GSR and LRP8 transcription by MYC-mediated activation and, thereby, (ii) the regulation of GPX4 translation and activity.

[0087] Example 5. This example shows embodiments in which SAL-inhibited signaling is phenocopied by targeting MUC1-C with the GO-203 inhibitor. Cells were treated with the GO-203 inhibitor. GO-203 treatment decreased DU-145 cell survival in a concentration-dependent manner (Fig.5A), which was associated with induction of ferroptosis as evidenced by lipid peroxidation (Fig.5B) and TfR1 expression (Fig. 5C). Similar results were obtained in H660 cells (Figs.5H and 5I), confirming that, like silencing MUC1-C, GO-203 induces ferroptosis. GO-203 treatment also downregulated (i) GSR transcripts and protein (Fig. 5D and 5K) and (ii) GSH levels (Fig. 5E and 5K). Moreover, GO-203 decreased (ii) LRP8 and GPX4 expression (Fig. 5F and 5L) and GPX activity (Fig. 5G and 5M). These findings indicate that GO-203 phenocopies SAL-mediated MUC1- C downregulation and silencing MUC1-C genetically in inducing ferroptosis.

[0088] Example 6. This example shows embodiments of the effects of SAL and targeting MUC1-C on CSC ferroptosis. DU-145 CSCs were enriched by serial passage (S1 to S13) of tumorspheres, which was associated with progressive increases in sphere forming efficiency (SFE) (Fig. 6A). Treatment of the enriched CSCs with SAL downregulated expression of MUC1-C and the PC stem cell marker CD13343 (Fig.6G). SAL treatment of the CSCs also decreased self-renewal capacity (Fig.6H), which was abrogated by the ferroptosis inhibitor Ferrostatin-1 (Fer-1) (Fig. 6I). Silencing MUC1-C in DU-145 CSCs similarly decreased self-renewal (Fig. 6B), which was blocked by Fer-1 (Fig.6C). Importantly, rescue of MUC1-C silencing with MUC1-C / CD reversed suppression of self- renewal (Fig. 6D). Treatment with GO-203 was associated with suppression of NF-^B (Fig. 6J), and NF-^B induced tumorsphere formation (Fig.6K). Targeting CSCs with GO-203 was also associated with loss of self-renewal capacity (Fig. 6E) and induction of ferroptosis (Fig.6F). Without being bound by therapy, these data show that MUC1-C induces self-renewal and ferroptosis resistance in enriched CSCs.

[0089] Example 7. This example shows embodiments in which SAL downregulates MUC1-C expression in association with suppression of tumorigenicity. DU-145 cells were treated with SAL / polymeric nanoparticles (SAL / NPs; HSB-1216), from which a concentration- and time- dependent inhibition of viability was observed (Fig. 7A). By contrast, empty polymeric nanoparticles (NPs) had little if any effect (Supplemental Fig. S7A). As shown for SAL, treatment with SAL / NPs suppressed MUC1-C and NF-^B expression (Fig.7B). SAL / NPs also downregulated (i) GSR, LRP8, and GPX4 levels (Fig. 7B) and (ii) induced ferroptosis (Figs.7C and 7D). Treatment of DU-145 tumor xenografts with SAL / NPs, but not empty NPs, suppressed tumorigenicity in the absence of body weight loss or other overt toxicities (Fig.7E; Supplemental Fig. S7B). Analysis of tumors from SAL / NP-treated mice further showed decreased expression of MUC1-C and GPX4, a negative regulator of ferroptosis (Fig.7F). Without being bound by theory, these data show that MUC1-C is a target of SAL in tumors.

[0090] Example 8. This example shows the methods and materials used in Examples 1-7.

[0091] Cell culture. DU145 CRPC cells (ATCC) and NCI-H660 NEPC cells (ATCC) were cultured in RPMI1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS. BT-549 TNBC cells (ATCC) were cultured in RPMI1640 medium containing 10% FBS and 10 µg / mL insulin. MDA-MB-468 TNBC cells were cultured in Leibovitz's L-15 Medium (Thermo Fisher Scientific) supplemented with 10% FBS. Cells were treated with the MUC1-C inhibitor GO- 203, salinomycin (S8129, SelleckChem, Houston, TX, USA), BAY11-7082 (SelleckChem) and salinomycin encapsulated in polymeric nanoparticles (SAL / NPs, HSB- 1216; HillstreamBiopharma, Bridgewater, NJ, USA). Cell viability was assessed using the Alamar Blue assay (Thermo Scientific, Rockford, IL, USA) in sextuplicate wells. The IC50 value was determined by nonlinear regression of the dose–response data using Prism 9.0 (GraphPad Software). Authentication of the cells was performed by short tandem repeat (STR) analysis. Cells were monitored for mycoplasma contamination using the MycoAlert®Mycoplasma Detection Kit (Lonza, Rockland, MA, USA). Cells were maintained for 3 months for performing experiments.

[0092] Gene silencing and rescue vectors. MUC1shRNA (MISSION shRNA TRCN0000122938; Sigma, St. Louis, MO, USA), MYCshRNA (MISSION shRNA TRCN0000039642; Sigma) or a control scrambled shRNA (CshRNA; Sigma) was inserted into the pLKO-tet-puro vector (Plasmid #21915; Addgene, Cambridge, MA, USA). The MUC1shRNA#2 (MISSION shRNA TRCN0000430218) was produced in HEK293T cells as described 54. Flag-tagged MUC1-CD wasinserted into pInducer20 (Plasmid #44012, Addgene). Cells transduced with the vectors were selected for growth in 1–2 ^g / ml puromycin. Cells were treated with 0.1% DMSO as the vehicle control or 500 ng / ml DOX (Millipore Sigma, Burlington, MA, USA).

[0093] qRT-PCR. Total cellular RNA was isolated using Trizol reagent (Thermo Fisher Scientific). cDNAs were synthesized using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Grand Island, NY, USA). The cDNA samples were amplified using the PowerUp™ SYBR Green PCR Master Mix (Applied Biosystems) and the CFX96 Real-Time PCR System (BIO- RAD, Hercules, CA, USA).

[0094] Immunoblot analysis. Total lysates prepared from non-confluent cells were subjected to immunoblot analysis using anti-MUC1-C (HM- 1630-P1ABX, 1:1000 dilution; Thermo Fisher Scientific), anti-^-actin (A5441, 1:5000 dilution; Sigma-Aldrich), anti-GAPDH (#2118, 1:1000; CST), anti-p65 (ab32536, 1:1000 dilution, Abcam, Cambridge, MA, USA), anti-GPX4 (#52455, 1:1000 dilution, CST), anti-GSR (18257-1-AP, 1:2000 dilution; PROTEINTECH, Rosemont, IL, USA), anti-LRP8 (NB100-2216, 1:1000 dilution; Novus Biologicals, Centennial, CO, USA) and anti-CD133 (#5860, 1:1000 dilution, CST).

[0095] Lipid peroxidation assay. Ratiometric measurement of lipid peroxidation was performed using the Lipid Peroxidation Assay Kit (ab243377; Abcam) according to the manufacturer’s instructions. Cells were analyzed by MACSQuant Analyzer 10 Flow Cytometer (Miltenyi Biotec, Waltham, MA, USA). Measurement of PE / FITC ratio was performed with FlowJo v10.6.2 (BD Biosciences, Franklin Lakes, NJ, USA) software.

[0096] Flow cytometry. Cells were blocked by incubation with 1% BSA / PBS for 20 minutes on ice. After washing with ice cold PBS, cells were incubated with anti-TfR1 (CD71) antibody (MABC1765, clone 3F3-FMA, 1:100 dilution; Millipore Sigma) or an IgG1 isotype control antibody (MOPC-21, 1:100 dilution; BioLegend, San Diego, CA, USA) for 40 min on ice. FITC-conjugated goat F(ab)2 anti-mouse immunoglobulin was used as the secondary reagent (115-096-146, 1:100 dilution, Jackson ImmunoResearch, West Grove, PA, USA). Dead cells were stained with eBioscience 7-AAD viability staining solution (00-6993-50, Invitrogen). Cell death rates were measured using propidium iodide (PI) (Thermo Scientific). Cells were analyzed by MACSQuant Analyzer 10 Flow Cytometer (Miltenyi Biotec). Measurement of geometric MFI (gMFI) was performed with FlowJo v10.6.2 (BD Biosciences) software.

[0097] Gene transcription assays. Newly synthesized RNA transcripts from the already existingRNA was obtained using Click-iT Nascent RNA Capture Kit (C10365; Thermo Fisher Scientific) according to the manufacturer’s instructions. The captured transcripts were analyzed using qRT- PCR.

[0098] ATAC-seq. ATAC-seq libraries were generated from three biologically independent replicates per condition. Chromatin accessibility was assessed using Integrative Genomics Viewer (IGV_2.13.0).

[0099] Chromatin accessibility assay. DNase I chromatin accessibility assays were performed.

[0100] Measurement of GSH levels. GSH (GSH-Glo Glutathione Assay, V6911; Promega, Madison, WI, USA) levels were determined according to the manufacturer’s instructions. Luminescence intensity was detected using FLUOstar Omega plate reader (BMG LABTECH, Cary, NC, USA).

[0101] Measurement of GPX activity levels. Assays for measurements of GPX activity (Glutathione Peroxidase Assay Kit, MAK437; Millipore Sigma) levels were performed according to the manufacturer’s instructions. The GPX activity level was measured using FLUOstar Omega plate reader (BMG LABTECH).

[0102] Tumorsphere formation assays. Cells (2-6 x 103) were seeded per well in 6-well ultra-low attachment culture plates (Corning Life Sciences, Corning, NY, USA) in DMEM / F1250 / 50 medium (Corning Life Sciences) with 20 ng / ml EGF (Millipore Sigma), 20 ng / ml bFGF (Millipore Sigma) and 1% B27 supplement (Gibco). Tumorspheres were counted under an inverted microscope in triplicate wells.

[0103] Mouse tumor xenograft studies. Six- to 8-week old nude mice (Taconic Farms, Germantown, NY, USA) were injected subcutaneously in the flank with 1 x 107DU-145 cells in 100 µl of a 1:1 solution of medium and Matrigel (BD Biosciences). When the mean tumor volume reached 150-200 mm3, mice were pair-matched into groups of 6 mice each. Mice were treated intraperitoneally with SAL / NPs (5 mg SAL / kg) or an equivalent amount of empty NPS each week x 5 weeks. Mice were sacrificed on day 35 when control tumors reached >2000 mm3as calculated by the formula: (width)2x length / 2. The resource equation method was used to determine the minimum number of mice for achieving significance.

[0104] Statistics. Each experiment was performed at least three times. Data are expressed as the mean±SD. The unpaired Mann-Whitney U test was used to determine differences between means of groups. A p-value of <0.05 denoted by an asterisk (*) was considered statistically significant.EQUIVALENTS

[0105] Various aspects of the present disclosure may be used alone, in combination, or in a variety of arrangements not specifically discussed in the embodiments described in the foregoing and is, therefore, not limited in its application to the details and arrangement of components set forth in the foregoing description or illustrated in the drawings. For example, aspects described in one embodiment may be combined in any manner with aspects described in other embodiments.

Claims

CLAIMS What is claimed:

1. An antibody-drug conjugate of the formula: A –S (Formula I), wherein: A is an antibody or fragment thereof that binds MUC1-C; and S is SAL, a derivative thereof, or a pharmaceutically acceptable salt thereof.

2. An antibody-drug conjugate of the formula: A – B – S (Formula II), wherein: A is an antibody or fragment thereof that binds MUC1-C; B is a drug delivery nanoparticle; S is SAL, a derivative thereof, or a pharmaceutically acceptable salt thereof; and wherein the drug delivery nanoparticle encapsulates the SAL, derivative thereof, or pharmaceutically acceptable salt thereof.

3. An antibody-drug conjugate of the formula: A –S (Formula III), wherein: A is an antibody means for binding MUC1-C; and S is SAL, a derivative thereof, or a pharmaceutically acceptable salt thereof.

4. An antibody-drug conjugate of the formula: A – B – S (Formula IV), wherein: A is an antibody means for binding MUC1-C; B is a drug delivery nanoparticle; S is SAL, a derivative thereof, or a pharmaceutically acceptable salt thereof; and wherein the drug delivery nanoparticle encapsulates the SAL, derivative thereof, or pharmaceutically acceptable salt thereof.

5. The antibody-drug conjugate of claim 2 or claim 4, wherein the drug delivery nanoparticle comprises polymers, a mixture of polymers and lipids, or lipids.

6. The antibody-drug conjugate of claim 5, wherein the antibody or fragment thereof islinked to the polymer or lipid.

7. The antibody-drug conjugate of any one of claims 1-6, wherein the antibody or fragment thereof is as described herein.

8. The antibody-drug conjugate of any one of claims 1-7, comprising an antibody that binds MUC1-C.

9. The antibody-drug conjugate of any one of claims 1-7, comprising an antibody fragment that binds MUC1-C.

10. The antibody-drug conjugate of any one of claims 1-9, wherein the antibody or fragment thereof binds SEQ ID NO:

57.

11. The antibody-drug conjugate of any one of claim 10, wherein antibody or fragment thereof binds SEQ ID NO: 58 or SEQ ID NO:

59.

12. The antibody-drug conjugate of any one of claims 1-11, wherein the antibody or fragment thereof comprises a variable domain and a constant domain, wherein the variable domain comprises a framework region and a complementary determining means for binding SEQ ID NO:

57.

13. The antibody-drug conjugate claim 12, wherein the complementary determining means for binding comprises binding SEQ ID NO: 58 or SEQ ID NO:

59.

14. The antibody-drug conjugate of claim 6, wherein the constant region is IgG1 or IgG2.

15. The antibody-drug conjugate of any one of claims 1-13, wherein the antibody or fragment thereof comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the HCVR comprises the complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3 and the LCVR comprises CDRs LCDR1, LCDR2 and LCDR3, wherein: a. the amino acid sequence of HCDR1 is SEQ ID NO: 15, the amino acid sequence of HCDR2 is SEQ ID NO: 16, the amino acid sequence of HCDR3 is SEQ ID NO: 17, the amino acid sequence of LCDR1 is SEQ ID NO: 18, the amino acid sequence of LCDR2 is SEQ ID NO: 19, and the amino acid sequence of LCDR3 is SEQ ID NO: 20; b. the amino acid sequence of HCDR1 is SEQ ID NO: 21, the amino acid sequence of HCDR2 is SEQ ID NO: 22, the amino acid sequence of HCDR3 is SEQ ID NO: 23, the amino acid sequence of LCDR1 is SEQ ID NO: 24, the amino acid sequence ofLCDR2 is SEQ ID NO: 25, and the amino acid sequence of LCDR3 is SEQ ID NO: 26; c. the amino acid sequence of HCDR1 is SEQ ID NO: 27, the amino acid sequence of HCDR2 is SEQ ID NO: 28, the amino acid sequence of HCDR3 is SEQ ID NO: 29, the amino acid sequence of LCDR1 is SEQ ID NO: 30, the amino acid sequence of LCDR2 is SEQ ID NO: 31, and the amino acid sequence of LCDR3 is SEQ ID NO: 32; d. the amino acid sequence of HCDR1 is SEQ ID NO: 33, the amino acid sequence of HCDR2 is SEQ ID NO: 34, the amino acid sequence of HCDR3 is SEQ ID NO: 35, the amino acid sequence of LCDR1 is SEQ ID NO: 36, the amino acid sequence of LCDR2 is SEQ ID NO: 37, and the amino acid sequence of LCDR3 is SEQ ID NO: 38; e. the amino acid sequence of HCDR1 is SEQ ID NO: 39, the amino acid sequence of HCDR2 is SEQ ID NO: 40, the amino acid sequence of HCDR3 is SEQ ID NO: 41, the amino acid sequence of LCDR1 is SEQ ID NO: 42, the amino acid sequence of LCDR2 is SEQ ID NO: 43, and the amino acid sequence of LCDR3 is SEQ ID NO: 44; f. the amino acid sequence of HCDR1 is SEQ ID NO: 45, the amino acid sequence of HCDR2 is SEQ ID NO: 46, the amino acid sequence of HCDR3 is SEQ ID NO: 47, the amino acid sequence of LCDR1 is SEQ ID NO:48, the amino acid sequence of LCDR2 is SEQ ID NO: 49, and the amino acid sequence of LCDR3 is SEQ ID NO: 50; or g. the amino acid sequence of HCDR1 is SEQ ID NO: 51, the amino acid sequence of HCDR2 is SEQ ID NO: 52, the amino acid sequence of HCDR3 is SEQ ID NO: 53, the amino acid sequence of LCDR1 is SEQ ID NO: 54, the amino acid sequence of LCDR2 is SEQ ID NO: 55, and the amino acid sequence of LCDR3 is SEQ ID NO:

56.

16. The antibody-drug conjugate of any one of claims 1-13, wherein the antibody or fragment thereof comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein: a. the amino acid sequence of the LCVR is SEQ ID NO: 1, and the amino acidsequence of the HCVR is SEQ ID NO: 2; b. the amino acid sequence of the LCVR is SEQ ID NO: 3, and the amino acid sequence of the HCVR is SEQ ID NO: 4; c. the amino acid sequence of the LCVR is SEQ ID NO: 5, and the amino acid sequence of the HCVR is SEQ ID NO: 6; d. the amino acid sequence of the LCVR is SEQ ID NO: 7, and the amino acid sequence of the HCVR is SEQ ID NO: 8; e. the amino acid sequence of the LCVR is SEQ ID NO: 9, and the amino acid sequence of the HCVR is SEQ ID NO: 10; f. the amino acid sequence of the LCVR is SEQ ID NO: 11, and the amino acid sequence of the HCVR is SEQ ID NO: 12; or g. the amino acid sequence of the LCVR is SEQ ID NO: 13, and the amino acid sequence of the HCVR is SEQ ID NO:

14.

17. The antibody-drug conjugate of any one of claims 1-7 and 9-13, wherein the antibody fragment that binds MUC1-C comprises a F(ab), comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the HCVR comprises the complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3 and the LCVR comprises LCDRs LCDR1, LCDR2 and LCDR3, wherein: a. the amino acid sequence of HCDR1 is SEQ ID NO: 15, the amino acid sequence of HCDR2 is SEQ ID NO: 16, the amino acid sequence of HCDR3 is SEQ ID NO: 17, the amino acid sequence of LCDR1 is SEQ ID NO: 18, the amino acid sequence of LCDR2 is SEQ ID NO: 19, and the amino acid sequence of LCDR3 is SEQ ID NO: 20; b. the amino acid sequence of HCDR1 is SEQ ID NO: 21, the amino acid sequence of HCDR2 is SEQ ID NO: 22, the amino acid sequence of HCDR3 is SEQ ID NO: 23, the amino acid sequence of LCDR1 is SEQ ID NO: 24, the amino acid sequence of LCDR2 is SEQ ID NO: 25, and the amino acid sequence of LCDR3 is SEQ ID NO: 26; c. the amino acid sequence of HCDR1 is SEQ ID NO: 27, the amino acid sequence of HCDR2 is SEQ ID NO: 28, the amino acid sequence of HCDR3 is SEQ ID NO: 29, the amino acid sequence of LCDR1 is SEQ ID NO: 30, the amino acidsequence of LCDR2 is SEQ ID NO: 31, and the amino acid sequence of LCDR3 is SEQ ID NO: 32; d. the amino acid sequence of HCDR1 is SEQ ID NO: 33, the amino acid sequence of HCDR2 is SEQ ID NO: 34, the amino acid sequence of HCDR3 is SEQ ID NO: 35, the amino acid sequence of LCDR1 is SEQ ID NO: 36, the amino acid sequence of LCDR2 is SEQ ID NO: 37, and the amino acid sequence of LCDR3 is SEQ ID NO: 38; e. the amino acid sequence of HCDR1 is SEQ ID NO: 39, the amino acid sequence of HCDR2 is SEQ ID NO: 40, the amino acid sequence of HCDR3 is SEQ ID NO: 41, the amino acid sequence of LCDR1 is SEQ ID NO: 42, the amino acid sequence of LCDR2 is SEQ ID NO: 43, and the amino acid sequence of LCDR3 is SEQ ID NO: 44; f. the amino acid sequence of HCDR1 is SEQ ID NO: 45, the amino acid sequence of HCDR2 is SEQ ID NO: 46, the amino acid sequence of HCDR3 is SEQ ID NO: 47, the amino acid sequence of LCDR1 is SEQ ID NO:48, the amino acid sequence of LCDR2 is SEQ ID NO: 49, and the amino acid sequence of LCDR3 is SEQ ID NO: 50; or g. the amino acid sequence of HCDR1 is SEQ ID NO: 51, the amino acid sequence of HCDR2 is SEQ ID NO: 52, the amino acid sequence of HCDR3 is SEQ ID NO: 53, the amino acid sequence of LCDR1 is SEQ ID NO: 54, the amino acid sequence of LCDR2 is SEQ ID NO: 55, and the amino acid sequence of LCDR3 is SEQ ID NO:

56.

18. The antibody-drug conjugate of any one of claims 1-7 and 9-13, wherein the antibody fragment that binds MUC1-C comprises an Fv or scFv, comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the HCVR comprises the complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3 and the LCVR comprises CDRs LCDR1, LCDR2 and LCDR3, wherein: a. the amino acid sequence of HCDR1 is SEQ ID NO: 15, the amino acid sequence of HCDR2 is SEQ ID NO: 16, the amino acid sequence of HCDR3 is SEQ ID NO: 17, the amino acid sequence of LCDR1 is SEQ ID NO: 18, the amino acid sequence of LCDR2 is SEQ ID NO: 19, and the amino acid sequence of LCDR3is SEQ ID NO: 20; b. the amino acid sequence of HCDR1 is SEQ ID NO: 21, the amino acid sequence of HCDR2 is SEQ ID NO: 22, the amino acid sequence of HCDR3 is SEQ ID NO: 23, the amino acid sequence of LCDR1 is SEQ ID NO: 24, the amino acid sequence of LCDR2 is SEQ ID NO: 25, and the amino acid sequence of LCDR3 is SEQ ID NO: 26; c. the amino acid sequence of HCDR1 is SEQ ID NO: 27, the amino acid sequence of HCDR2 is SEQ ID NO: 28, the amino acid sequence of HCDR3 is SEQ ID NO: 29, the amino acid sequence of LCDR1 is SEQ ID NO: 30, the amino acid sequence of LCDR2 is SEQ ID NO: 31, and the amino acid sequence of LCDR3 is SEQ ID NO: 32; d. the amino acid sequence of HCDR1 is SEQ ID NO: 33, the amino acid sequence of HCDR2 is SEQ ID NO: 34, the amino acid sequence of HCDR3 is SEQ ID NO: 35, the amino acid sequence of LCDR1 is SEQ ID NO: 36, the amino acid sequence of LCDR2 is SEQ ID NO: 37, and the amino acid sequence of LCDR3 is SEQ ID NO: 38; e. the amino acid sequence of HCDR1 is SEQ ID NO: 39, the amino acid sequence of HCDR2 is SEQ ID NO: 40, the amino acid sequence of HCDR3 is SEQ ID NO: 41, the amino acid sequence of LCDR1 is SEQ ID NO: 42, the amino acid sequence of LCDR2 is SEQ ID NO: 43, and the amino acid sequence of LCDR3 is SEQ ID NO: 44; f. the amino acid sequence of HCDR1 is SEQ ID NO: 45, the amino acid sequence of HCDR2 is SEQ ID NO: 46, the amino acid sequence of HCDR3 is SEQ ID NO: 47, the amino acid sequence of LCDR1 is SEQ ID NO:48, the amino acid sequence of LCDR2 is SEQ ID NO: 49, and the amino acid sequence of LCDR3 is SEQ ID NO: 50; or g. the amino acid sequence of HCDR1 is SEQ ID NO: 51, the amino acid sequence of HCDR2 is SEQ ID NO: 52, the amino acid sequence of HCDR3 is SEQ ID NO: 53, the amino acid sequence of LCDR1 is SEQ ID NO: 54, the amino acid sequence of LCDR2 is SEQ ID NO: 55, and the amino acid sequence of LCDR3 is SEQ ID NO: 56.

19. The antibody-drug conjugate of any one of claims 1-7 and 9-13, wherein the antibody fragment that binds MUC1-C comprises a VhH, comprising a heavy chain variable region (HCVR), wherein the HCVR comprises the complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3, wherein: a. the amino acid sequence of HCDR1 is SEQ ID NO: 15, the amino acid sequence of HCDR2 is SEQ ID NO: 16, and the amino acid sequence of HCDR3 is SEQ ID NO: 17; b. the amino acid sequence of HCDR1 is SEQ ID NO: 21, the amino acid sequence of HCDR2 is SEQ ID NO: 22, and the amino acid sequence of HCDR3 is SEQ ID NO: 23; c. the amino acid sequence of HCDR1 is SEQ ID NO: 27, the amino acid sequence of HCDR2 is SEQ ID NO: 28, and the amino acid sequence of HCDR3 is SEQ ID NO: 29; d. the amino acid sequence of HCDR1 is SEQ ID NO: 33, the amino acid sequence of HCDR2 is SEQ ID NO: 34, and the amino acid sequence of HCDR3 is SEQ ID NO: 35; e. the amino acid sequence of HCDR1 is SEQ ID NO: 39, the amino acid sequence of HCDR2 is SEQ ID NO: 40, and the amino acid sequence of HCDR3 is SEQ ID NO: 41; f. the amino acid sequence of HCDR1 is SEQ ID NO: 45, the amino acid sequence of HCDR2 is SEQ ID NO: 46, and the amino acid sequence of HCDR3 is SEQ ID NO: 47; or g. the amino acid sequence of HCDR1 is SEQ ID NO: 51, the amino acid sequence of HCDR2 is SEQ ID NO: 52, and the amino acid sequence of HCDR3 is SEQ ID NO:

53.

20. A pharmaceutical composition comprising the antibody-drug conjugate of any one of claims 1-19, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

21. A method of treating cancer in a patient in need thereof, comprising administering to the patient (a) the antibody-drug conjugate of any one of claims 1-19, or (b) the pharmaceutical composition of claim 20.

22. The method of treating cancer in a patient in need thereof of claim 21, wherein the cancer comprises cancers that express MUC1-C on their cell surface, renal cancer, leukemia, acute myeloid leukemia, lymphoma, cutaneous T cell lymphoma, multiple myeloma, breast cancer, triple negative breast cancer, Merkel cell carcinoma, small cell lung cancer, non-small cell lung cancer, castration-resistant prostate cancer, neuroendocrine prostate cancer, urothelial carcinoma, or colorectal cancer.

23. A method of inducing ferroptosis in a subject, comprising administering to the subject (a) the antibody-drug conjugate of any one of claims 1-19, or (b) the pharmaceutical composition of claim 20.

24. Use of (a) the antibody-drug conjugate of any one of claims 1-19, or (b) the pharmaceutical composition of claim 20 for treating cancer.

25. The use of claim 24, wherein the cancer comprises cancers that express MUC1-C on their cell surface, renal cancer, leukemia, acute myeloid leukemia, lymphoma, cutaneous T cell lymphoma, multiple myeloma, breast cancer, triple negative breast cancer, Merkel cell carcinoma, small cell lung cancer, non-small cell lung cancer, castration-resistant prostate cancer, neuroendocrine prostate cancer, urothelial carcinoma, or colorectal cancer.

26. The antibody-drug conjugate of any one of claims 1-19, or the pharmaceutical composition of claim 20 for use in therapy.

27. The antibody-drug conjugate of any one of claims 1-19, or the pharmaceutical composition of claim 20 for use in treating cancer.

28. The antibody-drug conjugate, or the pharmaceutical composition of claim 27, wherein the cancer comprises cancers that express MUC1-C on their cell surface, renal cancer, leukemia, acute myeloid leukemia, lymphoma, cutaneous T cell lymphoma, multiple myeloma, breast cancer, triple negative breast cancer, Merkel cell carcinoma, small cell lung cancer, non-small cell lung cancer, castration-resistant prostate cancer, neuroendocrine prostate cancer, urothelial carcinoma, or colorectal cancer.

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