MUC1-c as a target for treatment resistant HR+ / her2- breast cancer

Abstract

The present invention provides methods for treating HR+ / HER2- breast cancer that is resistant to endocrine therapy and / or CDK4 / 6 inhibitor treatment by targeting MUC1-C.

Description

[0001]

[0002] MUC1-C AS A TARGET FOR TREATMENT RESISTANT HR+ / HER2- BREAST CANCER

[0003] CROSS-REFERENCE TO RELATED APPLICATION

[0004] This application claims the benefit of and priority to U.S. Provisional Patent Application 63 / 790,805, filed April 18, 2025, which is incorporated by reference herein in its entirety.

[0005] SEQUENCE LISTING

[0006] This application contains a Sequence Listing electronically submitted via Patent Center to the United States Patent and Trademark Office as an XML file entitled “0680.003674W001. xml” having a size of 160,587 bytes and created on April 17, 2026. The information contained in the Sequence Listing is incorporated by reference herein.

[0007] GOVERNMENT INTERESTS

[0008] This invention was made with government support under CA097098 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0009] FIELD

[0010] The present disclosure is in the field of treating patients with HR+ / HER2- breast cancer resistant to CDK4 / 6 inhibitor and / or endocrine therapy treatment. The present disclosure is in the field of a treatment of drug-resistant HR+ / HER2- breast cancer by targeting MUC1-C alone and in combination with standard of care treatment.

[0011] BACKGROUND

[0012] Hormone receptor (HR)-positive, HER2 -negative breast cancer (HR+ / HER2- BC) accounts for 70% of breast cancers. HR+ / HER2- BC is conventionally treated with endocrine therapy (ET) comprised of aromatase inhibitors and selective endocrine receptor (ER) modulators / degraders (SERMs / SERDs). ET reduces the risk of recurrence in the adjuvant setting and improves survival in patients with metastatic disease. Nonetheless, resistance to ET is an inevitable outcome. Mutations in the ESRI gene are a common mechanism for ET resistance, often

[0013]

[0014] occurring in the estrogen receptor alpha (ER) ligand binding domain at amino acids Y537 and D538 (Robinson, D.R. etal., Nat Genet, 45, 2013).

[0015] HR+ / HER2- BCs are also treated with agents, such as palbociclib, abemaciclib and ribociclib, which are small molecules inhibitors that target ER-mediated cyclin-dependent kinase 4 (CDK4) / CDK6 signaling. Upregulation of the CDK4 / 6 pathway in HR+ / HER2- BCs drives the Gl / S phase transition by inactivating the retinoblastoma (RB) protein and inducing E2F transcription factor (TF) functions (Watt, A. C. et al., Breast Cancer Res, 24, 2022). Combining agents that target ER signaling with CDK4 / 6 inhibitors has improved clinical outcomes (Finn, R. S. et al, Breast Cancer Res Treat, 184, 2020). However, HR+ / HER2- BCs invariably develop resistance to CDK4 / 6 inhibitors by mechanisms that have been associated with loss of functional RB, increased CDK6 levels, activation of the cyclin E / CDK2 axis, and / or upregulation of receptor tyrosine kinase (RTK) and PI3K signaling.

[0016] The MUCIN 1 (MUC1) gene, identified based on its overexpression in human breast cancers, evolved in mammals to promote placentation and protection of barrier tissues. MUC1 encodes an oncogenic C-terminal (MUC1-C) subunit that is activated in response to the loss of homeostasis and promotes proliferative, inflammatory, and remodeling responses necessary for wound repair. However, in contrast to its protective function, prolonged activation of MUC1-C by chronic inflammation drives cancer progression (Kufe, D., Cancers, 14, 2022).

[0017] MUC1 is overexpressed in HR+ BCs and associated with poor clinical outcomes. Direct interaction of ER and MUC1-C suppresses ER ubiquitylation and degradation by mechanisms unknown. Treatment of HR+ / HER2- BC is limited by resistance to ET and CDK4 / 6 inhibitors. To date, there is no known common pathway that confers resistance to these agents. Not known is whether (i) MUC1-C is necessary for resistance ofHR+ / HER2-BCs to ET and CDK4 / 6 inhibitors, and (ii) MUC1-C is a potential target for the treatment of refractory HR+ / HER2- BC.

[0018] SUMMARY

[0019] In the present disclosure, MUC1-C is shown to be a target for treatment of ET / CDK4 / 6- resistant HR+HER2- BCs. It is reported that (i) the MUC1 gene is upregulated in HR+ / HER2- BCs and (ii) the MUC1-C protein regulates estrogen receptor alpha (ER)-driven transcriptomes. Mechanistically, it is demonstrated that MUC1-C is necessary for expression of SRC-3 and MED1 coactivators that drive ER-mediated target gene transcription. Cells with ESRI mutations that

[0020]

[0021] confer ET resistance, as well as cells with acquired resistance to the CDK4 / 6 inhibitor abemaciclib, are dependent on MUC1-C for (i) expression of these coactivators and ER target genes, (ii) survival, and (iii) self-renewal capacity. In support of these results, it is shown that treatment of HR+ / HER2- BC cells with an anti-MUCl-C antibody-drug conjugate (ADC) effectively inhibits survival, self-renewal and tumorgenicity. These findings indicate that MUC1-C is a common effector of drug-resistant HR+ / HER2- BC cells and is a potential target for their treatment.

[0022] The present disclosure provides a method to treat cancer comprising administering to a subject in need thereof at least one endocrine therapy, at least one CDK4 / 6 inhibitor, and at least one MUC1-C inhibitor. In a preferred embodiment, the cancer is HR+ / HER2- breast cancer. In a preferred embodiment, the MUC1-C inhibitor is an antibody drug conjugate (ADC). In a more preferred embodiment, the ADC is 3D1-MMAE.

[0023] The present disclosure provides a method to treat cancer comprising administering to a subject in need thereof at least one MUC1-C inhibitor, wherein the cancer presents a mutation in the ESR-1 gene. In a preferred embodiment, the mutation in the ESR-1 gene is independently selected from Y537 and D538G.

[0024] The present disclosure provides a method to treat breast cancer comprising administering to a subject in need thereof at least one MUC1-C inhibitor, wherein the breast cancer is resistant to at least one CDK4 / 6 inhibitor. In another embodiment, the breast cancer is resistant to at least one endocrine therapy treatment. In another embodiment, the breast cancer is resistant to both endocrine therapy treatment and CDK4 / 6 inhibitor treatment. In a preferred embodiment, the breast cancer is HR+ / HER2- breast cancer. In another embodiment, the MUC1-C inhibitor is an ADC. In a preferred embodiment, the ADC is 3D1-MMAE. In another embodiment, the MUC1- C inhibitor is a peptide. In a preferred embodiment, the peptide is GO-203. In another embodiment, the MUC1-C inhibitor is a small molecule. In a preferred embodiment, the small molecule is salinomycin. In another embodiment, the MUC1-C inhibitor is a sRNA molecule. In a preferred embodiment, the sRNA molecule is a siRNA, shRNA, and sgRNA.

[0025] The present disclosure provides a means for treating HR+ / HER2- breast cancer comprising a means for inhibiting MUC1-C, wherein the HR+ / HER2- breast cancer is resistant to 1 or more of a means for inhibiting CDK4 / 6 and a means for modulating hormone receptors. In the present disclosure, a means for inhibiting CDK4 / 6 are described and are well known in the art. Also, in

[0026]

[0027] the present disclosure, a means for modulating hormone receptors is described as various endocrine therapy (ET) treatments and are well known in the art.

[0028] Various CDK4 / 6 inhibitor means are included in the present disclosure. In embodiments, the CDK4 / 6 inhibitor means of the present disclosure comprises a small molecule or large molecule that inhibits CDK4 / 6. In some embodiments, the CDK4 / 6 inhibitor means may be palbociclib, ribociclib, abemaciclib, or is a molecule that inhibits CDK4 / 6.

[0029] Various ET means are included in the present disclosure. In embodiments, the ET means of the present disclosure comprises a small molecule or large molecule that modulates hormone receptors. In some embodiments, the HR modulator means is independently selected from a SERM, SERD, SERCA, ShERPA, aromatase inhibitor, and GnRH agonist means. In some embodiments, the SERM means may be tamoxifen or toremifene. In some embodiments, the SERD means may be fulvestrant or elacestrant. In some embodiments, the GnRH means may be leuprolide or goserelin. In some embodiments, the aromatase inhibitor means may be anastrozole, letrozole, or exemestane.

[0030] Various MUC1-C inhibitor means are disclosed. This MUC1-C inhibitor means is a small molecule drug, peptide, sRNA, an antibody-drug conjugate (ADC) or a combination thereof that is capable of inhibiting MUC1-C. In some embodiments, the small molecule drug means is salinomycin. In some embodiments, the peptide means is GO-203. In some embodiments, the peptide means is GO-201. In some embodiments, the ADC means is 3D1-MMAE.

[0031] The present disclosure further provides pharmaceutical compositions comprising a MUC1- C inhibitor (either an ADC of the present disclosure or a small molecule inhibitor of MUC1-C, or a MUC1-C inhibitor in the form of a peptide or sRNA molecule), a CDK4 / 6 inhibitor, and an endocrine therapy.

[0032] DRAWINGS

[0033] The drawings illustrate generally, by way of example, but not by way of limitation, various embodiments discussed herein.

[0034] Figure 1. MUC1 is upregulated in HR+ / HER2- BCs and regulates ER-driven gene transcriptomes, a and b. Analysis of the TCGA BRCA dataset for MUC1 expression in HR+ / HER2- BCs vs normal breast tissue (a) and HR+ / HER2- vs ER- / HER2- BCs (b). c. MCF-7 and T47D cell membrane (MEM), cytosolic (CYTO) and nucleoplasm (NP) were immunoblotted

[0035]

[0036] with antibodies against the indicated proteins, d. Chromatin from MCF-7 and T47D cells was immunoblotted with antibodies against the indicated proteins, e. Lysates from MCF-7 / tet- MUClshRNA and T47D / tet-shMUClshRNA cells treated with vehicle or DOX for 7 days were immunoblotted with antibodies against the indicated proteins, f and g. GSEAof RNA-seq data from MCF-7 cells (f) and T47D cells (g) with MUC1-C silencing using the HALLMARK ESTROGEN RESPONSE EARLY (upper panels) and HALLMARK ESTROGEN RESPONSE LATE (lower panels) gene signatures, h. Common downregulated ERGs and LRGs in MCF-7 and T47D cells with MUC1-C silencing, i and j. MCF-7 / tet-MUClshRNA (i) and T47D / tet- MUClshRNA (j) cells treated with vehicle or DOX for 7 days were analyzed for MUC1-C, TFF1, MYB and BCL2 transcripts by qRT-PCR using primers listed in Supplemental Table S2. The results (mean±SD of 4 determinations) are expressed as relative levels compared to that obtained for vehicle-treated cells (assigned a value of 1).

[0037] Figure 2. MUC1-C regulates ER coactivator SRC-3 by an MK2-mediated mechanism, a. Nuclear lysates from MCF-7 cells treated with 100 nM 17P-estradiol (E2) for 3 hours were precipitated with anti-MUCl-C or a control IgG. The precipitates and input lysate were immunoblotted with antibodies against the indicated proteins, b. Nuclear lysates from MCF-7 cells treated with 100 nM E2 for 3 hours were precipitated with anti-SRC-3 or a control IgG. The precipitates and input lysate were immunoblotted with antibodies against the indicated proteins, c and d. Lysates of MCF-7 / tet-MUClshRNAand T47D / tet-MUClshRNA cells treated with vehicle or DOX for 7 days were immunoblotted with antibodies against the indicated proteins, e. MCF-7 / CshRNA and MCF-7 / SRC-3shRNA#l cells were analyzed for the indicated transcripts by qRT-PCR. The results (mean±SD of 4 determinations) are expressed as relative levels compared to that obtained for CshRNA cells (assigned a value of 1). f. MCF-7 and T47D cells treated with vehicle or 5 pM GO-203 were analyzed for colony formation. Shown are representative photomicrographs of stained colonies (left). The results (mean±SD of three determinations) are expressed as relative colony formation compared to that for vector cells (assigned a value of l)(right). g. MCF-7 / CshRNA, MCF-7 / SRC-3shRNA#l and MCF-7 / SRC- 3shRNA#2 cells were analyzed for colony formation. Shown are representative photomicrographs of stained colonies (left). The results (mean±SD of three determinations) are expressed as relative colony formation compared to that for vector cells (assigned a value of 1) (right).

[0038]

[0039] Figure 3. MUC1-C regulates ER coactivator MED1 by a CDK7-mediated mechanism, a. Nuclear lysates from MCF-7 cells treated with 100 nM 170-estradiol (E2) for 3 hours were precipitated with anti-MUCl-C or a control IgG. The precipitates and input lysate were immunoblotted with antibodies against the indicated proteins, b. Nuclear lysates from MCF-7 cells treated with 100 nM E2 for 3 hours were precipitated with anti-MEDl or a control IgG. The precipitates and input lysate were immunoblotted with antibodies against the indicated proteins, c and d. Lysates from MCF-7 / tet-MUClshRNA and T47D / tet-MUClshRNA cells treated with vehicle or DOX for 5 days were immunoblotted with antibodies against the indicated proteins, e. GSEAof RNA-seq data from MCF-7 and T47D cells with MUC1-C silencing using the UDAYAKUMAR MED1 TARGETS UP gene signature, f. MCF-7 / CshRNA, MCF-7 / MED1 shRNA# 1 and MCF-7 / MEDlshRNA#2 cells were analyzed for MED1, ESRI, TFF1, MYB and BCL2 transcripts by qRT-PCR. The results (mean±SD of four determinations) are expressed as relative levels compared to that obtained for CshRNA cells (assigned a value of 1). g. MCF-7 / CshRNA, MCF-7 / MED 1 shRNA# 1 and MCF-7 / MEDlshRNA#2 cells were analyzed for colony formation. Shown are representative photomicrographs of stained colonies (left). The results (mean±SD of three determinations) are expressed as relative colony formation compared to that for CshRNA cells (assigned a value of 1) (right).

[0040] Figure 4. ER(Y537S) mutant cells are MUC1-C dependent, a. Nuclear lysates from MCF-7(Y537S) cells grown in hormone deprived conditions for 2 days were precipitated with anti-MUCl-C or a control IgG. The precipitates and input lysate were immunoblotted with antibodies against the indicated proteins, b. Lysates from MCF-7(Y537S) / tet-MUClshRNA cells treated with vehicle or DOX for 7 days were immunoblotted with antibodies against the indicated proteins, c. Lysates from MCF-7(Y537S) cells treated with vehicle or 5 pM GO-203 for 2 days were immunoblotted with antibodies against the indicated proteins, d and e.

[0041] Chromatin from MCF-7 / tet-MUCl shRNA (d) and MCF-7 / ER(Y537) / tet-MUClshRNA (e) cells treated with vehicle or DOX for 11 days were immunoblotted with antibodies against the indicated proteins, f. MCF-7(Y537S) / tet-MUClshRNA cells treated with vehicle or DOX for 7 days were analyzed for MUC1-C, TFF1, MYB and BCL2 transcripts 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). g. MCF-7 / ER(Y537S) / tet-MUClshRNA cells treated with vehicle or DOX were analyzed for colony formation. Shown are representative

[0042]

[0043] photomicrographs of stained colonies (left). The results (mean±SD of three determinations) are expressed as relative colony formation compared to that for vehicle treated cells (assigned a value of 1) (right), h. MCF-7(Y537S) cells treated with vehicle or 5 pM GO-203 were analyzed for colony formation. Shown are representative photomicrographs of stained colonies (left). The results (mean±SD of three determinations) are expressed as relative colony formation compared to that for vehicle-treated cells (assigned a value of 1) (right).

[0044] Figure 5. CDK4 / 6 inhibitor resistant HR+ / HER2- BC cells are MUCl-C-dependent. a. Lysates from MCF-7 / tet-MUClshRNAand T47D / tet-MUClshRNA cells treated with vehicle of DOX for 7 days were immunoblotted with antibodies against the indicated proteins, b. GSEA of RNA-seq data from MCF-7 cells and T47D cells with MUC1-C silencing using the HALLMARK E2F TARGETS gene signature, c. Parental and abemaciclib-resistant MCF-7-AR and T47D-AR cells were treated with the indicated concentrations of abemaciclib for 6 days and analyzed for cell viability by Alamar Blue staining. The results (mean±SD) are expressed as relative cell viability (% control) compared with that for untreated cells, d. Lysates from MCF-7 and T47D parental wild-type (WT) and AR cells were immunoblotted with antibodies against the indicated proteins, e. Chromatin from MCF-7 and T47D WT and AR cells was immunoblotted with antibodies against the indicated proteins, f. Lysates from T47D-AR / tet-MUClshRNA cells treated with vehicle of DOX for 7 days were immunoblotted with antibodies against the indicated proteins, g. Chromatin from T47D-AR / tet-MUClshRNA cells treated with vehicle or DOX for 14 days was immunoblotted with antibodies against the indicated proteins, h. T47D- AR / tet-MUClshRNA cells treated with vehicle or DOX were analyzed for colony formation. Shown are representative photomicrographs of stained colonies (left). The results (mean±SD of three determinations) are expressed as relative colony formation compared to that for vehicle- treated cells (assigned a value of 1) (right), i. T47D-AR / CshRNA and T47D- AR / MUClshRNA#2 cells were analyzed for colony formation. Shown are representative photomicrographs of stained colonies (left). The results (mean±SD of three determinations) are expressed as relative colony formation compared to that for CshRNA cells (assigned a value of 1) (right).

[0045] Figure 6. Anti-MUCl-C ADC is effective against treatment-resistant HR+ / HER2- BC cells and tumors, a. The designated MCF-7 cells were analyzed for cell surface MUC1-C

[0046]

[0047] expression by flow cytometry, b and c. MCF7, MCF-7(Y537S) and MCF-7(D538G) (b) and ZR- 75-1 (c) cells treated with 100 nM anti-MUCl-C ADC were analyzed for tumorsphere formation. Shown are representative photomicrographs of tumorspheres (left). The results (mean±SD of three determinations) are expressed as relative sphere formation efficiency (SFE) compared to that for control cells (assigned a value of 1) (right), d. Nude mice were injected with 1 * 107ZR- 75-1 cells in the 4thmammary fat pad. Mice randomized into two groups were treated with vehicle or 5 mg / kg anti-MUCl-C ADC weekly for 5 weeks. Tumor volumes are expressed as the mean±SEM. All 20 mice in the vehicle control group had progressive tumor growth with 10 sacrificed by day 78 and the remaining by day 144. The study was terminated at day 178 as tumor volumes remained static for multiple weeks. At that time, 6 of 10 mice in the ADC group were alive with only 2 bearing measurable tumors. Treatment with the ADC produced tumor regressions in all treated mice with all tumors being unmeasurable by day 31. P values were calculated on day 111. e. Mean body weight changes of the ZR-75-1 tumor bearing mice treated with vehicle or anti-MUCl-C ADC. The results are expressed as mean values for which the SEMs were <10% of the means, f. PDX1415 tumor samples were implanted subcutaneously in ovariectomized NOD-SCID-IL2Rgc- / - mice with E2 supplementation. Mice randomized into two groups when tumors reached 100 mm3 were treated with vehicle or 7.5 mg / kg anti-MUCl-C ADC weekly for 5 weeks. Tumor volumes are expressed as the mean±SEM. The asterisk (*) denotes sacrifice due to tumor volumes exceeding acceptable limits, g. Mean body weight changes of the PDX1415 tumor bearing mice treated with vehicle or anti-MUCl-C ADC. The results are expressed as mean values for which the SEMs were <10% of the means.

[0048] Figure 7. Expression of MUC1-C in HR+ / HER2- BCs with ESRI mutations and refractory to CDK4 / 6 inhibitor treatment, a. Kaplan-Meier analysis of the TCGABRCA Luminal B dataset for progression free survival as a function of MUCl-high vs MUCl-low tumors, b. Representative examples of apical membranous, diffuse membranous and cytoplasmic staining for MUC1-C by IHC. c-e. IHC score of MUC1-C levels on membranous, cytoplasmic or apical membranous regions were scored as 0, 1+, 2+ or 3+ based on the highest intensity occupying >10% of the evaluated area. The representative IHC score for HR+ / HER2- (n=18) (c), ESRI mutant (n=l 1) (d) and CDK4 / 6 inhibitor resistant (n=5) (e) BCs was determined by adopting the maximum score of each region, f. IHC staining for MUC1-C expression of a HR+ / HER2- BC metastatic to pleura pre-treatment (left) and post-treatment with ET in

[0049]

[0050] combination with CDK4 / 6 inhibition in a setting of progressive disease (right), g. Schema depicting MUCl-C-mediated regulation of ER signaling in ER wild-type, ER mutant and CDK4 / 6 inhibitor resistant cells. Our results demonstrate that MUC1-C forms nuclear complexes with SRC-3 and MED1 and that targeting MUC1-C genetically and pharmacologically decreases SRC-3 and MED1 expression. Importantly, we further show that targeting MUC1-C suppresses ER, SRC-3 and MED1 levels in chromatin. We also demonstrate mechanistically that MUC1-C is necessary for expression of (i) MK2, which stabilizes SRC-3, and (ii) CDK7, which stabilizes MEDE These findings uncover the previously unrecognized role of MUC1-C in regulating SRC- 3 and MED1 as effectors of drug resistance. Identification of this common pathway in drug resistant HR+ / HER2- BC cells formed the basis for the findings that an anti-MUCl-C ADC is effective for their treatment.

[0051] Figure 8. Association of MUC1 with ESRI expression and HALLMARK gene signatures, a. Analysis of the TCGABRCA dataset for MUC1 expression in (i) ER+ vs ER-, and (ii) HER2+ vs HER2- BCs. b. Association o MUCl with expression of ESRI and ER target genes TFF1 anAMYB in the TCGA BRC A dataset, c. Schema of MUC1-C structure with the 58 aa extracellular domain (ED), 28 aa transmembrane domain (TD) and 72 aa cytoplasmic domain (CD). Modification of the ED by N-glycosylation at the NLT motif functions as galectin-3 binding site and thereby interactions with RTKs at the cell membrane, as well as intracellular effectors. N-glycosylated MUC1-C is detectable as a -23-25 kDa glycoprotein (left).

[0052] Unglycosylated MUC1-C is detectable as a 17 kDa protein (right). The MUC1-C ED alpha-3 helix is the ADC target, d. Lysates from ZR-75-l / tet-MUClshRNA cells treated with vehicle or DOX for 7 days were immunoblotted with antibodies against the indicated proteins, e. Lysates from MCF-7 / CshRNA and MCF-7 / MUClshRNA#2 cells were immunoblotted with antibodies against the indicated proteins, f. Lysates from MCF-7 cells treated with 2.5 pM GO-203 for 3 days were immunoblotted with antibodies against the indicated proteins, g and h. GSEA of RNA-seq data from MCF-7 (g) and T47D (h) cells with MUC1-C silencing using the indicated HALLMARK gene signatures.

[0053] Figure 9. MUC1-C regulates SRC-3 expression, a. Lysates from ZR-75-l / tet- MUClshRNA cells treated with vehicle or DOX for 7 days were immunoblotted with antibodies against the indicated proteins, b. Lysates from MCF-7 / CshRNAand MCF-7 / MUClshRNA#2

[0054]

[0055] cells were immunoblotted with antibodies against the indicated proteins, c. Lysates from MCF-7 cells treated with 5 pM GO-203 for 4 days were immunoblotted with antibodies against the indicated proteins, d. MCF-7 / tet-MUCl shRNA, T47D / tet-MUC 1 shRNA and ZR-75-l / tet- MUClshRNA cells treated with vehicle or DOX for 7 days were analyzed for SRC-3 transcripts by qRT-PCR. The results (mean±SD of 4 determinations) are expressed as relative levels compared to that obtained for vehicle-treated cells (assigned a value of 1). e. The indicated cells expressing tet-MUC 1 shRNA treated with vehicle or DOX for 7 days were analyzed for MK2 transcripts. The results (mean±SD of 4 determinations) are expressed as relative levels compared to that obtained for vehicle-treated cells (assigned a value of 1). f. MCF-7 cells treated with vehicle or 2.5 pM GO-203 for 4 days were analyzed for MK2 transcripts. The results (mean±SD of 4 determinations) are expressed as relative levels compared to that obtained for vehicle-treated cells (assigned a value of 1). g. Lysates from E2-stimulated MCF-7 cells were immunoprecipitated with anti-MUCl-C and a control IgG. The input (10% of total lysate) and precipitates were immunoblotted with anti-MK2. h. Association of MUC1 and MK2 in the TCGABRCA dataset, i. Lysates from MCF-7 treated with vehicle or 5 pM PF-3644022 for 24 hours were immunoblotted against the indicated proteins, j. MCF-7 / CshRNA and MCF-7 / SRC- 3shRNA#2 cells were analyzed for the indicated transcripts by qRT-PCR. The results (mean±SD of 4 determinations) are expressed as relative levels compared to that obtained for CshRNA cells (assigned a value of 1). k. MCF-7 cells treated with vehicle or 5 pM PF-3644022 were analyzed for colony formation. Shown are representative photomicrographs of stained colonies (left). The results (mean±SD of three determinations) are expressed as relative colony formation compared to that for vehicle-treated cells (assigned a value of 1) (right).

[0056] Figure 10. MUC1-C regulates MED1 expression, a. Lysates from ZR-75-l / tet- MUC1 shRNA cells treated with vehicle or DOX for 7 days were immunoblotted with antibodies against the indicated proteins, b. Lysates from MCF-7 cells treated with 5 pM GO-203 for 4 days were immunoblotted with antibodies against the indicated proteins, c. MCF-7 / tet-MUCl shRNA, T47D / tet-MUCl shRNA and ZR-75-l / tet-MUClshRNA cells treated with vehicle or DOX for 7 days were analyzed for MED1 transcripts by qRT-PCR. The results (mean±SD of 4 determinations) are expressed as relative levels compared to that obtained for vehicle-treated cells (assigned a value of 1). d. GSEA of RNA-seq data from MCF-7 and T47D cells with MUC1-C silencing using the UDAYAKUMAR MED1 TARGETS DN gene signature, e. Lysates

[0057]

[0058] from MCF-7 cells treated with vehicle or 1 pM samuraciclib for 48 hours were immunoblotted with antibodies against the indicated proteins.

[0059] Figure 11. MCF-7(D538G) cells are dependent on MUC1-C for survival, a. Nuclear lysates from MCF-7(Y537S) cells grown in hormone deprived conditions for 2 days and then stimulated with 100 nM E2 for 3 hours were precipitated with anti-MUCl-C or a control IgG. The precipitates and input lysate were immunoblotted with antibodies against the indicated proteins, b. MCF-7 / ER(Y537S) / CshRNA and MCF-7 / ER(Y537S) / MUClshRNA#2 cells were analyzed for colony formation. Shown are representative photomicrographs of stained colonies (left). The results (mean±SD of three determinations) are expressed as relative colony formation compared to that for CshRNA cells (assigned a value of 1 )(right). c. MCF-7 / ER(D538G) / tet- MUClshRNA cells treated with vehicle or DOX for 7 days were analyzed for the indicated transcripts by qRT-PCR. The results (mean±SD of 4 determinations) are expressed as relative levels compared to that obtained for vehicle-treated cells (assigned a value of 1). d. MCF- 7 / ER(D538G) / tet-MUClshRNA cells treated with vehicle or DOX for 7 days were analyzed for colony formation. Shown are representative photomicrographs of stained colonies (left). The results (mean±SD of three determinations) are expressed as relative colony formation compared to that for vehicle-treated cells (assigned a value of 1) (right), e. MCF-7 / ER(D538G) cells treated with vehicle or 5 pM GO-203 were analyzed for colony formation. Shown are representative photomicrographs of stained colonies (left). The results (mean±SD of three determinations) are expressed as relative colony formation compared to that for vehicle-treated cells (assigned a value of 1) (right).

[0060] Figure 12. MCF-7-AR and T47D-AR cells are MUC1-C dependent, a. Lysates from ZR-75-l / tet-MUClshRNA cells treated with vehicle or DOX for 7 days were immunoblotted with antibodies against the indicated proteins, b and c. GSEAof RNA-seq data from MCF-7 cells (b) and T47D cells (c) with MUC1-C silencing using the indicated HALLMARK gene signatures, d and e. MCF-7-AR (d) and T47D-AR (e) cells treated with vehicle or 5 pM GO-203 for 3 days were immunoblotted with antibodies against the indicated proteins, f. T47D-AR / tet- MUClshRNA cells treated with vehicle or DOX for 7 days were analyzed for the indicated transcripts by qRT-PCR. The results (mean±SD of 4 determinations) are expressed as relative levels compared to that obtained for vehicle-treated cells (assigned a value of 1). g and h. MCF-

[0061]

[0062] 7- AR (g) and T47D-AR (h) cells treated with vehicle or 5 pM GO-203 were analyzed for colony formation. Shown are representative photomicrographs of stained colonies (left). The results (mean±SD of three determinations) are expressed as relative colony formation compared to that for vehicle-treated cells (assigned a value of 1) (right).

[0063] Figure 13. Cell surface MUC1-C expression and anti-MUCl-C ADC dose-response curves, a. The designated MCF-7 cells were treated with the indicated concentrations of anti- MUC1-C ADC for 7 days and analyzed for cell viability by Alamar Blue staining. The results (mean±SD of three determinations) are expressed as relative cell viability (% control) compared with that for untreated cells, b. The designated MCF-7 cells were analyzed for cell surface MUC1-C expression by flow cytometry, c. The designated MCF-7 cells were treated with the indicated concentrations of anti-MUCl-C ADC for 7 days and analyzed for cell viability by Alamar Blue staining. The results (mean ± SD of three determinations) are expressed as relative cell viability (% control) compared with that for untreated cells, d. The designated T47D cells were analyzed for cell surface MUC1-C expression by flow cytometry, e. The designated T47D cells were treated with the indicated concentrations of anti-MUCl-C ADC for 7 days and analyzed for cell viability by Alamar Blue staining. The results (mean ± SD of three determinations) are expressed as relative cell viability (% control) compared with that for untreated cells, f and g. The designated MCF-7 (f) and T47D (g) cells treated with 100 nM anti- MUCl-C ADC were analyzed for tumorsphere formation. Shown are representative photomicrographs of tumorspheres (left). The results (mean±SD of three determinations) are expressed as relative sphere formation efficiency (SFE) compared to that for control cells (assigned a value of 1) (right).

[0064] Supplemental Table SI. Common down-regulated ERGs and LRGs in MCF-7 and T47D cells with MUC1-C silencing.

[0065] ERGs LRGs

[0066] CA12 CA12 CD44 CD44 ELOVL2 KIF20A GFRA1 MYB

[0067]

[0068]

[0069] GREB1 RAB31 MUC1 SLC7A5

[0070] MYB T0P2A

[0071] MYBL1

[0072] RAB31

[0073] SLC7A5

[0074] UGCG

[0075]

[0076] Supplemental Table S2. Primers used for qRT-PCR.

[0077] TACCGATCGTAGCCCCTATG MUC1-C FWD

[0078] SEQ ID NO: 75 CTCACCAGCCCAAACAGG REV SEQ ID NO: 76 CCCTCCCAGTGTGCAAATAAG TFF1 FWD

[0079] SEQ ID NO: 77 GAACGGTGTCGTCGAAACAG REV SEQ ID NO:78 GAAAGCGTCACTTGGGGAAAA MYB FWD SEQ ID NO:79 TGTTCGATTCGGGAGATAATTGG REV SEQ ID NO: 80 GGTGGGGTCATGTGTGTGG BCL2 FWD

[0080] SEQ ID NO: 81 CGGTTCAGGTACTCAGTCATCC REV SEQ ID NO: 82 AGACGGGAGCAGGAAAGTAAA SRC-3 FWD

[0081] SEQ ID NO: 83 GTAAAAGCGGTCCTAAGGAGTC REV SEQ ID NO: 84 CGCAGTTCCACGTCAAGTC MK2 FWD

[0082] SEQ ID NO: 85 GGGCGAATTTCTCCTGGGTC REV SEQ ID NO: 86 CTGGAACGGCTCCATGCAA MED1 FWD

[0083] SEQ ID NO: 87 CTTCTCCATGACTTGACGCAC REV SEQ ID NO: 88 GATGAGATTGGCATGGCTTT

[0084] P-actin FWD

[0085] SEQ ID NO: 89

[0086]

[0087] REV CACCTTCACCGTTCCAGTTT

[0088]

[0089]

[0090] SEQ ID NO:90

[0091] Supplemental Table S3. Clinical characteristics of patients with HR+ / HER2- BC tumors stained for MUC1-C expression by IHC. Data are expressed as number (n) and percentage (%) of tumors unless otherwise specified.

[0092] a. HR+ / HER2- BC surgical specimens (n=18)

[0093] Age at surgery (mean ± SD) 53.4 ± 14.0 male / female 0(0) / 18(100)

[0094] T is / 1 / 2 / 3 0(0) / 13(72) / 4(22) / 1(5.6) NO / 1 18(100) / 0(0)

[0095] M 0 / 1 18(100) / 0(0) Histology

[0096] 20(100) / 0(0) Invasive Ductal Carcinoma / others

[0097] Neoadjuvant treatment

[0098] 0(0) / 18(100) yes / no

[0099] MUC1-C staining score

[0100] 2(11) / 0(0) / 3(17) / 13(72) Apical 0 / 1+ / 2+ / 3+

[0101] 4(22) / 7(39) / 7(39) / 0(0) Membrane 0 / 1+ / 2+ / 3+

[0102] 1(5.6) / 8(44) / 7(39) / 3(17) Cytoplasm 0 / 1+ / 2+ / 3+

[0103]

[0104] b. ER mutant recurrent / metastatic tumor biopsy specimens (n=ll)

[0105] Age at biopsy (mean ± SD) 51.2 ± 10.7 male / female 0(0) / 11(100) ESRI mutation

[0106] 10(91) / 1(9.1) Y537S / L536R

[0107] Biopsy site

[0108] 4(36) / 3(27) / 4(36) skin / lymph node / liver

[0109] Previous ET

[0110] 11(100) / 0(0) yes / no

[0111]

[0112]

[0113] MUC1-C staining score

[0114] 2(18) / 1(9.1) / 2(18) / 6(55) Apical 0 / 1+ / 2+ / 3+

[0115] 2(18) / 2(18) / 5(45) / 2(18) Membrane 0 / 1+ / 2+ / 3+

[0116] 0(0) / 4(36) / 4(36) / 3(27) Cy topi asm 0 / 1+ / 2+ / 3+

[0117]

[0118] c. CDK4 / 6i refractory tumor biopsy specimens (n=5)

[0119] CDK4 / 6i starting age (mean ± SD) 51.2 ± 10.14

[0120] male / female 0(0) / 5(100) CDK4 / 6i used

[0121] Palbociclib / Abemaciclib / sequencial(P^>A) 2(40) / 0(0) / 3(60) ET used in combination with CDK4 / 6i

[0122] F ul vestrant / Letrozol e 3(60) / 2(40) CDK4 / 6i administration duration

[0123] 224 (median, days)

[0124] Previous ET

[0125] yes / no 4(80) / 1(20) Previous chemotherapy

[0126] yes / no 3(60) / 2(40) MUC1-C staining score pre-CDK4 / 6i

[0127] Apical 0 / 1+ / 2+ / 3+ 1(20) / 0(0) / 1(20) / 3(60) Membrane 0 / 1+ / 2+ / 3+ 3(60) / 0(0) / 1(20) / 1(20) Cytoplasm 0 / 1+ / 2+ / 3+ 2(40) / 1(20) / 2(40) / 0(0) MUC1-C staining score post-CDK4 / 6i

[0128] Apical 0 / 1+ / 2+ / 3+ 0(0) / 2(40) / 2(40) / 1(20) Membrane 0 / 1+ / 2+ / 3+ 0(0) / 1(20) / 3(60) / 1(20) Cytoplasm 0 / 1+ / 2+ / 3+ 0(0) / 0(0) / 0(0) / 5(100)

[0129]

[0130] DESCRIPTIONThe present invention demonstrates that MUC1-C is a common effector of HR+ / HER2- BC cell resistance to agents targeting ER and CDK4 / 6 signaling. It is reported herein that MUC1-C confers resistance to these agents by regulating the ER coactivators (i) steroid receptor co-activator 3 (SRC-3 / AIB1 / NCOA3), and (ii) mediator subunit 1 (MED1). The identification of this common pathway of HR+ / HER2- BC resistance lends support for MUC1-C as a target for the treatment of refractory disease, which has limited therapeutic options. Antibodydrug conjugates (ADCs) are approved for the treatment of metastatic BC, including HR+ / HER2-low and HR+ / HER2- (Chang, H. L. et al., J Clin Invest, 133, 2023; Grammoustianou, M. et al., Cancers (Basel), 16, 2024; Modi, S. et al., N Engl J Med, 387, 2022; Rugo, H. S. et al., J Clin Oncol, 40, 2022). In extending MUC1-C dependency of drug-resistant HR+ / HER2- BCs, it is demonstrated herein that an anti-MUCl-C ADC is effective against these recalcitrant cancers, uncovering a potential new approach for their treatment.

[0131] Reference will now be made in detail to certain embodiments of the disclosed subject matter. While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the exemplified subject matter is not intended to limit the claims to the disclosed subject matter.

[0132] Definitions

[0133] The following definitions are included to provide a clear and consistent understanding of the specification and claims. As used herein, the recited terms have the following meanings. All other terms and phrases used in this specification have their ordinary meanings as one of skill in the art would understand. Such ordinary meanings may be obtained by reference to technical dictionaries, such as Hawley's Condensed Chemical Dictionary 16th Edition, by M. Larranga, R Lewis Sr., and R Lewis, New York, N.Y., 2016.

[0134] References in the specification to "one embodiment," "an embodiment," etc., indicate that the embodiment described may include a particular aspect, feature, structure, moiety, or characteristic, but not every embodiment necessarily includes that aspect, feature, structure, moiety, or characteristic. Moreover, such phrases may, but do not necessarily, refer to the same embodiment referred to in other portions of the specification. Further, when a particular aspect, feature, structure, moiety, or characteristic is described in connection with an embodiment, it is within the knowledge of one skilled in the art to affect or connect such aspect, feature, structure, moiety, or characteristic with other embodiments, whether or not explicitly described. As used

[0135]

[0136] herein, the term "in some embodiments" refers to embodiments of all aspects of the disclosure, unless the context clearly indicates otherwise.

[0137] The singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a compound" includes a plurality of such compounds, so that a compound X includes a plurality of compounds X. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for the use of exclusive terminology, such as "solely," "only," and the like, in connection with any element described herein, and / or the recitation of claim elements or use of "negative" limitations.

[0138] The term "and / or" means any one of the items, any combination of the items, or all of the items with which this term is associated. The phrase "one or more" is readily understood by one of skill in the art, particularly when read in context of its usage. For example, one or more substituents on a phenyl ring refers to one to five, or one to four, for example if the phenyl ring is di -substituted.

[0139] As used herein, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating a listing of items, “and / or” or “or” shall be interpreted as being inclusive, e.g., the inclusion of at least one, but also including more than one of a number of items, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.”

[0140] As used herein, the terms “including,” “includes,” “having,” “has,” “with,” or variants thereof, are intended to be inclusive similar to the term “comprising.”

[0141] The term "about" can refer to a variation of ± 5%, ± 10%, ± 20%, or ± 25% of the value specified. For example, "about 50" percent can in some embodiments carry a variation from 45 to 55 percent. For integer ranges, the term "about" can include one or two integers greater than and / or less than a recited integer at each end of the range. Unless indicated otherwise herein, the term "about" is intended to include values, e.g., weight percentages, proximate to the recited range that are equivalent in terms of the functionality of the individual ingredient, the composition, or the

[0142]

[0143] embodiment. The term about can also modify the endpoints of a recited range as discuss above in this paragraph.

[0144] As will be understood by the skilled artisan, all numbers, including those expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, are approximations and are understood as being optionally modified in all instances by the term "about." These values can vary depending upon the desired properties sought to be obtained by those skilled in the art utilizing the teachings of the descriptions herein. It is also understood that such values inherently contain variability necessarily resulting from the standard deviations found in their respective testing measurements.

[0145] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges recited herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof, as well as the individual values making up the range, particularly integer values. A recited range (e.g., weight percentages or carbon groups) includes each specific value, integer, decimal, or identity within the range. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, or tenths. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art, all language such as "up to," "at least," "greater than," "less than," "more than," "or more," and the like, include the number recited and such terms refer to ranges that can be subsequently broken down into sub-ranges as discussed above. In the same manner, all ratios recited herein also include all sub-ratios falling within the broader ratio. Accordingly, specific values recited for radicals, substituents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for radicals and substituents.

[0146] One skilled in the art will also readily recognize that where members are grouped together in a common manner, such as in a Markush group, the invention encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group.

[0147] Additionally, for all purposes, the invention encompasses not only the main group, but also the main group absent one or more of the group members. The invention therefore envisages the explicit exclusion of any one or more of members of a recited group. Accordingly, provisos may

[0148]

[0149] apply to any of the disclosed categories or embodiments whereby any one or more of the recited elements, species, or embodiments, may be excluded from such categories or embodiments, for example, for use in an explicit negative limitation.

[0150] The term "at least" prior to a number or series of numbers (e.g., "at least two") is understood to include the number adjacent to the term "at least," and all subsequent numbers or integers that could logically be included, as clear from context. When "at least" is present before a series of numbers or a range, it is understood that "at least" can modify each of the numbers in the series or range.

[0151] The term “CDK4 inhibitor” refers to an inhibitor of cyclin-dependent kinase 4.

[0152] The term “CDK6 inhibitor” refers to an inhibitor of cyclin-dependent kinase 6.

[0153] The term “endocrine therapy” refers to a therapy or treatment useful for modulating (e.g. regulating, reducing, blocking, or inhibiting) the effects of the expression, a level, or an amount of one or more hormones found to cause or otherwise cause progression of a breast cancer as described herein. Endocrine therapy as described herein may include but is not limited to agents known in the art as a non-hormone and hormone therapies such as a selective estrogen receptor modulator (SERM), a selective estrogen receptor degrader (SERD), a gonadotropin-releasing hormone (GnRH) agonist, a selective estrogen receptor covalent antagonist (SERCA), a selective human estrogen receptor partial agonist (ShERPA), an aromatase inhibitor (Al) or a combination thereof.

[0154] 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).

[0155] In embodiments, the CDRs are interspersed with FRs. For example, antibodies can 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.

[0156] 2018)).

[0157] 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

[0158]

[0159] 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 antibodies 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 Diibel eds., 2d ed. 2010)).

[0160] 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., IgGl, IgG2, IgG3, IgG4. In a particular embodiment, the antibodies of the present disclosure are IgM, IgG, IgGl, 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.

[0161] 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.

[0162] 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 extracellular domain (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.

[0163] 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),

[0164]

[0165] glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) phenylalanine (F), tyrosine (Y), tryptophan (W).

[0166] 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.

[0167] 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)).

[0168] 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 KD of less than 10'6molar, such as less than 10’7molar, less than 10‘8molar, less than 10’9molar, or less than IO’10molar.

[0169] 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

[0170]

[0171] physician or caregiver, prescribing the agent for administration and thereby causing the application of an agent to a subject, through ingestion, infusion, injection, or any other means, whether selfadministered 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, breast cancer, HR+ breast cancer, HR+ / HER2- breast cancer, or refractory HR+ / HER2- breast cancer.

[0172] 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, breast cancer, HR+ breast cancer, HR+ / HER2- breast cancer, or refractory HR+ / HER2- breast 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 improvement in one or more cancer endpoints, such as 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 or the Approval of Cancer Drugs and Biologies: 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).

[0173] 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, breast cancer, HR+ breast cancer, HR+ / HER2- breast cancer, or refractory HR+ / HER2- breast cancer. Such an antibody-drug conjugate has a KD of 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

[0174]

[0175] the therapeutic beneficial effects. Many anti-cancer therapies include tamoxifen and other aromatase inhibitors.

[0176] 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 antibodydrug conjugates comprising antibody fragments are routine (see, e.g., Antibody Engineering: Methods and Protocols (Damien Nevoltris and Patrick Chames eds., 3d ed. 2018)).

[0177] The antibodies can be 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, 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)).

[0178] 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). In some aspects, the complementary binding means functions through the same epitope as the disclosed antibodies.

[0179] 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

[0180]

[0181] ameliorate a cancer, such as cancers that express MUC1-C on their cell surface, including breast cancer, HR+ breast cancer, HR+ / HER2- breast cancer, or refractory HR+ / HER2- breast cancer.

[0182] "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, including breast cancer, HR+ breast cancer, HR+ / HER2- breast cancer, or refractory HR+ / HER2- breast cancer.

[0183] 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.

[0184] 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.

[0185] A pharmaceutical composition of the present disclosure contains an "effective" or "therapeutically effective" amount, as used interchangeably herein, of a MUC1-C inhibitor 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 MUC1-C inhibitor to elicit a desired response in the individual. An effective amount is also one in which any toxic or detrimental effects of the MUC1-C inhibitor of the present disclosure are outweighed by the therapeutically beneficial effects.

[0186]

[0187] The term "identity," as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as determined by the match between strings of such sequences "Identity" and "similarity" can be readily calculated by known methods, including, but not limited to, those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, vonHeinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., Siam J. Applied Math., 48:1073 (1988). Typical methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Typical computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and PASTA (Atschul, S. F. et al., J. Molec. Biol. 215 :403-410 (1990). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBINLMNIH Bethesda, Md. 20894: Altschul, S., etal., J. Mol. Biol. 215 :403-410 (1990). The well-known Smith Waterman algorithm may also be used to determine identity.

[0188] The term “standard,” as used herein, refers to something used for comparison. For example, it can be a known standard agent or compound which is administered and used for comparing results when administering a test compound, or it can be a standard parameter or function which is measured to obtain a control value when measuring an effect of an agent or compound on a parameter or function. Standard can also refer to an “internal standard”, such as an agent or compound which is added at known amounts to a sample and is useful in determining such things as purification or recovery rates when a sample is processed or subjected to purification or extraction procedures before a marker of interest is measured. Internal standards are often a purified marker of interest which has been labeled, such as with a radioactive isotope, allowing it to be distinguished from an endogenous marker.

[0189]

[0190] The term "contacting" refers to the act of touching, making contact, or of bringing to immediate or close proximity, including at the cellular or molecular level, for example, to bring about a physiological reaction, a chemical reaction, or a physical change, e.g., in a solution, in a reaction mixture, in vitro , or in vivo.

[0191] The term "in need thereof means that the subject has been identified or suspected as having a need for the particular method or treatment. In some embodiments, the identification can be by any means of diagnosis or observation. In any of the methods described herein, the subject can be in need thereof.

[0192] As used herein, a “subject in need thereof’ is a patient, who will benefit from the method of this invention. The terms "subject" refers to an animal, such as a mammalian species (e.g., human). A subject can be a healthy individual, an individual that has or is suspected of having a disease or a predisposition to the disease, or an individual that needs therapy or suspected of needing therapy. The terms "individual" or "patient" are intended to be interchangeable with "subject."

[0193] As used herein, the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof, are intended to be inclusive similar to the term “comprising.”

[0194] The terms “comprises,” “comprising,” and the like can have the meaning ascribed to them in U.S. Patent Law and can mean “includes,” “including” and the like. As used herein, “including” or “includes” or the like means including, without limitation.

[0195] Methods involving conventional molecular biology techniques are described herein. Such techniques are generally known in the art and are described in detail in methodology treatises, such as Molecular Cloning: A Laboratory Manual, 4th ed., Green and Sambrook, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y, 2014; and Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1992 (with periodic updates). Methods for chemical synthesis of nucleic acids are discussed, for example, in Beaucage and Carruthers, Tetra. Letts. 22: 1859-1862, 1981, and Matteucci et al., J. Am. Chem. Soc.

[0196] 103:3185, 1981.

[0197] MUC1 / MUC1-C

[0198] MUC1 is a mucin-type glycoprotein that is expressed on the apical borders of normal secretory epithelial cells (Kufe et al., 1984). MUC1 forms a heterodimer following synthesis as a single polypeptide and cleavage of the precursor into two subunits in the endoplasmic reticulum

[0199]

[0200] (Ligtenberg et al., 1992). The cleavage may be mediated by an autocatalytic process (Levitan et al., 2005). The >250 kDa MUC1 N-terminal (MUC1 N-ter, MUC1-N) subunit contains variable numbers of 20 amino acid tandem repeats that are imperfect with highly conserved variations and are modified by O-linked glycans (Gendler et al., 1988; Siddiqui et al., 1988). MUC1-N is tethered to the cell surface by dimerization with the ~23 kDa C-terminal subunit (MUC1 C-ter, MUC1-C), which includes a 58 amino acid extracellular region, a 28 amino acid transmembrane domain and a 72-amino acid cytoplasmic domain (CD) (Merlo et al., 1989). It is the 58 amino acid portion of the MUC1-C / ECD (italics) to which antibodies of the present invention bind. The human MUC1- C sequence is shown below:

[0201] SVVVQLTLAFREGTINVHDVETQb'NOYKTEAASRYNLTlSDVSVSDVPbP F£42 G^GVPGWGIALLVLVCVLVALAIVYLIALAVCQCRRKNYGQLDIF PARDTYHPMSEYPTYHTHGRYVPPSSTDRSPYEKVSAGNGGSSLSYTNPA VAATSANL

[0202] (SEQ ID NO:91

[0203] (The bold sequence indicates the CD.) With transformation of normal epithelia to carcinomas, MUC1 is aberrantly overexpressed in the cytosol and over the entire cell membrane (Kufe et al., 1984; Perey et al., 1992). Cell membrane-associated MUC1 is targeted to endosomes by clathrin- mediated endocytosis (Kinlough et al., 2004). In addition, MUC1-C, but not MUC1-N, is targeted to the nucleus (Baldus et al., 2004; Huang et al., 2003; Li et al., 2003a; Li et al., 2003b; Li et al., 2003c; Wei et al., 2005; Wen et al., 2003) and mitochondria (Ren et al., 2004)).

[0204] MUC1-C interacts with members of the ErbB receptor family (Li et al., 2001b; Li et al., 2003c; Schroeder et al., 2001) and with the Wnt effector, P-catenin (Yamamoto et al., 1997). The epidermal growth factor receptor and c-Src phosphorylate the MUC1 cytoplasmic domain (MUC1- CD) on Y-46 and thereby increase binding of MUC1 and P-catenin (Li et al., 2001a; Li et al., 2001b). Binding of MUC1 and P-catenin is also regulated by glycogen synthase kinase 3P and protein kinase C8 (Li et al., 1998; Ren et al., 2002). MUC1 colocalizes with P-catenin in the nucleus (Baldus et al., 2004; Li et al., 2003a; Li et al., 2003c; Wen et al., 2003) and coactivates transcription of Wnt target genes (Huang et al., 2003). Other studies have shown that MUC1 also binds directly to p53 and regulates transcription of p53 target genes (Wei et al., 2005). Notably,

[0205]

[0206] overexpression of MUC1-C is sufficient to induce anchorage-independent growth and tumorigenicity (Huang et al., 2003; Li et al., 2003b; Ren et al., 2002; Schroeder et al., 2004). MUC1 Inhibitors

[0207] Small Molecule Inhibitors of MUC1 / MUC1-C

[0208] Small molecule inhibitors of MUC1 include salinomycin (CAS #: 53003-10-4). Other approaches to inhibit MUC 1 include using sRNAs or anti-cancer peptides. Other approaches to inhibit MUC1 include using PROTACs.

[0209] Small RNA molecule Inhibitors

[0210] sRNA sequences are designed to target specific gene sequences such as the mRNA of MUC1-C. sRNAs that inhibit cell invasion, metastasis or suppress translation of MUC1 may be selected from siRNA, shRNA, and sgRNA. In embodiments, MUClshRNA is used in the invention which inhibits MUC1-C by silencing its gene expression.

[0211] Peptide Inhibitors

[0212] Anti-cancer peptides that are functional against MUC1 include GO-203 and GO-201 (US Pat No 11136410).

[0213] GO-203 is a cell-penetrating peptide inhibitor specifically designed to target the oncogenic protein MUC1-C. By inhibiting MUC1-C, GO-203 disrupts its interaction with other signaling molecules and transcription factors, thereby impeding cancer cell growth and survival. IUPAC Condensed sequence of GO-203 is:

[0214] RRRRRRRRR-CQCRRKN (SEQ ID NO:92, all D amino acids).

[0215] Condensed sequence of GO-201:

[0216] D-Arg-D-Arg- D-Arg- D-Arg- D-Arg- D-Arg- D-Arg- D-Arg- D-Arg-Cys-Gln-Cys-Arg-Arg- Lys-Asn-Tyr-Gly-Gln-Leu-Asp-Ile-Phe-Pro (SEQ ID NO:93).

[0217] MUC1 Antibodies and conjugates thereof (ADC)

[0218] Antibody Drug Conjugates or ADCs are a class of highly potent biopharmaceutical drugs designed as a targeted therapy for the treatment of people with cancer. ADCs are complex molecules composed of an antibody (a whole mAb or an antibody fragment such as a single-chain variable fragment, or scFv) linked, via a stable chemical linker with labile bonds, to a biological active cytotoxic (anticancer) payload or drug. Antibody Drug Conjugates are examples of bioconjugates and immunoconjugates.

[0219]

[0220] By combining the unique targeting capabilities of monoclonal antibodies with the cancerkilling ability of cytotoxic drugs, antibody-drug conjugates allow sensitive discrimination between healthy and diseased tissue. This means that, in contrast to traditional chemotherapeutic agents, antibody-drug conjugates target and attack the cancer cell so that healthy cells are less severely affected.

[0221] In the development ADC-based anti-tumor therapies, an anticancer drug (e.g., a cell toxin or cytotoxin) is coupled to an antibody that specifically targets a certain tumor marker (e.g., a protein that, ideally, is only to be found in or on tumor cells and is not shed from the cancer cell membrane; in this case MUC1-C). Antibodies track these proteins down in the body and attach themselves to the surface of cancer cells. The biochemical reaction between the antibody and the target protein (antigen) triggers a signal in the tumor cell, which then absorbs or internalizes the antibody together with the cytotoxin. After the ADC is internalized, the cytotoxic drug is released and kills the cancer. Due to this targeting, ideally the drug has lower side effects and gives a wider therapeutic window than other chemotherapeutic agents.

[0222] A stable link between the antibody and cytotoxic (anti-cancer) agent is an aspect of an ADC. Linkers are based on chemical motifs including disulfides, hydrazones or peptides (cleavable), or thioethers (noncleavable) and control the distribution and delivery of the cytotoxic agent to the target cell. Cleavable and noncleavable types of linkers have been proven to be safe in preclinical and clinical trials.

[0223] The availability of more stable linkers has changed the function of the chemical bond. The type of linker, cleavable or non-cleavable, lends specific properties to the cytotoxic (anti-cancer) drug. For example, a non-cleavable linker keeps the drug within the cell. As a result, the entire antibody, linker and cytotoxic (anti -cancer) agent enter the targeted cancer cell where the antibody is degraded to the level of an amino acid. The resulting complex — amino acid, linker and cytotoxic agent — now becomes the active drug. In contrast, cleavable linkers are catalyzed by enzymes in the cancer cell where it releases the cytotoxic agent. The difference is that the cytotoxic payload delivered via a cleavable linker can escape from the targeted cell and, in a process called “bystander killing,” attack neighboring cancer cells.

[0224] Another type of cleavable linker, currently in development, adds an extra molecule between the cytotoxic drug and the cleavage site. This linker technology allows researchers to create ADCs with more flexibility without worrying about changing cleavage kinetics. Researchers are also

[0225]

[0226] developing a new method of peptide cleavage based on Edman degradation, a method of sequencing amino acids in a peptide. Future direction in the development of ADCs also include the development of site-specific conjugation (TDCs) to further improve stability and therapeutic index and a emitting immunoconjugates and antibody-conjugated nanoparticles.

[0227] Provided herein are antibody-drug conjugates (ADCs). In embodiments, the provided ADCs are of the formulas:

[0228] Formula I. A-D

[0229] wherein:

[0230] A is an antibody or fragment thereof that binds MUC1-C; and

[0231] D is small molecule drug / cytotoxic, a derivative thereof, or a pharmaceutically acceptable salt thereof.

[0232] In embodiments, the antibody or fragment thereof that binds MUC1-C (A) is conjugated, or linked, through a linkage to the small molecule drug, a derivative thereof, or a pharmaceutically acceptable salt thereof (D) of Formula I.

[0233] Various linkages are possible for embodiments of Formula I. Conventionally, ADCs are linked to antibodies or fragments thereof through lysine or cysteine amine or sulfhydryl groups 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 P-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.

[0234] Additional linkages that are useful in embodiments of ADCs of the present disclosure can be found in US Patent Publication No. US 10,617,773 and Antibody-Mediated Drug Delivery Systems (Pathak and Benita, eds., 2012), both of which are incorporated by reference in their entireties.

[0235] Some embodiments can also include nanoparticles comprising polymers, a mixture of polymers and lipids, or lipids. Polymers contemplated in the present disclosure include polyethylenimine (PEI), poly(lactic acid) (PLA), poly(glutamic acid) (PGLA), poly (cyanoacrylate), chitosan, poly(ethylene glycol) (PEG), polypropylene glycol) (PPG), and

[0236]

[0237] derivatives thereof. Lipids contemplated in the present disclosure include cholesterols or saturated or unsaturated phospholipids, which can be functionalized with various polymers, such as PEG and derivatives thereof.

[0238] In embodiments, the nanoparticle 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. US 2020 / 0046648, which is incorporated by reference in its entirety.

[0239] Antibodies binding to MUC1-C extracellular domain (ECD)

[0240] 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

[0241] (SEQ ID NO: 57, MUC1-C / ECD)

[0242] In embodiments, the antibody or fragment thereof binds the MUC1-C / ECD at the a3 helix (VHDVETQFNQY, SEQ ID NO: 58) or a4 helix (TEAASPYRN, SEQ ID NO: 59).

[0243] 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 IgGl. In embodiments, the constant region is IgG2. In embodiments, the constant region is IgG2a.

[0244] 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. Tables 1 and 2 includes example sequences that can be used in the present disclosure.

[0245] Table 1. Example sequences for use in the ADCs of the present disclosure.

[0246]

[0247] Clone HCVR, I.CVR CDR1 CDR2 CDR3 name

[0248] 8E1 / 8F1 HCVR EYEFPSHO INSDGOST VRLYYGNVM EVQLVESGGGLVa^ESLKLSCESWEYEFPSHDMSWV D¥ RKTPEKRLELVAAINSDGGSTYYPDTMERRFHSRDNTKK SEO ® NO: SEO ® NO: TI.Y1.QMSSIRSEDTAI.¥¥€VRL.Y¥GNVMD¥WGQ.GTSV IS 16 SEO ID NO: WSS 17

[0249] SEO ® NO: 1

[0250] tom QSIVHSNG KVSNRFS SOSTHVH.T DWMTQTPISLWSLGDQMISCMSQSLVHS^GHIYL NTY ¥¥¥¥lQKPGQSPKLlDfKVSNNFSGVPDRFSS8GSGTDFT SEO ID NG: SEO 10 ND: LKISRVEAEDLGVffOStlSIHVPLITGAGIlCtELK SEOJD NO: 19 20 SEO ® NO: 2 18

[0251] SAS HCVR GFSinYG iWSQGST AKNYLGSLDY OyQLKESGPGlVAPSaSLSMTCTVSGFSLTTYGVHWV RDFPGKGkEWl.WI¥¥SDGSH¥NSPlKSRISISRDNSKS SEO KI NG: SEO ID NG: SEQIO NO: QVFI.KMNSLQAEJDTAIYYCAKNYLGSLDYWGQGTSV’I’ 21 22 23 VSS SEO ® NO: 3

[0252] LCVR OSIVHNN KVSNRFS SQTTHVPLT

[0253] D¥ / VLWPL$LPVSlGE?OASISCRSSD.SLVHNNGEm / LH GDTY IVYLatPGQSPRLGYKVSNRFSGVPSRFSGSGSGTGFTF SEO ID NO: SEO ® NO: KISRVEAEDlGVYFGSQTmVPlTFGAGl^LElK SEO.® NO: 25 26

[0254] SEO ® NO: 4 24

[0255] 2H11 HCVR GYTFTGYS WINTETGEP GTGGDD

[0256]

[0257] MH TYDDFKGQIQLVQSGPELKKPGEWKTSCKASGYTFTGYSMHWVK SEQ 1D N0: <MPGKGLKWMGWi^TETGEP7¥A00FKGRFALSLETS SEQJDNO: SEQsQ NO: 29 ASn¥lQiN«LKNEDTAI¥FCVRGTGGDDWGQGTTLT 27 28

[0258] VSSAKHP SEQID NO: 5

[0259] LCVR RSSQSLVH KVSWS SOGTHVPPT EWMTQIPLSLPySLGDQASiSCRSSQSLVHSNGNTEL SNGNTVLH HW¥LQKPGQSPi(Lli¥KVS«RFSGVPDRFSG$GSGTDF SEO ID NO: SEO ID NG: TLKiNR¥€A.EDLGV¥FCSG.GTHVPPfFGGGTKL£lKRAQ SEO IQ ND: 31 32 AAFTV 30

[0260] SEO ID NO: 6

[0261] HCVR GFTFN¥FW EILPGTGST ¥D¥TSSMD¥ QVaiQQSGAELMKPGASVKJSCKAIGFTFNYFWlEWV IE NWKFKG KQRPGHGLEWIGEIlPGTGSWNEKFKGKAIFrADTSS SEQ IQ NO: NTA¥MQLRSITSEDSAV¥YCVR¥D¥TS$MD¥WGQGTS SEQJDNO: SEOJD NQ: 35 VIVSS 33 34

[0262] SEG :□ NO: 7

[0263] LCVR CKASENVG GASNRVT GOSYSYPWI NIVMTG6PKSMSMSVGERV1LTCKASENVGTWSWY I¥VS QQKPEQSPKLLI¥GASNR¥TGVPNRFTGSGSATDFTLTI SEO; IO NO: SEO IQ NO: SSVQAEDLAOY¥CGDS¥S¥PWTFGGGTiClEIK SEQIDNO: 37 38 SEQ ID NO: 8 3b

[0264] HCVR GHTF-TSYW EINPSNGRT OGLHVSGFA QVaiQ(^GAaWPGME.KlSCKASGHTFTS¥WMHW MH Y' / NENFKT ¥ VKORPGQGLEWIGE1NPSNGRTYYNENFKIXAH3VDK ¥SSSASMQLRSLTSEDSAV¥¥CASDGD¥\«GFA¥WGQ. SEO IQ NG: SEO: i 0 NO: SEO. ID NG: GTT1TVSS 39 40 41 SEQ ID NO: 9

[0265] LCVR CRA5ESVQ GASNVET QQNWKVP DIVLTQSPGSUVSLGOSVTISCRASESVOYSGTSLMHW YSGTSLMH WT YQQKPGQPPKLUYGASNVETGWARFSGSGSGTDFSL SEO ID NO:

[0266] NlHPVEEDDlAMYFCQQNWKVPWTFGGGriCLEIK SE0JDNO: 43 SEOJD NO: SECOO NG: W 42 44 h7B8-l(VH-Vl.>-hFc-h€D3(Vl-¥H|-scFv

[0267] Leader sequem:e-7B8-i heavy chain variable region •• (G4S)3-7B8-1 Light chain variable region- G4S Human igGl Fc G4S-CD3 light chain variable region (G4S)3 CD^ heavy chain variable region

[0268] SEO ID ND: 60

[0269] MEI^tSWFLVALFRC^(X:£V01VQSGAEVKKPGESm$€KGSGn'FN¥F-WEWVRaMPGKGLEWMG EILPGTGSW^EKFKGQVriSAQKSSTA¥LaWS$LKASDTAMW<^RW¥TS$MDYWGQGTLVTVSSGG GGSGGGGSG^GSEIVLTQSPATUISPGERAUSCRASESVQYSGT^LMHWYQQKPGQAPRLUYGASHV ETGIPARFSGSG^WmilSSLEPEDFAWYCaQFIWKVPWTFGaSTKVElKGGGGSEPKSCOCTHTCPPC ?APEAAGGiGVFLFPPKPRDTLMGRTPEVTCVVVDVSHEDPEVKFNW¥VDGVEVHNAKTKPREEO¥NS1Y RWSVLTVLHQDWLNGKE¥KCmNKALGAPiEKTlSKAKGOPREPO.WIlPPSRDELrKNQVSlSCAVKGF ¥iJSDiAVEWESNGQPENN¥DnPmDSDGSFFLVSKETVDKSRWQQGNVF$CSVMHEALHNH¥TQKSl.S

[0270]

[0271] LSPGKGGGGS81QMTQSPSSiSASVG&RVHTCRAS0DIRN¥lNW¥OTPGKAPKLLI¥¥TSRLESGVPSRF

[0272]

[0273] SGSGSGTQYTLTiSSLQPEDFArVYCQQGhlTLPWTFGQGTKVElKGGGGSGGGGSGGGGSEVQLVESGGG lVaPGG$lRL$CAMG¥SFTG¥TMNWVRDAPGKGlEWVAUNP¥KGVTT¥ADSVKGRFTISVDK$mTA¥L QM?4SLRAEDTA¥¥¥CARSG¥¥GDSDW¥FD¥WGQ.GTL¥T¥SS

[0274] h7B84(yH-VLf-hFe-ssr:Fv

[0275] leader seqaenw-788-1 heavy chain variable- region - (G4S)3-7B84 Light chain variable reglon- G4S-Huffian::gbl Fc

[0276] SEQ ID NO: 61 MEFGLSWFLVALFRGVQCEVaiVQSGAEVKiCPGESLKiSCKGSGFTF?i¥FWIEWyRQMPGi(GL£WMG E:LPGrGSTN¥NFKFkbQ¥T:SADKSIS'A¥lQWbSLKASD'AMtYCAR¥G¥lSSMD¥WG$2GTlVT¥SSGG GGSGGGGSGGGGSElVLTQSPATlSLSPG£RAn.SCRASESVQ¥SGrSlMHW¥QaKPGQAPRlU¥GASHV eGlPARFSGSG.SGTOFTlTISSlEPEDFAm€mHWKVPWFG»TKVEIKGGG&SEPKSCOKTHTCPPC PAPEAAGGPSVFlFPPKPmnMISRTPE¥TCV¥WV$HEaPEWFHW¥VDGVEVHfiAKnCPREEQ¥N$l¥ RWS¥LTVLHQDWLNGiCE¥K€ikVSHKALGAPIEK'nSKAitGQ?’REPQ¥¥TLPPSRDELTKHQVSLWQ¥KG FWSDiAV£W£SHGQPEHWKTEPPVLKSOGSFFL¥SKLni>KSRWCaGHVFXSVMH£ALHHH¥TCmSl SLSPGK

[0277] h 788 - 1( ¥L-VH|-hFc-CD3 (VH- ¥l.?-s:s;Fv

[0278] leader eeqsn?nne-7BS-l Light chairs variable reginn-{G4S)37B8-1 heavy chain variable region - G4S-Human IgGl Fc-G4S-€D3 heavy chain variable regton-(G4S|3-W3 light chain variable region

[0279] SEO ID NO: 62 MK¥LLPTAAAGLLLLMOPAMAEIVLTQSPATLSLSPGERATL.SCRASESVU¥SGTSLMHW¥QQKPGaAPR ILiYGASHVETGIPAFtFSGSGSGTDFTtTISSlLPLOFAWVCGQNWKyPWTFGQGTKVEiKGGGGSGGGG SGGGGSE¥QLVQSGAEVKi(PSESLKiSCKGSGFTFNYFWi:EWVR.QMPGKGLEWMGEILPGTGSTN¥NESF KGQVFISADKSISTAUQWSSIJCASOTM^WCARYDYrSSMDYWGaGTEVTVSSGGGGSEPKSCOnHTC PPCPAPEAAGGPSVFIFPPK?KOTLM1SRTPE¥TCWVDVSHEDPEWFHWW»GVEVHMAK?KPREEQ¥ NST¥RWSyLWLHQryM^GK£¥KCKVS^KALGAPIEKTISKAKGaPREPamLPPSRl>ELTKNa¥SLSCA VKGP7PSDIAyEWE$NGaPENN¥DTTPP¥LDSDGSFFl¥SKLT¥DKSRWaaGFWFS€5VMHEALH^H¥T qKSLSLSPGKGGGGSEVa.VESGGGlVQPGGSLRlSCAASG¥SFTG¥TM^WRQAPGKGLEWVAL.iHP¥K GVmAD$VWRniSVQKSWAYL^HSLRAE0TAV¥YCARSGWGmDWF»WGQGTLVWSSt3G GGSGGGGSGGGGSDIQ;MTQ$PSSlSASVGDhVTiTCFtA$QDIRN¥LNWYQaKPGKAPKlLi¥¥T$RLESGV PSRFSGSGSGTD¥TlTiS^.QPEDFAl¥¥CaQGN7LPWTFGqGnVEiK _

[0280] h78S- (VL-VHhhFc-seFv

[0281] Leader sequen<ce-788-l light chain variable region-(G4S}3-7BB-l heavy chain variable region - GAS- Human IgGl Fc

[0282] SFQ ID NO: 63 MK¥LLPlAAAGLLLLMQPAMAEiynQSPATiSlS?GERATlSCRASESyQ¥SGFSLMHW¥aaKPGaAPR lLI¥GASNyETGiPARFSGSGSGTQFTLTISSLEPE0FAy¥¥€UQNWK¥P¥VTFGQGTKV£IKGGGGSGGGG S^GG$EVQLVaSSAEVKKPGE$LKiSCKG$GFTFmFWiEWVRQMP6KGlEWMGEIlPGTGSW¥«EKF KGQ¥TISADKSISTA¥L€WSStKASDTAMY¥CAR¥D¥TSSMGWGQ:GTLY,T¥-SSGGGGSEFKS€DKTHTC ?PCPAPEMGGP$VFtF??KPK»TLMISRT?mCW¥DV5H£DPEVi<FHWWDGVEVH«AKTKPREEQ¥ N;$mWS¥lWLH(M>¥Yl«GKE¥KCKVSMAlGAPiEKB$KAKGa>REPQVnLPPSROEl3K^QVSLWa yKGF¥PSL>LA¥EWESNGQPENN¥KrfPP¥LKSDGSFFL¥SKLr¥T}KSRWaaGN¥FSCS¥MHEALHNH¥|-Q KSLSL5PGK

[0283] 67884 (yH-CHlhmh?:c-hCD3 iVl-VHrscFv

[0284] VH of humanized aatl-MUC-l antibody 7B&-1, CHI of human IgGl, mhFc of human IgGl, Linker ~ Anti-human CD3 VI, Linker ~ Antl-hmnan CDS VH

[0285]

[0286] SEQJD HG: 64EVOIVQSGAEVKKPGESLKISCKGSGFEFNYFWIEWWQMPGKGLEWMGEILPGTGSTNYNEKFKGQVH $ADK5ISTAYLQW$5LmDTAMY\TAR¥EPmSMDn¥GaGTL¥I¥SSASTKGPSWPLAPSSKST$GGTA AlGaVKDYFPEPVWSV^SGALTSGVHTFPAViaSSGLYSL^WWPSSSlGTQTYICNVNHKPSNTKVQ KKVEPKSWKIYTGPPGPAPELLGGPSVFCFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWmXWE VHNAKTKPREEOyNSTYRWSVLTVLHQDWt^GKEYKCKVSNKALPAPIECTiSKAKGQCREPQVYRPPSR DEITKNQVSLRCHVKGFYPSDIAVEWESNGQPEFINYKTTKPVIDSDGSFFIYSKTVDKSRWQQGNVFSCS VLHEGLHNHYTCMSLSlSPGKGGGGSDIQMTQSPSSlSASVGDRfZFnCRASC^iRNYLNWYQQKPGKAPK LLiYYISRLESGVPSRFSGSGSGTDYrLTISSLQPEOFATYYCQQGNTLPWrFGQGTKVElKGGGGSGGGGS GGGGSEVQLVESGGGLVaPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVmAOSV KGRFH$VDKSKNTA%.C^4N$LRAEDTAVY¥CARSGYYGDSOWYFOVWGQGTIVTV^

[0287] h788-l (VL-Cq

[0288] VI of humanized enli-MDC- 1 antibody 7B8 1.. CL of human IgGl

[0289] SEO ID NO: 65 EIVLTQSPATLSLSPGERATlSCRASESVQ¥SGTSLMHWYQQKPGaAPRLli¥GASNVnGIPARFS6SGSGT DFiniSSlEPEDFAmCQQNWWPWTFGQGIWEIKRIVAAP5VFtFPPSDEQLKSGTASWaENNF¥PR EMVaWRVDNM.QSGNSQ£SVTECM3SKDSI¥SLSSTLTESKAD¥EKHKVYA€EVrHQGLSSPVFKSFNRG£ C

[0290] H€W< NFWMN QIYPGDGDT SYYR5AWFA aVaiQQSGAELVRPGSSVKIXKrSGYAFSNFWMNW NYNGKFKG Y ¥K0:RPCO3LEWlGDJYPGDGDTN¥NGKFKGi<ATLTAEi SEO ID NO:

[0291] SSSSTA¥MQL5SLTSEASAV¥FCARSY¥RSAWFA¥WGQ 45 $EG. ID NO: SEDJD NO: GTLVSVSA 46 47 SEO ID MO: 11

[0292] LCVR: RASOSIGTS YASESIS QQSNNWPL QILLTQSPAILSVSPGERVSFSCRASCtSIGTSIHWYQQRT IH T NGSPRLLIKYASESiSGIPSRFSGSGSGTDFRSINSyESED SEO ID NO: lADYYCQQSNNWPLTFGAGTKLELK SEO: ID NO; 49 SEDJO NO: SEC 10 NO: 12 48 50 h.3Dl(VH-yL)-hFc-h€D3(Vl-VHHcFv

[0293] leader sequence-301 heavy d»tn variable region - (G4$|3-3D1 Light chain variable reglon- G4S-Human IgGl Fc -G4S-CD3 light chain variable reglon-{G4S)3 -CD3 heavy chain variable region

[0294] SEQ ID NO: 66 MEFGLSWVFLVAlFRGVGCEVQLVOSGAEVaPGESmSCKGSGYAFSNFWMNWVRQMPGKGLEWM GDjYPGOGDTNYNGKFKGQV^SADKSISTA¥lQWSSLKASDTAM¥¥CARS¥¥R$AWFA¥WGQGTLVTVS LGGGGSGGGGSGGGGSEmT(^PDFaSVTPKEKVTIT€RASaSIGTSIHW¥CK^PDmPKLLlRYASESiSG VPSRFSGSGSGTDFnilNSlEMDAATYYCQC^NNWKTFGQGTKyElKGGGGSEPKSCOKTHTCPPCPA? EAAGGP$VF^PPKPK»nMISRTPE¥TCVW»VSHEDPEVKFNWWDGV£VHNAKTKPREEQYNST¥RW WLTYLHQDWLNGKEYKCmNKAlGAPiEiCriSRAKGQPREPQWTlPPSRDELTICNQYSLSCAVKGFYPS DIAVEWESNGaPENNYDTTPPVLDSDGSFFLVSKLWDICSRWQC^NVFSCSVMHEAlHNHYI-aKSlSlSP GKGGGGSDIWTQSPSSLSASVGDRVriTCRASQDIRNYLNWYQQKPGKAPKUiWTSRLESGWSRFSGS G»TDYTlTISSLQPEDFAT¥¥CQQGNTLPWlTGC>GTKVEIWGGG5GGGG$Ga3GSEVa.VESGGGLVQ PGGSLRL5CAASG¥SFTCYt7MNWRaAPGi?:GL£¥WALINPYKGVn¥’ADSVKGRFTS¥DICSKNTAYLQM NSLRAEDTAV¥¥CARSG¥¥GDSDW¥FQVWGQGTl¥n?SS

[0295] h301(YH-VL)-hFe-$cFv

[0296] Leader sequeme-3Dl heavy chain variable region - (G4S0-3D1 Light chain variable region-

[0297]

[0298] G4S-Hnntan IgGl Fe

[0299]

[0300] SEQ ID NO; 67 MEFGI^WVFlVALFRGVQCEVQLVaSGAEVKKPGESlKISCItGSGYAFSWWMfiWVRQMPGKGlEWM C^llYPGIX^DrNYNGKFKGUVTiSADKSiS’l'AYLQWSSLKASiyi'AMYYCARSYYRSAWFAYWGCMSTlVTVS LGGGGSGGGGSGGGGSEIVlIOSPDFQSVrPKEKVTITCRASQSIGTSIHWYQQKPQOSPKLlhCYASESiSG VPSRFSGSGSGTDFTi;NNSI.EAEDAAIY¥aMBNNWPLI¥GQGTKVEIKGGGG8EPKSCDKTHTCPPCPAP EAAGGPSVFLFPPKPKDUMiSRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV SVLTVLHQOWLNGKEYKCKVSNKALGAPIEKTISKAKGaPREPQVYn.PPSRDElTKHa^LWaVKGFYPS DIAVEWESHGClPENN¥mPPVi.KSDGSFFt¥SKlW>KSRWCKlGNVFS€5VMHEALHNH¥T0XSlSi.SP GK

[0301] h3miVi-VHH'Fc-hCD3(VH-Vi i-5<:Fe

[0302] Leader sequence-301 light chain variable region- (G4S)3 - 3D1 heavy chain variable region - G4$-Human ig<31 Fc -G4S-C03 heavy chain variable reglon-(G4S)3 -CD3 light chain variable region

[0303] SEO ID NO: 68 MKYLLPTAAAGLLLLAAaPAMAEIVLTQSPDFQSVTPKEmiTCRASOSIGTSIHWyaaKPDOSPaLmYA SESISGVPSRFSGS^GTDFKTimLEAEDAAWYCQQSNNWPLTFGQGTKVElKGGGGS^GGSGGGGS EVUVQSGAEVKKPGESLKISCKGSGYAFSHFWMNWVRQMPGKGLEWMGWYPGDGDT^YHGKFKGQ VTiSADKSISTAYLQWSSLKASDYAM¥Y(ARSYYi?5AWFAYWGQGTlVlVSi.GGGGSEPKSCDKTHTCPPC PAPEAAGGPSVFLFPPKPKDTLMiSRTPEVTCVWaVSHEDPEVKFNWyVDGVEVHNAKTKPREEQYNSTY RWSVLIVLHaDWLNGKEYKCKVSN KMWIEKTISKAKGORE PQVniPPSROELTKNQVSlSCAVKGF YRSDIAVEW£SNGQPENNYDTTPPVlOSDG5FFLVSKL.TVDK8RWCLaGNVF8CSyMHEALHNHYTQKSL& t.$PGKGGGGS£VQtVESGGGLVUPGGSLRtSCAASGYSFTGYlMNWVRQAPGKSLEWVALiNPYKG¥rr YADSVKGRFTiSVDKSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSBWYFQVWGQGTLVTVSSGGGGSG GGGSGGGS8DlOA1TO5PSSLSAS¥G0RVTiICRASO,Dl RNYLNWYQQKPG KAPKLUYYFSRL .ESGVPSRFS G5GSGTDnLTI.SSLaP£DFArf¥CaQGNTLPWTFGQGTRVEIK

[0304] h3Dl(VL-VH)-hFe&cFv

[0305] Leader sequence-301 Light chain variable reglon-(G4S|3- 3D1 heavy chain variable region - G4S- Human IgGl Fc

[0306] SEQID NO: 69 MKYLLPTAA4GLLLLA4O.PAMAEIVLTQSPDFQSVTPK£K¥TiTCRASOSIGT$iHWQOKPDD,SPKlLh(¥A SEStSGVPSRFSGSGSGTDFTlTINSLEAEDAATYYCQQSNNWPLTFGQGTKVElKGGGGSGGGGSGGGGS EVQLVQSGAEVKKPGESLKlSCKGSGYAFSHFWMMWVRQMPGKGLEWMGQiYPGDGDTIMYHGKFKGQ VTISADKSISTAYLQWSSLKASDTAMWCARSYYRSAWFAYWGQGTLVTVSLGGGGSEPI^CDKTtfi'CPPC PAPEMGGPSVFlFPPKPKDTLMiSRTPPZTCVWDVSHEDPEVKFNWYVDGVEVHNAKWREEaYNSTY RWSVLTVLHQnWLNGICE¥KCK¥SNi<AMSAPEKTISKAKGaPREPQmLPPSRDElTKDaVSLWaVKG F¥PSDiAVEWESNGQPENN¥KnPP¥lKSDGSFFL¥SKLTVDKSRWQQGNVF$C$VMHEALHNH¥TQK$L SLSPGK VH of humanized ant»-MUC-l antibody 3D1{CHI of human IgGl, Fc of human IgGl with LALA- PG mutations,, ticker ~ Anti-human CDS VI, Linker ~ Anti ■human CD3 V H

[0307] SECHD NO; 70 EVDLVQSGAFVKKPGESLKiSCKGSGYAFSNFWNINWVRQMPGKGLEWMGQiYPGDGDTNYNGKFKGO. VTISADKSISTAYLQWSSLiCASDTAMYYCARS'YYRSAWFAYWGQGTLVTVSLASTKGPSVFPLAPSSKSlSG GTAALGaVK»YFP£PVTVSWNSGALTSGVHFFPAVl.QSSGl¥SLSSWTV^S$LGTQl¥lCNVHHKP$NT KVDOVEPKSCDKTHTGPPCPAPEAAGGPSVFLFPPKPKOTLMISRFPEVTCVWOVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRWSVLWLHQDWLNGKEYKCKV^KALGAPiEKTISKAKGQPREPQVYTL

[0308]

[0309] PPSROELTKNQVSLTCLVKGF¥PSDIAVEWESI''iGOPENN¥KTTPPVI.DS[XiSFFL¥SKnVDKSRWQQGNV

[0310]

[0311] F$CSVMi4EALHNil¥TGK5L5LSPGKGGGGSD4G.MTQ$P5St5ASVC<3RVTiT€RA$QDiRN¥LNW¥QQKF GKAPKLLiWSRlESGVPSRFSGSGSGTOYTLTlSSLQPEDFATfYCQClGNTlPWTFGQGTKVEiKGGGGSG GGGSGGGGSEVQLVtSGGGL¥QPGGSLFG5CAASG¥5FTG¥7MNWVROAPGKGl.LW¥AGNP¥KGVTTY AOWKGRHISVDKSK«TAYLQMNSlRA£DTA¥¥¥CARSG¥¥GDSDW¥FaVWG€^TlVlVSS hBDI (VL-F.L}

[0312] VI of humanized anti-MUC-1 antibody 3D1, CL of human igGl

[0313] SEQ IE) NO: 71

[0314] 5GSGTDFTI.T1 NSLLAEE^mCOQSNNWPETFGacaKVEiKRWAAPSVFiFPPSOEQLKSGTASWCLL? ^FYPRESKVQ WKVDNALQSGflSGEStGEQDSKGSVfSLSSTLTLSKAQYEKilhWACEyTI-iQGLSSPVTKS FNRGEE 4G5 HCVR GFSLSTSG HiYWDDQK GVSSWFPY aiTLKESGPGiLOPSQTlSLTCSFSGFSLSTSGMGVSWIR MGVS RW5LK5 QPSGKGLEWLSHi¥WDE?DKR¥NPSLK$RLSiSKEffSRN SEQ iD NO: QVFlKiTSVDTA0TAIY¥€APGVSSWFP¥WGPGTlVTV SEQ iD GO: SEQ i O NO: 53

[0315] SA 51 52

[0316] SEQ H.) NO: 13

[0317] LCVR CKASQ.SVG FASNRYS QQHYiFPYT XP / M7Q7PKFLPVSAGGRVTVTCmQSVGN¥VAW¥Q NWA QKPGQSPKLUYFASNRYSGVPD SEOJD NO: SEQ ID NO: RFTGSGSGTDFTFFSSVO.VEEiLAVYFCQCH-QlFPWFGS SEQiG NG: 55 56 GTKLEiK 54

[0318] SEQ ID NG: 14 _

[0319]

[0320] 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.

[0321] 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

[0322]

[0323] 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, 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.

[0324] 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

[0325]

[0326] 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, 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.

[0327] 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

[0328]

[0329] 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, 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.

[0330] 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.

[0331] 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

[0332]

[0333] HCDR3. Tn embodiments, the amino acid sequence of HCDR1 is SEQ TD 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 of HCDR2 is SEQ ID NO: 52, and the amino acid sequence of HCDR3 is SEQ ID NO: 53.

[0334] Table 2

[0335] Antibody CDR Sequences

[0336] Antibody Heavy Chain Light Chain

[0337] 6A6 CDR1 GFSLTTYG. .. QSLVHNNGDTY.

[0338] SEQ ID NO: 103 SEQ ID NO: 109

[0339] 6A6 CDR2 IWSDGST... KVSNRFS...

[0340] SEQ ID NO: 104 SEQ ID NO: 110

[0341] 6A6 CDR3 AKNYLGSLDY SQTTHVPLT

[0342] SEQ ID NO: 105 SEQ ID NO: 111 8F1 / 8E1 CDR1 EYEFPSHD. .. QSLVHSNGNTY...

[0343] SEQ ID NO: 106 SEQ ID NO: 112 8F1 / 8E1 CDR2 INSDGGST... KVSNRFS...

[0344] SEQ ID NO: 107 SEQ ID NO: 113 8F1 / 8E1 CDR3 VRLYYGNVMDY SQSTHVPLT

[0345]

[0346] SEQ ID NO: 108 SEQ ID NO: 114 H11 CDR1 GYTFTGYSMH RSSQSLVHSNGNTYLH SEQ ID NO: 127 SEQ ID NO: 130 H11 CDR3 WINTETGEPTYDDFKG KVSNRFS

[0347] SEQ ID NO: 128 SEQ ID NO: 131 H11 CDR3 GTGGDD SQGTHVPPT

[0348] SEQ ID NO: 129 SEQ ID NO: 132 B8 CDR1 GHTFTSYWMH CRASESVQYSGTSLMH SEQ ID NO: 142 SEQ ID NO: 151 B8 CDR3 EINP SNGRT YYNENFKT GASNVET

[0349] SEQ ID NO: 143 SEQ ID NO: 152 B8 CDR3 DGDYVSGFAY QQNWKVPWT SEQ ID NO: 144 SEQ ID NO: 153 G5 CDR1 GFSLSTSGMGVS CKASQSVGNYVA SEQ ID NO: 145 SEQ ID NO: 154 G5 CDR2 HIYWDDDKRYNPSLKS FASNRYS

[0350] SEQ ID NO: 146 SEQ ID NO: 155 G5 CDR3 GVSSWFPY QQHYIFPYT

[0351] SEQ ID NO: 147 SEQ ID NO: 156 B11 CDR1 GFTFNYFWIE CKASENVGTYVS SEQ ID NO: 148 SEQ ID NO: 157 B11 CDR2 EILPGTGSTNYNEKFKG GASNRYT

[0352] SEQ ID NO: 149 SEQ ID NO: 158 B11 CDR3 YDYTSSMDY GQSYSYPWT

[0353] SEQ ID NO: 150 SEQ ID NO: 159 D1 CDR1 NFWMN RASQSIGTSIH

[0354] SEQ ID NO: 176 SEQ ID NO: 179 D1 CDR2 QIYPGDGDTNYNGKFKG YASESIS

[0355] SEQ ID NO: 177 SEQ ID NO: 180 D1 CDR3 SYYRSAWFAY QQSNNWPLT

[0356]

[0357]

[0358] SEQ ID NO: 178 SEQ ID NO: 181

[0359]

[0360] An exemplary heavy chain amino acid and nucleotide sequences are shown below: EVQLVESGGGLVQPGESLKLSCESNEYEFPSHDMSWVRKTPEKRLELVAA INSDGGSTYYPDTMERRFIISRDNTKKTLYLQMSSLRSEDTALYYCVRLY YGNVMDYWGQGTSVTVSS (SEQ ID NO: 115) GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGAGTC CCTGAAACTCTCCTGTGAATCCAATGAATACGAATTCCCTTCCCATGACA TGTCTTGGGTCCGCAAGACTCCGGAGAAGAGGCTGGAGTTGGTCGCAGCC ATTAATAGTGATGGTGGTAGCACCTACTATCCAGACACCATGGAGAGACG ATTCATCATCTCCAGAGACAATACCAAGAAGACCCTGTACCTGCAAATGA GCAGTCTGAGGTCTGAGGACACAGCCTTGTATTACTGTGTAAGACTCTAC TATGGTAATGTTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTC CTCA(SEQ ID NO: 116)

[0361] An exemplary light chain amino acid and nucleotide sequences are shown below: DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLYWYLQKPGQSPK LLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVP LTFGAGTKLELK (SEQ ID NO: 119)

[0362] TCAGAGCCTTGTACACAGTAATGGAAACACCTATTTATATTGGTACCTAC AGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGA TTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTT CACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCT GCTCTCAAAGTACACATGTTCCTCTCACGTTCGGTGCTGGGACCAAGCTG GAGCTGAAAC (SEQ ID NO: 120)

[0363] An exemplary heavy chain amino acid and nucleotide sequences are show below:

[0364]

[0365] QVQLKESGPGLVAPSQSLSMTCTVSGFSLTTYGVHWVRQPPGKGLEWLVV IWSDGSTTYNSPLKSRLSISRDNSKSQVFLKMNSLQADDTAIYYCAKNYL GSLDYWGQGTSVTVSS (SEQ ID NO: 117) CAGGTGCAGCTGAAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCGCAGAG CCTGTCCATGACATGCACCGTCTCAGGGTTTTCATTAACTACCTATGGTG TTCACTGGGTTCGCCAGCCTCCAGGAAAGGGTCTGGAGTGGCTGGTAGTG ATATGGAGTGATGGAAGCACAACCTATAATTCACCTCTCAAGTCCAGACT GAGCATCAGCAGGGACAACTCCAAGAGCCAAGTATTCTTAAAAATGAACA GTCTCCAAGCTGATGACACAGCCATCTACTACTGTGCCAAAAATTACCTC GGTAGTCTGGACTACTGGGGTCAGGGAACCTCAGTCACCGTCTCCTCA

[0366] (SEQ ID NO: 118)

[0367] An exemplary light chain amino acid and nucleotide sequences are shown below: DWLTQTPLSLPVSLGDQASISCRSSQSLVHNNGDTYLHWYLQKPGQSPK LLIYKVSNRFSGVPDRFSGSGSGTDFTFKISRVEAEDLGVYFCSQTTHVP LTFGAGTKLELK (SEQ ID NO: 121) GATGTTGTGTTGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGA TCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAATAATG GAGACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAG CTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTT CAGTGGCAGTGGATCAGGGACAGATTTCACATTCAAGATCAGCAGAGTGG AGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAACTACACATGTTCCG CTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAAC (SEQ ID NO: 122) An exemplary heavy chain amino acid and nucleotide sequences are shown below: QIQLVQSGPELKKPGETVKTSCKASGYTFTGYSMHWVKQAPGKGLKWMGW

[0368]

[0369] INTETGEPTYADDFKGRFALSLETSASTTYLQINNLKNEDTATYFCVRGT GGDDWGQGTTLTVSSAKTTP (SEQ ID NO: 123) CAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGAC AGTCAAGACCTCCTGCAAGGCCTCTGGTTATACCTTCACAGGCTATTCAA TGCACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGG ATAAACACTGAGACTGGTGAGCCAACATATGCAGATGACTTCAAGGGACG GTTTGCCTTGTCTCTGGAAACCTCTGCCAGCACTACCTATTTGCAGATCA ACAACCTCAAAAATGAGGACACGGCTACATATTTCTGTGTTAGGGGGACG GGGGGTGACGACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGCCAA AACGACACCC (SEQ ID NO: 124)

[0370] An exemplary light chain amino acid and nucleotide sequences are shown below: DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPK LLIYKVSNRFSGVPDRFSGSGSGTDFTLKINRVEAEDLGVYFCSQGTHVP PTFGGGTKLEIKRADAAPTV (SEQ ID NO: 125) GATGTTGTGATGACCCAAACTCCGCTCTCCCTGCCTGTCAGTCTTGGAGA TCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATG GAAACACCTATTTACATTGGTACCTTCAGAAGCCAGGCCAGTCTCCAAAG CTCCTGATCTACAAAGTTTCCAACCGGTTTTCTGGGGTCCCAGACAGGTT CAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAACAGAGTGG AGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAGGTACACATGTTCCT CCGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCTGATGCTGC ACCAACTGTA (SEQ ID NO: 126)

[0371] An exemplary heavy chain amino acid and nucleotide sequences are shown below: OVOLQQSGAELMKPGASVKISCKAIGFTFNYFWIEWVKORPGHGLEWIGE

[0372]

[0373] ILPGTGSTNYNEEFKGK AIFTADT S SNTAYMQLRSLTSED S AVYYC VRYD YTSSMDYNGOGTSVIVSS (SEQ ID NO: 160) CAGGTTCAGCTGCAGCAGTCTGGAGCTGAGCTGATGAAGCCTGGGGCCTC AGTGAAAATTTCCTGCAAGGCTATTGGCTTCACATTCAATTACTTCTGGA TAGAGTGGGTAAAACAGAGGCCTGGGCATGGCCTTGAGTGGATTGGAGAG ATTTTACCTGGAACTGGTAGTACTAACTACAATGAGAAGTTCAAGGGCAA GGCCATATTCACTGCAGATACATCCTCCAACACAGCCTACATGCAACTCC GCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTGTAAGATACGAC TATACCTCTTCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTC CTCAG(SEQ ID NO:161)

[0374] An exemplary light chain amino acid and nucleotide sequences are shown below: NIVMTQPKSMSMSVGERVTLTCKASENVGTYVSWYWKPEQSPKLLIYGAS NRYTGVPNRFTGSGSATDFTLTISSVOAEDLADYYCGQSYSYPWTFGGGT KLEIK (SEQ ID NO: 162) AACATTGTAATGACCCAATCTCCCAAATCCATGTCCATGTCAGTAGGAGA GAGGGTCACCTTGACCTGCAAGGCCAGTGAGAATGTGGGTACTTATGTAT CCTGGTATCAACAGAAACCAGAGCAGTCTCCTAAACTACTGATATACGGG GCATCCAACCGGTACACTGGGGTCCCCAATCGCTTCACGGGCAGTGGATC TGCAACAGATTTCACTCTGACCATCAGCAGTGTGCAGGCTGAAGACCTTG CAGATTATTACTGTGGACAGAGTTACAGCTATCCGTGGACGTTCGGTGGA GGCACCAAGCTGGAAATCAAAC (SEQ ID NO: 163)

[0375] An exemplary heavy chain amino acid and nucleotide sequences are shown below: QITLKESGPGILOPSOTLSLTCSFSGFSLSTSGMGVSWIRQPSGKGLEWL SHIYWDDDKRYNPSLDSRLSISKDTSRNOVFLKITSVDTADTATYYCAPG

[0376]

[0377] VSSWFPYWGPGTLVTVSA (SEQ ID NO: 164) CAGATTACTCTGAAAGAGTCTGGCCCTGGGATATTGCAGCCCTCCCAGAC CCTCAGTCTGACTTGTTCTTTCTCTGGGTTTTCACTGAGCACTTCTGGTA TGGGTGTGAGCTGGATTCGTCAGCCTTCAGGAAAGGGTCTGGAATGGCTG TCACACATTTACTGGGATGATGACAAGCGCTATAACCCATCCCTGAAGAG CCGACTCTCAATCTCCAAGGATACCTCCAGAAACCAGGTATTCCTCAAGA TCACCAGTGTGGACACTGCAGATACTGCCACATACTACTGTGCTCCCGGC GTATCCTCATGGTTTCCTTACTGGGGCCCAGGGACTCTGGTCACTGTCTC TGCAG (SEQ ID NO: 165)

[0378] An exemplary light chain amino acid and nucleotide sequences are shown below: SIVMTOTPKFLPVSAGDRVTVTCKASOSVGNYVAWYOQKPGOSPKLLIYF ASNRYSGVPDRFTGSGSGTDFTFTISSVQVEDLAVYFCOQHYIFPYTFGS GTKLEIK (SEQ ID NO: 166) AGTATTGTGATGACCCAGACTCCCAAATTCCTGCCTGTATCAGCAGGAGA CAGGGTTACCGTGACCTGCAAGGCCAGTCAGAGTGTGGGTAATTATGTAG CCTGGTACCAACAGAAGCCAGGACAGTCTCCTAAACTACTGATATACTTT GCATCCAATCGCTATAGTGGAGTCCCTGATCGCTTCACTGGCAGTGGATC TGGGACAGATTTCACTTTCACCATCAGCAGTGTGCAGGTTGAAGACCTGG CAGTTTATTTCTGTCAGCAGCATTATATCTTTCCGTATACGTTCGGATCG GGGACCAAGCTGGAAATAAAAC (SEQ ID NO: 167)

[0379] An exemplary heavy chain amino acid and nucleotide sequences are shown below: OVOLOOPGAELVKPGASEKLSCKASGHTFTSYWMHWVKORPGOGLEWIGE INP SNGRT YYNENFKTK ATLT VDK YS S S ASMQLRSLT SEDSAVYYCASDG DYVSGFAYWGOGTTLTVSS (SEQ ID NO: 168)

[0380]

[0381] CAGGTCCAACTGCAGCAGCCTGGGGCTGAACTGGTGAAGCCTGGGGCTTC AGAGAAGCTGTCCTGCAAGGCTTCTGGGCACACCTTCACCAGCTACTGGA TGCACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGAG ATTAATCCTAGCAACGGTCGTACTTACTACAATGAGAACTTCAAGACCAA GGCCACACTGACTGTAGACAAATATTCCAGCTCAGCCTCCATGCAACTCC GCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGTGATGGT GACTACGTCTCGGGCTTTGCCTACTGGGGCCAAGGCACCACTCTCACAGT CTCCTCAG (SEQ ID NO: 169)

[0382] An exemplary light chain amino acid and nucleotide sequences are shown below: DIVLTOSPGSLAVSLGOSVTISCRASESVOYSGTSLMHWYOQKPGOPPKL LIYGASNVETGVPARFSGSGSGTDFSLNIHPVEEDDIAMYFCGQNWKVPW TFGGGTKLEIK (SEQ ID NO: 170) GACATTGTGCTCACCCAATCTCCAGGTTCTTTGGCTGTGTCTCTAGGGCA GAGTGTCACCATCTCCTGCAGAGCCAGTGAAAGTGTTCAATATTCTGGCA CTAGTTTAATGCACTGGTATCAACAGAAACCAGGACAGCCACCCAAACTC CTCATCTATGGTGCATCCAACGTAGAGACTGGGGTCCCTGCCAGGTTTAG TGGCAGTGGGTCTGGGACAGACTTCAGCCTCAACATCCATCCTGTGGAGG AGGATGATATTGCAATGTATTTCTGTCAGCAAAATTGGAAGGTTCCTTGG ACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAC (SEQ ID NO: 171)

[0383] An exemplary heavy chain nucleotide and amino acid sequences are shown below: CAGGTTCAGCTGCAGCAGTCTGGGGCTGAGCTGGTGAGGCCTGGGTCCTC AGTGAAGATTTCCTGCAAGACTTCTGGCTATGCATTCAGTAACTTCTGGA TGAACTGGGTGAAGCAGAGGCCTGGACAGGGTCTAGAGTGGATTGGACAG

[0384]

[0385] ATTTATCCTGGAGATGGTGACACTAACTACAATGGAAAGTTCAAGGGTAA AGCCACACTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAGCTCA GCAGTCTAACATCTGAGGCCTCTGCGGTCTATTTCTGTGCAAGGTCCTAC TATAGGTCGGCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCTCTGT CTCTGCA (SEQ ID NO : 172) OVOLOQSGAELVRPGSSVKISCKTSGYAFSNFWMNWVKORPGOGLEWIGQ TYPGDGDTNYNGKFKGKATLTADKSSSTAYMQLS SLTSEA S AVYFC ARSY YRSAWFAYWGOGTLVSVSA (SEQ ID NO: 173)

[0386] An exemplary heavy chain nucleotide and amino acid sequences are shown below:

[0387] GACATCTTGCTAACTCAGTCTCCAGCCATCCTGTCTGTGAGTCCAGGAGA AAGAGTCAGTTTCTCCTGCAGGGCCAGTCAGAGCATTGGCACAAGCATAC ACTGGTATCAGCAAAGAACAAATGGTTCTCCAAGGCTTCTCATAAAGTAT GCTTCTGAGTCTATCTCTGGGATCCCTTCCAGGTTTAGTGGCAGTGGATC AGGGACAGATTTTACTCTTAGCATCAACAGTGTGGAGTCTGAAGATATTG CAGATTATTACTGTCAACAAAGTAATAACTGGCCACTCACGTTCGGTGCT GGGACCAAGCTGGAGCTGAAA (SEQ ID NO: 174)

[0388] DILLTQ SPAIL S VSPGERVSF SCRASQ SIGT SIHWYOORTNGSPRLLIKY ASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCOQSNNWPLTFGA GTKLELK (SEQ ID NO: 175)

[0389] Therefore, provided herein is an antibody that binds selectively to MUCl-C / extracellular domain (MUC1-C / ECD) wherein said antibody comprises a variable heavy chain comprising CDR1, CDR2 and CDR3 regions each having 90% or more homology to SEQ ID NOs: 3’, 4’, and 5’, or 6’, 7’ and 8’, or 27’, 28’ and 29’, or 42’, 43’ and 44’, or 45’, 46’ and 47’, or 48’, 49’ and 50’, and a variable light chain comprising CDR1, CDR2 and CDR3 regions each having 90% or more

[0390]

[0391] homology to SEQ ID NOs: 9’, 10’ and 11’ or 12’, 13’ and 14’, or 30’, 31’ and 32’, or 51’, 52’, and 53’, or 54’, 55’ and 56’, or 57’, 58’ and 59’, respectively;

[0392] a variable heavy chain having 80% or more homology to SEQ ID NO: 15’, 19”, 23, 60’, 64’ or 68’, and a variable light chain having 80% or more homology to SEQ ID NO: 17’, 21’, 25’, 62’, 66’, 70’, respectively; and / or

[0393] a variable heavy chain encoded by a nucleic acid having 70% or more homology to SEQ ID NO: 16’, 20’, 24’, 61’, 65’ or 69’, and a variable light chain encoded by a nucleic acid having 70% or more homology to SEQ ID NO: 18’, 2’2, 26’, 63’, 67’ or 71’, respectively.

[0394] Furthermore, the antibodies sequences may vary from the sequences provided above (e.g., Table 1 and 2). For example, amino sequences may vary from those set out above in that (a) the variable regions may be segregated away from the constant domains of the light chains, (b) the amino acids may vary from those set out above while not drastically affecting the chemical properties of the residues thereby (e.g., conservative substitutions), (c) the amino acids may vary from those set out above by a given percentage, e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology. Alternatively, the nucleic acids encoding the antibodies may (a) be segregated away from the constant domains of the light chains, (b) vary from those set out above while not changing the residues coded thereby, (c) may vary from those set out above by a given percentage, e.g., 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology, or (d) vary from those set out above by virtue of the ability to hybridize under high stringency conditions, as exemplified by low salt and / or high temperature conditions, such as provided by about 0.02 M to about 0.15 M NaCl at temperatures of about 50° C. to about 70° C.

[0395] As an example, the heavy and light chains may have 85%, 90%, 95% or 99% homology to SEQ ID NO:15, 19, 23, 60, 64 or 68, and 17, 21, 25, 62, 66 or 70, respectively. The heavy and light chains may be encoded by nucleic acids having 85%, 90%, 95% or 99% homology to SEQ ID NO: 16, 20, 24, 61, 65 or 69, and 18, 22, 26, 63, 67 or 71, respectively. In one embodiment, the antibody does not bind to SEQ ID NO: 33, 34 or 35.

[0396] Provided for use herein are Single Chain Variable Fragment (scFv). scFv is a fusion of the variable regions of the heavy and light chains of immunoglobulins, linked together with a short (usually serine, glycine) linker. This chimeric molecule, also known as a single domain antibody, retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of a linker peptide. This modification usually leaves the specificity unaltered.

[0397]

[0398] These molecules were created historically to facilitate phage display where it is highly convenient to express the antigen binding domain as a single peptide. Alternatively, scFv can be created directly from subcloned heavy and light chains derived from a hybridoma. Single domain or single chain variable fragments lack the constant Fc region found in complete antibody molecules, and thus, the common binding sites (e.g., protein A / G) used to purify antibodies (single chain antibodies include the Fc region). These fragments can often be purified / immobilized using Protein L since Protein L interacts with the variable region of kappa light chains.

[0399] Additional antibodies and fragments thereof that are useful in embodiments of ADCs of the present disclosure can be found in US Patent Publication Nos. US 10,617,773, US 10,059,775, US 11,136,410 and US 2023 / 0265208A1, each of which is incorporated by reference in its entirety.

[0400] Drug conjugates

[0401] Provided herein are antibody-drug conjugates (ADCs). In embodiments, the provided ADCs are of the formulas:

[0402] Formula I. A-D

[0403] wherein:

[0404] A is an antibody or fragment thereof that binds MUC1-C; and

[0405] D is small molecule drug / cytotoxic agent, a derivative thereof, or a pharmaceutically acceptable salt thereof.

[0406] Examples of cytotoxic agents for use in the invention provided herein include, but are not limited to, microtubule inhibitors, DNA-damaging agents, topoisomerase inhibitors or a combination thereof. Examples, include, but are not limited to, Monomethyl auristatin E (MMAE), Monomethyl Auristatin F (MMAF), Maytansine Derivatives (DM1 and DM4), Calicheamicin, Doxorubicin Derivatives and / or Pyrrol obenzodi azepine (PBD) Dimers.

[0407] In embodiments, 3D 1 -MMAE is an ADC used in the invention to inhibit MUC1-C expression.

[0408] CDK4 / 6 Inhibitors

[0409] Cyclin-dependent kinases 4 and 6 (CDK4 / 6) are enzymes that regulate the cell cycle and its progression via phosphorylation, playing a vital role in cell division and growth. In cancer, especially breast cancers, these proteins can undergo mutations, become overactive, and contribute to uncontrolled cancer cell growth.

[0410]

[0411] Due to the prevalence of CDK4 / 6 mutations in cancer, especially breast cancer, inhibitors of CDK4 / 6 have been developed, approved, and are commonly used in the treatment of HER+ / HER2- BC. Inhibitors of CDK4 / 6 include, but are not limited to, palbociclib, ribociclib, and abemaciclib.

[0412] Palbociclib, a selective inhibitor of CDK4 and CDK6, is a well-known drug sold in the United States under the name IB RANCE® with the chemical name of 6-Acetyl-8-cyclopentyl-5- methyl-2-{[5-(l-piperazinyl)-2-pyridinyl]amino}pyrido[2,3-d]pyrimidin-7(8 / / )-one (CAS # 571190-30-2). Ribociclib, is a selective inhibitor of CDK4 and CDK6, is a well-known drug sold in the United States under the name KISQALI® with the chemical name of 7-Cyclopentyl-A,A- dimethyl-2-{[5-(l-piperazinyl)-2-pyridinyl]amino}-7Z / -pyrrolo[2,3-d]pyrimidine-6-carboxamide (CAS # 1211441-98-3). Abemaciclib, a selective inhibitor of CDK4 and CDK6, is a well-known drug sold in the United states under the name VERZENIO® with the chemical name of A-[5-[(4- Ethyl-l-piperazinyl)methyl]-2-pyridinyl]-5-fluoro-4-[4-fluoro-2-methyl-l-(l-methylethyl)-l / / - benzimidazol-6-yl]-2-pyrimidinamine (CAS #: 1231929-97-7).

[0413] Endocrine Therapy

[0414] Endocrine therapy, also known as hormone therapy, is a well-known method of treating cancer, especially breast cancer, which uses hormones or hormone-blocking agents to slow or inhibit the progression of cancer cell proliferation via blocking hormone production or blocking hormone receptors on cancer cells. Hormone therapy is a particularly prevalent treatment for ER+ breast cancer as the cancer cells are dependent on estrogen in order to grow.

[0415] Several types of endocrine therapy are approved for treatment of breast cancers that are hormone sensitive or hormone receptor positive (HR+) including but not limited to SERMs, SERDs, SERCAs, ShERPAs, aromatase inhibitors, and GnRH agonists.

[0416] SERMs bind to estrogen receptors and prevent estrogen from binding. They are also known to mimic the effects of estrogen in other parts of the body. Tamoxifen and toremifene are well- known FDA approved SERMs for the treatment of breast cancer. Tamoxifen is sold in the United States under the name NOLVADEX® with a chemical name of (Z)-2-[4-(l,2-Diphenylbut-l- enyl)phenoxy]-A,A-dimethylethanamine (CAS #: 10540-29-1). Toremifene is sold in the United States under the name FARESTON® with a chemical name of 2-[4-[(lZ)-4-Chloro-l,2-diphenyl- but-l-en-l-yl]phenoxy]-A',Af-dimethylethanamine (CAS #: 89778-26-7).

[0417] SERDs also bind to estrogen receptors and block estrogen from binding yet differ in that they are anti-estrogen in that when bound to the ER, the receptor is targeted for destruction or

[0418]

[0419] degradation. Fulvestrant and Elacestrant are well-known FDA approved SERDs for the treatment of breast cancer. Fulvestrant is sold in the United States under the name FASLODEX® with a chemical name of (JR, 87?, 95,135, 145, 175)-13-methyl-7-[9-(4, 4, 5,5,5- pentafluoropentylsulfinyl)nonyl]-6,7,8,9, 11,12,14, 15,16, 17- decahydrocyclopenta[a]phenanthrene-3,17-diol (CAS #: 129453-61-8). Elacestrant is sold in the United States under the name ORSERDU® with a chemical name of (67?)-6-{2-[Ethyl({4-[2- (ethylamino)ethyl]phenyl}methyl)amino]-4-methoxyphenyl}-5,6,7,8-tetrahydronaphthalen-2-ol (CAS # 722533-56-4).

[0420] GnRH agonists, also known as luteinizing hormone-releasing hormone (LHRH) agonists suppress ovarian function by interfering with signals that stimulate the ovaries to produce estrogen. Leuprorelin and goserelin are well-known FDA approved GnRH agonists for the treatment of breast cancer. Leuprorelin or leuprolide is sold in the United States under the name LUPRON® with a chemical name of / V-[l-[[l -[[!-[[! -[[I -[[l-[[5-(di aminomethylideneamino)-! - [2-(ethylcarbamoyl)pyrrolidin-l-yl]-l-oxo-pentan-2-yl]carbamoyl]-3-methyl-butyl]carbamoyl]- 3-methyl-butyl]carbamoyl]-2-(4-hydroxyphenyl)ethyl]carbamoyl]-2-hydroxy-ethyl]carbamoyl]- 2-(177-indol-3-yl)ethyl]carbamoyl]-2-(3 / 7-imidazol-4-yl)ethyl]-5-oxo-pyrrolidine-2-carboxamide (CAS # 53714-56-0). Goserelin is sold in the United States under the name ZOLADEX® with a chemical name of N-(21-((lH-indol-3-yl)methyl)-l,l-diamino-12-(tert-butoxymethyl)-6-(2-(2- carbamoylhydrazinecarbonyl)cyclopentanecarbonyl)-15-(4-hydroxybenzyl)-18- (hydroxymethyl)-25-(lH-imidazol-5-yl)-9-isobutyl-8,ll,14,17,20,23-hexaoxo- 2,7,10,13,16,19,22-heptaazapentacos- 1 -en-24-yl)-5-oxopyrrolidine-2-carboxamide (CAS # 65807-02-5).

[0421] Aromatase inhibitors block the activity of the enzyme aromatase, which the body uses to make estrogen. Anastrozole, letrozole, and exemestane are well-known FDA approved aromatase inhibitors for the treatment of breast cancer. Anastrozole is sold in the United States under the name ARIMIDEX® with a chemical name of 2,2'-[5-(l / 7-l,2,4-triazol-l-ylmethyl)-l,3- phenylene]bis(2 -methylpropanenitrile) (CAS # 120511-73-1). Letrozole is sold in the United States under the name FEMARA® with a chemical name of 4,4'-((l / -l,2,4-triazol-l- yl)methylene)dibenzonitrile (CAS # 112809-51-5). Exemestane is sold in the United States under the name AROMASIN® with a chemical name of 6-Methylideneandrosta-l,4-diene-3, 17-dione (CAS #: 107868-30-4).

[0422]

[0423] Cancers may become resistant / refractory to treatment. Provided herein are methods to overcome resistant to CDK4 / 6 inhibition and endocrine therapy.

[0424] Cancer / Methods of Treating

[0425] The present disclosure further provides pharmaceutical compositions comprising a MUC1- C inhibitor (either an ADC of the present disclosure or a small molecule inhibitor of MUC1-C, or a MUC1-C inhibitor in the form of a peptide or sRNA molecule), a CDK4 / 6 inhibitor, and an endocrine therapy. The present disclosure of all of those potential targeting of MUC1-C (either genetically and / or pharmacologically) with CDK4 / 6 inhibitors and / or endocrine therapy is useful for treating cancers. The present disclosure shows the results of using an ADC MUC1-C inhibitor (3D1-MMAE) with treatment-resistant HR+ BC cells with low levels of HER2 (ZR-75-1) provides effective suppression of cell line growth and self-renewal capacity. Thus, by using an agent inhibiting or targeting MUC1-C with HR+ / HER2- BCs with resistance to ET and CDK4 / 6 inhibition, treatments of cancer are improved. In one embodiment, the MUC1-C inhibitor is an ADC. In a preferred embodiment, the ADC is 3D1-MMAE. In another embodiment, the MUC1- C inhibitor is a peptide. In a preferred embodiment, the peptide is GO-203. In another embodiment, the MUC1-C inhibitor is a small molecule. In a preferred embodiment, the small molecule is salinomycin. In another embodiment, the MUC1-C inhibitor is a sRNA. In a preferred embodiment, the sRNA is independently selected from siRNA, shRNA, and sgRNA.

[0426] The present disclosure also provides a CDK4 / 6 inhibitor and / or an ET and a MUC1-C inhibitor 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 at least one of a MUC1-C inhibitor (either an ADC of the present disclosure, a peptide inhibitor, a sRNA molecule, or a small molecule inhibitor of MUC1-C) and at least one of a CDK4 / 6 inhibitor and / or at least one of an ET. In another embodiment, the present disclosure provides a method of treating cancer, comprising administering an effective amount of a MUC 1 -C inhibitor (either an ADC of the present disclosure, a peptide inhibitor, a sRNA molecule, or a small molecule inhibitor of MUC1-C) and a CDK4 / 6 inhibitor and / or an ET. Particularly, the present disclosure provides a method of treating a MUC1- positive cancer, selected from cancers that express MUC1-C on their cell surface, such as breast cancer, HR+ breast cancer, HR+ / HER2- breast cancer, or refractory HR+ / HER2- breast cancer,

[0427]

[0428] comprising administering to said patient an effective amount of a MUC1-C inhibitor (either an ADC of the present disclosure, a peptide inhibitor, a sRNA molecule, or a small molecule inhibitor of MUC1-C) and a CDK4 / 6 inhibitor and / or an ET or an effective amount of a pharmaceutical composition comprising the same.

[0429] In another embodiment, the present disclosure provides a method of inhibiting cancer cell self-renewal capacity in a patient, comprising administering to a patient in need thereof a pharmaceutical composition comprising a MUC1-C inhibitor (either an ADC of the present disclosure, a peptide inhibitor, a sRNA molecule, or a small molecule inhibitor of MUC1-C) and a CDK4 / 6 inhibitor and / or an ET. In another embodiment, the present disclosure provides a method of inhibiting cancer cell self-renewal capacity in a patient, comprising administering to the patient an effective amount of a MUC1-C inhibitor (either an ADC of the present disclosure, a peptide inhibitor, a sRNA molecule, or a small molecule inhibitor of MUC1-C) and a CDK4 / 6 inhibitor and / or an ET. Particularly, the present disclosure provides a method of inhibiting cancer cell selfrenewal capacity in a patient with a MUC1 -positive cancer, selected from cancers that express MUC1-C on their cell surface, such as breast cancer, HR+ breast cancer, HR+ / HER2- breast cancer, or refractory HR+ / HER2- breast cancer, comprising administering to said patient an effective amount of a MUC1-C inhibitor (either an ADC of the present disclosure, a peptide inhibitor, a sRNA molecule, or a small molecule inhibitor of MUC1-C) and a CDK4 / 6 inhibitor and / or an ET or an effective amount of a pharmaceutical composition comprising the same.

[0430] The present disclosure also provides comprising a MUC1-C inhibitor (either an ADC of the present disclosure, a peptide inhibitor, a sRNA molecule, or a small molecule inhibitor of MUC1-C) and a CDK4 / 6 inhibitor and / or an ET for use in decreasing tumor cell viability. In an embodiment, the present disclosure provides a MUC1-C inhibitor (either an ADC of the present disclosure, a peptide inhibitor, a sRNA molecule, or a small molecule inhibitor of MUC1-C) and a CDK4 / 6 inhibitor and / or an ET for use in decreasing tumor cell viability. In another embodiment, the present disclosure provides a MUC1-C inhibitor (either an ADC of the present disclosure, a peptide inhibitor, a sRNA molecule, or a small molecule inhibitor of MUC1-C) and a CDK4 / 6 inhibitor and / or an ET for use in decreasing tumor cell viability in MUC1 -positive cancer, selected from cancers that express MUC1-C on their cell surface, such as breast cancer, HR+ breast cancer, HR+ / HER2- breast cancer, or refractory HR+ / HER2- breast cancer, comprising administering to said patient an effective amount of a MUC1-C inhibitor (either an ADC of the present disclosure,

[0431]

[0432] a peptide inhibitor, a sRNA molecule, or a small molecule inhibitor of MUC1-C) and a CDK4 / 6 inhibitor and / or an ET or an effective amount of a pharmaceutical composition comprising the same.

[0433] The present disclosure also provides a MUC1-C inhibitor (either an ADC of the present disclosure, a peptide inhibitor, a sRNA molecule, or a small molecule inhibitor of MUC1-C) and a CDK4 / 6 inhibitor and / or an ET or a pharmaceutical composition comprising the same for use in reversing resistance to anti-cancer therapy. In an embodiment, the present disclosure provides a MUC1-C inhibitor (either an ADC of the present disclosure, a peptide inhibitor, a sRNA molecule, or a small molecule inhibitor of MUC1-C) and a CDK4 / 6 inhibitor and / or an ET for use in reversing resistance to anti-cancer therapy. In another embodiment, the present disclosure provides a MLTC1-C inhibitor (either an ADC of the present disclosure, a peptide inhibitor, a sRNA molecule, or a small molecule inhibitor of MUC1-C) and a CDK4 / 6 inhibitor and / or an ET for use in reversing resistance to anti-cancer therapy in MUC1 -positive cancer, selected from cancers that express MUC1-C on their cell surface, breast cancer, HR+ breast cancer, HR+ / HER2- breast cancer, or refractory HR+ / HER2- breast cancer.

[0434] The present disclosure shows that MUC1-C confers resistance to CDK4 / 6 inhibition and / or ET in HR+ / HER2- BC, thereby potentially limiting these treatments. However, targeting MUC1- C restores sensitivity and reverses resistance to treatment with CDK4 / 6 inhibitors and / or an ET. Thus, in a setting of CDK4 / 6 inhibitor and / or ET resistance, one could treat with an anti-MUCl- C agent alone or in combination with a CDK4 / 6 inhibitor and / or an ET.

[0435] Formulation and Administration

[0436] The present invention provides pharmaceutical compositions comprising MUC1-C inhibitors and / or CDK4 / 6 inhibitors and / or an ET. In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Other suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, saline, dextrose, gelatin, malt, rice, flour,

[0437]

[0438] chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like.

[0439] The compositions can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, tri ethyl amine, 2-ethylamino ethanol, histidine, procaine, etc.

[0440] The inhibitors of the present invention may include classic pharmaceutical preparations. Administration of these compositions according to the present invention will be via any common route so long as the target tissue is available via that route. This includes oral, nasal, buccal, rectal, vaginal or topical. Alternatively, administration may be by intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions, described supra. Of particular interest is direct intratumoral administration, perfusion of a tumor, or administration local or regional to a tumor, for example, in the local or regional vasculature or lymphatic system, or in a resected tumor bed.

[0441] The active compounds may also be administered parenterally or intraperitoneally. Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0442] The present disclosure provides a method to treat cancer comprising administering to a subject in need thereof at least one endocrine therapy, at least one CDK4 / 6 inhibitor, and at least one MUC1-C inhibitor. In a preferred embodiment, the cancer is HR+ / HER2- breast cancer. In a preferred embodiment, the MUC1-C inhibitor is an antibody drug conjugate (ADC). In a more preferred embodiment, the ADC is 3D1-MMAE.

[0443] The present disclosure provides a method to treat cancer comprising administering to a subject in need thereof at least one MUC1-C inhibitor, wherein the cancer presents a mutation in the ESR-1 gene. In a preferred embodiment, the mutation in the ESR-1 gene is independently selected from Y537 and D538G.

[0444]

[0445] The present disclosure provides a method to treat breast cancer comprising administering to a subject in need thereof at least one MUC1-C inhibitor, wherein the breast cancer is resistant to at least one CDK4 / 6 inhibitor. In another embodiment, the breast cancer is resistant to at least one endocrine therapy treatment. In another embodiment, the breast cancer is resistant to both endocrine therapy treatment and CDK4 / 6 inhibitor treatment. In a preferred embodiment, the breast cancer is HR+ / HER2- breast cancer. In another embodiment, the MUC1-C inhibitor is an ADC. In a preferred embodiment, the ADC is 3D1-MMAE. In another embodiment, the MUC1- C inhibitor is a peptide. In a preferred embodiment, the peptide is GO-203. In another embodiment, the MUC1-C inhibitor is a small molecule. In a preferred embodiment, the small molecule is salinomycin. In another embodiment, the MUC1-C inhibitor is a sRNA molecule. In a preferred embodiment, the sRNA molecule is a siRNA, shRNA, and sgRNA.

[0446] The present disclosure provides a means for treating HR+ / HER2- breast cancer comprising a means for inhibiting MUC1-C, wherein the HR+ / HER2- breast cancer is resistant to 1 or more of a means for inhibiting CDK4 / 6 and a means for modulating hormone receptors. In the present disclosure, a means for inhibiting CDK4 / 6 are described and are well known in the art. Also, in the present disclosure, a means for modulating hormone receptors are described as various endocrine therapy (ET) treatments and are well known in the art.

[0447] Various CDK4 / 6 inhibitor means are included in the present disclosure. In embodiments, the CDK4 / 6 inhibitor means of the present disclosure comprises a small molecule or large molecule that inhibits CDK4 / 6. In some embodiments, the CDK4 / 6 inhibitor means may be palbociclib, ribociclib, abemaciclib, or is a molecule that inhibits CDK4 / 6.

[0448] Various ET means are included in the present disclosure. In embodiments, the ET means of the present disclosure comprises a small molecule or large molecule that modulates hormone receptors. In some embodiments, the HR modulator means is independently selected from a SERM, SERD, SERCA, ShERPA, aromatase inhibitor, and GnRH agonist means. In some embodiments, the SERM means may be tamoxifen or toremifene. In some embodiments, the SERD means may be fulvestrant or elacestrant. In some embodiments, the GnRH means may be leuprolide or goserelin. In some embodiments, the aromatase inhibitor means may be anastrozole, letrozole, or exemestane.

[0449] Various MUC1-C inhibitor means are disclosed. This MUC1-C inhibitor means is a small molecule drug, peptide, sRNA, an antibody-drug conjugate (ADC) or a combination thereof that

[0450]

[0451] is capable of inhibiting MUC1-C. In some embodiments, the small molecule drug means is salinomycin. In some embodiments, the peptide means is GO-203. In some embodiments, the peptide means is GO-201. In some embodiments, the ADC means is 3D1-MMAE.

[0452] Discussion

[0453] HR+ / HER2- BC is conventionally treated with ET and CDK4 / 6 inhibitors (Burstein, H. J., N Engl J Med, 383, 2020; McAndrew, N. P. et al., JCO Oncol Pract, 18, 2022; Ferro, A. el al., J Clin Med, 13, 2024; Finn, R. S. et al., N Engl J Med, 375, 2016). Combining ET with CDK4 / 6 inhibition has improved clinical outcomes (Burstein, H. J., N Engl J Med, 383, 2020; Turner, N. C. et al., N Engl J Med, 373, 2015; Finn, R. S. et al., Breast Cancer Res Treat, 184, 2020). Nonetheless, HR+ / HER2- BCs invariably develop resistance to these therapies by pleotropic mechanisms that limit further treatment (Watt, A. C. et al., Breast Cancer Res, 24, 2022). The present work uncovers MUC1-C as a common dependency of treatment resistant HR+ / HER2- BC cells. MUC1 is overexpressed in HR+ BCs (Pitroda, S. et al., Proc. Natl. Acad. Sci. USA, 106, 2009; Khodarev, N. et al., Cancer Res, 69, 2009); however, little is known about function of the oncogenic MUC1-C protein in the resistant disease setting. In addressing this limitation, we found in parental MCF-7 and T47D cells that MUC1-C is necessary for expression of ER early and late response genes. These results suggested that MUC1-C, which forms complexes with ER (Wei, X. et al., Mol. Cell, 21, 2006), plays a role in the ER transactivation function (Fig. 7g). Our results demonstrate that MUC1-C forms complexes with ER that include the SRC-3 and MED1 coactivators. These coactivators are overexpressed in HR+ BCs and contribute to their progression (Varisli, L. et al., Cancers (Basel), 15, 2023; Kiliti, A. et al., Front Endocrinol (Lausanne), 14, 2023; Kang, Y. K. et al., Proc Natl Acad Sci U S A, 99, 2002; Nagalingam, A. et al., Carcinogenesis, 33, 2012). SRC-3 and MED1 contain LXXLL motifs that interact with ER at LYDLL (aa 536-540, SEQ ID NO:94) which is mutated in association with ET resistance (Robinson, D. etal., Nat Genet, 45, 2013; Toy, W. et al., Nat Genet, 45, 2013; Merenbakh-Lamin, K. et al., Cancer Res, 73, 2013; Li, S. et cd., Cell Rep, 4, 2013; Jeselsohn, R. et al., Clin Cancer Res, 20, 2014; Jeselsohn, r. et al., Clin Cancer Res, 22, 2016). MUC1-C is necessary for SRC-3 and MED1 expression (Fig. 7g). Regarding SRC-3, silencing MUC1-C decreased expression of MK2, which stabilizes SRC-3 by phosphorylation at S857 (Shrestha, A. etal., Sci Rep, 10, 2020). We also found that MUC1-C regulates activation of CDK7 that stabilizes MED1 by phosphorylation atT1457 (Rasool, R. U. etal., Cancer Discov, 9, 2019; Guarducci, C. etal., Clin

[0454]

[0455] Cancer Res, 30, 2024). These results shed new light on previously unrecognized MUC1-C dependent regulation of ER target gene expression by SRC-3- and MEDl-mediated mechanisms (Fig- 7g).

[0456] Having identified these MUC1-C dependencies in drug-sensitive HR+ / HER2- BC cells, we focused on MCF-7 / ER(Y537S) mutant cells that exhibit constitutive ligand-independent activity and resistance to ET (Toy, W. et al., Nat Genet, 45, 2013; Fanning, S. W. et al., EHfe, 5, 2016). The ER(Y537S) mutant recruits SRC-3 in the absence of E2 stimulation by altering conformational dynamics of the ER LBD and stabilizing an agonist state (Fanning, S. W. et al., Elife, 5, 2016). In MCF-7 / ER(Y537S) mutant cells, we found that MUC1-C (i) forms complexes with ER(Y537S) and SRC-3, and (ii) is necessary for SRC-3 expression. Similar results were obtained for MED1, indicating that, as found in parental cells, MUC1-C regulates SRC-3 and MED1 in the setting of the ER(Y537S) mutation (Fig. 7g). In support of this dependency, targeting MUC1-C in MCF-7 / ER(Y537S) cells downregulated ER target gene expression and inhibited cell survival. To extend these studies, we generated abemaciclib-resistant MCF-7-AR and T47D-AR cells, which compared to their parental counterparts, exhibited upregulation of MUC1-C, SRC-3 and MED1 expression. Targeting MUC1-C in MCF-7-AR and T47D-AR cells downregulated (i) MK2 and SRC-3 and (ii) CDK7 and MED1, in support of dependency on MUC1-C expression. Resistance to CDK4 / 6 inhibitors has been attributed to increased CDK6 levels (Watt, A. C. et al., Breast Cancer Res, 24, 2022). Unlike the SRC-3 and MED1 axes, MUC1-C had no apparent effect on CDK4 or CDK6. Nonetheless, targeting MUC1-C in the MCF-7-AR and T47D-AR cells suppressed ER target gene expression and clonogenic survival. These findings indicated that in the setting of CDK4 / 6 resistance, MUC1-C regulates the SRC-3 and MED1 coactivators and thereby the ER transactivation function necessary for survival (Fig. 7g). The SRC-3 and MED1 coactivators can contribute to ER-mediated gene transcription by additional mechanisms including liquid-liquid phase separation and the formation of condensates on enhancers and super enhancers (SEs) (Boija, A. et al., Cell, 175, 2018; Bick, G. et al., Cancer Drug Resist, 5, 2022; Takayama, K. I. et al., Front Oncol, 12, 2022). Among these, ESRI and ER target genes are driven by SEs in MCF-7 cells (Sheng, Z. Z. etal., Nucleic Acids Res, 50, 2022; Hnisz, D. et al., Mol Cell, 58, 2015). Noteworthy in this regard is that MUC1-C (i) has an intrinsically disordered cytoplasmic domain and other characteristics that contribute to condensates, and (ii) is necessary for the formation of paraspeckles and other membrane-less condensates (Bhattacharya, A. et al., Oncogene, 43, 2024).

[0457]

[0458] Patients with HR+ / HER2- BCs with acquired resistance to ET and CDK4 / 6 inhibitors have limited therapeutic options. The present studies demonstrate that HR+ / HER2- BC cells resistant to these agents are dependent on MUC1-C signaling as a common mechanism for survival (Fig.

[0459] 7g). MUC1-C drives the CSC state in pan-cancers, including TNBC; whereas less is known about such a role in HR+ / HER2- BCs (Kufe, D. et al., Cancers (Basel), 14, 2022; Yamashita, N. et al., IntJMol Sci, 23, 2022). Treatment of MCF-7 and MCF-7 / ER(Y537S) cells with the anti-MUCl- C ADC was effective in inhibiting self-renewal capacity, indicating that these HR+ / HER2- BC CSCs are MUC1-C -dependent. The ER(D538G) mutation is, like ER(Y537S), prevalent in HR+ / HER2- BCs that are resistant to ET (Robinson, D. R. et al., Nat Genet, 45, 2013; Toy, W. et al., Nat Genet, 45, 2013; Merenbakh-Lamin, K. etal., Cancer Res, 73, 2013; Li. S. etal., Cell Rep, 4, 2013; Jeselsohn, R. et al., Clin Cancer Res, 20, 2014; Jeselsohn, R. etal., Clin Cancer Res, 22, 2016). We found that treatment of MCF-7 / ER(D538G) cells with the anti-MUCl-C ADC also suppresses self-renewal capacity, indicating that this agent is a potential candidate for patients with HR+ / HER2- BC harboring the highly prevalent ER(Y537S) and ER(D538G) mutations. Consistent with MUC1-C dependency of MCF-7-AR and T47D-AR cells for ER signaling, treatment of these models with the anti-MUCl-C ADC demonstrated loss of self-renewal capacity, indicating that MUC1-C is necessary for the CSC state across HR+ / HER2- BCs with pleotropic mechanisms of acquired treatment resistance (Fig. 7g). Furthermore, the anti-MUCl-C ADC was effective in suppressing growth of HR+ / HER2- BC cell line and PDX tumor xenografts. Of importance clinically, we found that MUC1-C is widely expressed in HR+ / HER2- BCs that harbor ESRI mutations, as well as those refractory to CDK4 / 6 inhibitors. We also found that MUC1-C expression in HR+ / HER2- BCs is heterogenous, which could be a factor for effectiveness of the anti-MUCl-C ADC.

[0460] EXAMPLES

[0461] The disclosure can be better understood by reference to the following examples which are offered by way of illustration. The disclosure is not limited to the examples given herein.

[0462] MUC1 is upregulated in HR+ / HER2- BCs and regulates ER-driven gene transcriptomes. Analysis of the TCGABRC A dataset demonstrated that ()MUC1 is significantly upregulated in (i) ER+ vs ER-, and (ii) HER2+ vs HER2- BCs ( Fig. 8a). MUC1 expression in all HER2+ tumors (ER+ and ER- subtypes) vs ER+ / HER2- tumors was not significantly different;

[0463]

[0464] whereas, comparison of ER+ / HER2- vs ER- / HER2+ tumors was statistically significant (Fig. 8a). We also found that MUC1 significantly correlates with expression of ESRI and the ER target genes TFF1 and MYB (Fig. 8b). Further

[0465] analysis identified upregulation o MUCl in (i) ER+ / HER2- BCs vs normal breast tissue (Fig. la), and (ii) ER+ / HER2- vs ER- / HER2- BCs (Fig. lb). MUC1 encodes an oncogenic MUC1-C protein comprised of 58 aa extracellular, 28 aa transmembrane and 72 aa cytoplasmic domains (Fig. 8c). MUC1-C is expressed as ~25 kDa glycosylated and 17 kDa unglycosylated proteins (Fig. 8c). The MUC1-C 25 kDa glycoprotein was detectable in the cell membrane and nucleoplasm of HR+ / HER2- MCF-7 and T47D cells (Fig. 1c). Consistent with recent studies in NSCLC and HNSCC cells (Haratake, N. et al., J Thorac Oncol, 2024; Nakashoji, A. et al., Cancer Res Commun, 4, 2024), we found that the MUC1-C 17 kDa protein, but not the 25 kDa glycoprotein, localizes to chromatin in MCF-7 and T47D cells (Fig. Id). In chromatin, the MUC1-C 17 kDa protein interacts with transcription factors (TFs) and

[0466] epigenomic effectors to regulate gene expression (Haratake, N. et al., J Thorac Oncol, 2024; Nakashoji, A. et al., Cancer Res Commun, 4, 2024). In this way, MUC1-C forms an E2-dependent complex with ER (Wei, X. et al., Mol. Cell, 21, 2006); however, the functional significance of this interaction, particularly in resistant HR+ / HER2- BCs, has remained unclear (Kufe, D. et al., Cancers Basel, 14, 2022). Here, we found that inducible silencing of MUC1-C in MCF-7 and T47D, as well as ZR-75-1 HR+ / HER2-, cells is associated with downregulation of ER expression (Fig. le; Fig. 8d). ER suppression was also observed when targeting MUC1-C with (i) stable silencing using a second MUClshRNA#2 (Fig. 8e), and (ii) the GO-203 inhibitor, which blocks the MUC1-C CQC motif in the cytoplasmic domain that is necessary for nuclear import and function (Fig. 8f) (Kufe, D. et al., Cancers Basel, 14, 2022). Based on these results, RNA-seq was performed on DOX-treated (i) MCF-7 / tet-MUClshRNA (Fig. If; Fig. 8g) and (ii) T47D / tet- MUClshRNA (Fig. 1g; Fig. 8h) cells which demonstrated that MUC1-C silencing associates with downregulation of the HALLMARK ESTROGEN RESPONSE EARLY and HALLMARK ESTROGEN RESPONSE LATE, as well as the HALLMARK E2F TARGETS and HALLMARK G2M CHECKPOINT, gene signatures. Among the ER Early Response Genes (ERGs), 11 were commonly suppressed in MCF-7 and T47D cells (FDR<0.05) (Fig. Ih; Supplemental Table SI). Additionally, 7 ER Late Response Genes (LRGs) were downregulated in both cell lines (Fig. Ih; Supplemental Table SI). Suppression of ER target genes TFF1 (Green, K. etal., Nat Rev Cancer,

[0467]

[0468] 7, 2007), MYB (Drabsch, Y. et al., Proc Natl Acad Sci USA, 104, 2007) and BCL2 (Merino, D. et al., Oncogene, 35, 2016) located in the vicinity of super enhancers (SEs) (Zheng, Z. Z. et al, Nucleic Acids Res, 50, 2022) was confirmed in MCF-7 and T47D cells with MUC1-C silencing (Figs, li and Ij). These results in HR+ / HER2- BC cells indicate that MUC1-C is of importance in regulating the (i) ER early and late response, and (ii) proliferative E2F and G2M signaling pathways.

[0469] MUC1-C regulates the ER coactivator SRC-3. The SRC -3 ER coactivator is overexpressed in HR+ BCs and contributes to their progression (Chen, H. et al., Cell, 90, 1997; Varisli, L. etal., Cancers (Basel), 15, 2023; Kiliti, A. J. et al., Front Endocrinol (Lausanne), 14, 2023). To investigate if MUC1-C regulates ER target genes by an SRC-3 -mediated mechanism, we first performed nuclear coimmunoprecipitation studies in MCF-7 cells, which demonstrated that MUC1-C forms complexes with ER and SRC-3 (Fig. 2a). These results were extended by analyzing anti-SRC-3 precipitates that detected nuclear complexes with ER and MUC1-C (Fig.

[0470] 2b). Surprisingly, we found that silencing MUC1-C in MCF-7, T47D and ZR-75-1 cells downregulates SRC -3 expression (Fig. 2c;Fig. 9a). Similar results demonstrating suppression of SRC-3 were obtained when targeting MUC1-C with (i) MUClshRNA#2 (Fig. 9b), and (ii) the GO-203 inhibitor (Fig 9c). Silencing MUC1-C in these cell models had modest effects on SRC-3 mRNA levels (Supplemental Fig. S2d), supporting potential regulation by a post-translational mechanism. SRC-3 is stabilized by mitogen-activated protein (MAP) kinase-activated protein kinase 2 (MAPKAPK2; MK2)-mediated phosphorylation at S857 (Shrestha, A. etal., SciRep, 10, 2020). We found that (i) targeting MUC 1-C with silencing and GO-203 treatment suppresses MK2 mRNA and protein levels (Fig. 2d; Figs. 9e and 9f) and (ii) MUC 1-C forms a complex with MK2 (Fig. 9g). In addition, analysis of the TCGA dataset revealed that MUC1 significantly associates with MK2, but not SRC-3, expression in HR+ / HER2- tumors (Fig. 9h), consistent with MUC1-C- mediated regulation of SRC-3 by a posttranscriptional MK2-dependent mechanism. In further support for a MUCl-C^MK2^SRC-3 pathway, targeting MK2 with the PF-3644022 inhibitor (Mourey, R. J. et al., J Pharmacol Exp Ther, 333, 2010) confirmed downregulation of SRC-3 expression (Fig 9i). By extension, silencing SRC-3 with two different SRC-3 shRNAs decreased TFF1, MYB and BCL2 levels (Fig. 2e; Fig. 9j). Furthermore, targeting MUC1-C (Fig. 2f), MK2 (Fig. 9k) and SRC-3 (Fig. 2g) inhibited clonogenicity, in support of a MUC 1-C^MK2^ SRC-3 pathway that regulates ER-driven function and survival.

[0471]

[0472] MUC1-C regulates CDK7->MED1 signaling. ER has a LYDLL (SEQ ID NO:94) motif (aa 536-540) that functions as a binding site for SRC -3 and other coactivators (Fanning, S. W. et al., Elife, 5, 2016). Mediator subunit 1 (MED1) is an ER coactivator with an LXXLL motif that confers interactions between ER and Mediator (Kang, Y. K. et al., Proc Natl Acad Sci USA, 99, 2002; Malik, S. et al., Nat Rev Genet, 11, 2010). As found for SRC-3, MUC1-C formed nuclear complexes with ER and MED1 (Figs. 3a and 3b). We also found that targeting MUC1-C in MCF- 7, T47D and ZR-75-1 cells downregulates pMEDl(T1457) and MED1 levels (Fig. 3c; Figs. 10a and 10b) in the absence of a pronounced effect on MED1 transcripts (Fig. 10c). MED1 is stabilized by CDK7-dependent phosphorylation at T1457 (Rasool, R. U. et al., Cancer Discov, 9, 2019; Guarducci, C. etal., Clin Cancer Res, 30, 2024). Consistent with MED 1 downregulation, targeting MUC1-C suppressed activation of CDK7, as evidenced by decreases in pCDK7(T170) and not CDK7 levels (Gong, Y. etal., Cell Commun Signal, 22, 2024) (Fig. 3d). We also found that MUC1- C associates with regulation of the UDAYAKUMAR MED1 UP and DN genes signatures in MCF- 7 and T47D cells (Fig. 3e; Fig. lOd). In concert with a MUCl-C^CDK7^pMEDl(T1457) pathway, targeting CDK7 with the samuraciclib inhibitor (Guarducci, C. et al., Clin Cancer Res, 30, 2024; Patel, H. et al., Mol Cancer Ther, 17, 2018) confirmed downregulation of pMEDl(T1457) and MED1 expression and, as a control, pPol II(S7) levels (Fig. lOe). In extending these studies, we silenced MED1 (Fig. 3f, left panel) and found (i) downregulation of ER, and ER target gene expression (Fig. 3f, right 4 panels) and (ii) inhibition of clonogenic survival (Fig. 3g), indicating that, in addition to SRC-3, MUC1-C regulates the CDK7— >MED1 pathway in HR+ / HER2- BC cells.

[0473] ER mutant cells are dependent on MUC1-C for ER signaling. Binding of E2 to ER uncovers a surface for interactions with coactivators necessary for ER function (Toy, W. etal., Nat Genet, 45, 2013; Fanning S. W. et al., Elife, 5, 2016). Somatic ER mutations involving predominantly Y537S and D538G recruit coactivators in the absence of E2 and confer antiestrogen resistance (Toy, W. et al., Nat Genet, 45, 2013; Fanning S. W. et al., Elife, 5, 2016). Having found that MUC1-C regulates SRC-3 and MED1, we asked if MUC1-C has an effect on ER(Y537S) signaling. Analysis of nuclear lysates from MCF-7 / ER(Y537S) cells demonstrated that MUC1-C forms complexes with ER(Y537S), SRC-3 and MED1 in the absence and presence of E2 stimulation (Fig. 4a; Fig. Ila). Targeting MUC1-C in MCF-7 / ER(Y537S) cells genetically (Fig. 4b) and pharmacologically with GO-203 (Fig. 4c) decreased ER(Y537S), SRC-3 and MED1

[0474]

[0475] expression. Silencing MUC1-C in MCF-7 cells also decreased MUC1-C expression in chromatin, as well as ER, SRC-3 and MED1 levels (Fig. 4d). Similar results were obtained in MCF- 7 / ER(Y537S) cells (Fig. 4e), indicating dependence on MUC1-C for localization of ER and these co-activators in chromatin. Consistent with these observations, MCF-7 / ER(Y537S) cells grown under hormone deprived (HD) conditions exhibited dependence on MUC1-C for expression of ER target genes (Fig. 4f) and for clonogenic survival (Figs. 4g and 4h; Fig. lib). Mutation of the ER LYDLL (SEQ ID NO:94) motif to LYGLL (D538G, SEQ ID NO:95) also confers constitutive ligand-independent activity and resistance to ET (Toy, W. el al., Nat Genet, 45, 2013; Fanning S. W. et al., Elife, 5, 2016). As found for the ER(Y537S) mutant, studies of MCF-7 / ER(D538G) cells demonstrated that MUC1-C is necessary for regulation of ER target genes (Fig. 11c) and survival (Figs, lid and lie). These results indicate that ER(Y537S) and ER(D538G) mutant cells are dependent on MUC1-C for ER signaling.

[0476] MUC1-C dependence in CDK4 / 6 inhibitor resistant HR+ / HER2- BC cells. CDK4 / 6 inhibitors are integral to the treatment of HR+ / HER2- breast cancers by targeting ER-mediated cyclin-dependent signaling. Combining ET with CDK4 / 6 inhibitors has improved clinical outcomes (Burstein, H. T,N Engl JMed, 383, 2020; Ferro, A. etal., J Clin Med, 13, 2024; Turner, N. C. et al., N Engl J Med, 373, 2015; Finn. R. S. et al., Breast Cancer Res Treat, 184, 2020); however, HR+ / HER2- tumors invariably become resistant to these agents by unclear mechanisms. Therefore, to extend our studies on involvement of MUC1-C in resistance of HR+ / HER2- BCs, we turned to the CDK4 / 6 pathway. ER-dependent activation of CDK4 / 6 complexes promotes RB inactivation by phosphorylation on S780 and in turn release of E2Fs to promote cell proliferation (Janostiak, R. et al., Cancers (Basel), 14, 2022). Acquired resistance to CDK4 / 6 inhibitors is associated with loss of functional RB (Watt, A. C. etal., Breast Cancer Res, 23, 2022). In assessing regulation of RB signaling in parental MCF-7, T47D and ZR-75-1 cells, we found that targeting MUC1-C decreases pRB(S780) and E2F1 levels (Fig. 5a; Fig. 12a), indicating that MUC1-C regulates the CDK4 / 6-RB / E2F axis. In accordance with these results, silencing MUC1-C in MCF- 7 and T47D cells was associated with suppression of the HALLMARK E2F TARGETS (Fig. 5b), HALLMARK G2M CHECKPOINT and HALLMARK MITOTIC SPINDLE (Figs. 12b and 12c) gene signatures. To determine if these effects of MUC1-C on the RB / E2F axis extend to settings of CDK4 / 6 treatment resistance, we established MCF-7 and T47D cells for growth in the presence of increasing abemaciclib concentrations of up to 500 nM (Fig. 5c). Abemacliclib-resistant MCF-

[0477]

[0478] 7-AR and T47D-AR cells exhibited marked upregulation of MUC1-C expression in total lysates as compared to that in wild-type cells (Fig. 5d). We also found upregulation of MUC1-C in chromatin from MCF-7-AR and T47D-AR cells, which was associated with increases in pCDK7(T170), CDK7 and MED1, and by contrast suppression of SRC-3 levels (Fig. 5e). Additionally, MYC levels were increased in chromatin (Fig. 5e), which was of interest in that MYC contributes to CDK4 / 6 inhibitor resistance by regulating the RB / E2F axis (Patel, H. et al., Mol Cancer Ther, 17, 2018). Targeting MUC1-C genetically and with GO-203 treatment suppressed (i) pRB(S780) and E2F1, as well as (ii) ER, MK2, SRC-3, pCDK7(T170) and MED1 levels in total lysates (Fig. 5f; Figs. 12d and 12 e). MUC1-C was also necessary for expression of ER, SRC-3, MED1 and MYC in chromatin (Fig. 5g). Consistent with these results, silencing MUC1-C decreased TFF1, MYB and BCL2 expression (Fig. 12f). Furthermore, targeting MLTC1- C in AR cells decreased survival (Figs. 5h and 5i; Figs. 12g and 12h), indicating that HR+ / HER2- AR cells are MUC1-C dependent.

[0479] Targeting MUC1-C dependency in HR+ / HER2- BC cells with an anti-MUCl-C ADC.

[0480] Patients with HR+ / HER- BCs (i) harboring ER mutations resistant to ET, and (ii) unresponsiveness to CDK4 / 6 inhibitors have few treatment options (Burstein, H. J., W Engl J Med, 383, 2020; Ferro, A. et al., J Clin Med, 13, 2024; Gnant, M. et a!., Am Soc Clin Oncol Educ Book, 43, 2023). Having demonstrated that drug resistant HR+ / HER2- BC cells are MUC1-C dependent, we asked if MUC1-C is a potential target for their treatment. MUC1-C is expressed at lower levels on the apical borders of normal epithelia as compared to increased expression over the entire surface of depolarized breast and other types of cancer cells (Kufe, D. et al., Hybridoma, 3, 1984; Kufe, D., Cancers (Basel), 14, 2022; Panchamoorthy, G. et al, JCI Insight, 3, 2018). Given that there are no clinically effective agents against MUC1-C, we generated a monoclonal antibody (MAb), designated 3D1, against the MUC1-C ED alpha-3 helix (Fig. 8c) (Panchamoorthy, G. et al., JCI Insight, 3, 2018). Humanized huMAb 3D1 was conjugated using a maleimidocaproyl-valine- citrulline- / 2-aminobenzyloxycarbonyl (vc) cleavable linker to monomethyl auristatin E (MMAE) at a drug-antibody ratio (DAR) of ~4 (Panchamoorthy, G. et al., JCI Insight, 3, 2018). The anti- MUC1-C ADC was active against MUC1 -positive, but not MUC1 -negative, TNBC breast cancer cells and nontoxic in human MUC1 -transgenic (MUCl.Tg) mice (Panchamoorthy, G. et al., JCI Insight, 3, 2018). Here, flow cytometry studies demonstrated thatMCF-7 cell surface expression of the anti-MUCl-C epitope is similar in wild-type and mutant ER(Y537S) and ER(D538G) cells

[0481]

[0482] (Fig. 6a) (Panchamoorthy, G. et al., JCI Insight, 3, 2018). Sensitivity to the anti-MUCl-C ADC was also comparable in wild-type MCF-7 (IC50=6.3 nM), MCF-7 / ER(Y537S) (IC50=27 nM), and MCF-7 / ER(D538G) (IC50=1.8 nM) cells (Fig. 13a). Analysis of MCF-7 vs MCF-7-AR cells further demonstrated similar expression of the anti-MUCl-C epitope (Fig. 13b) and sensitivity to the anti-MUCl-C ADC (Fig. 13c). Moreover, T47D and T47D-AR cells exhibited comparable expression levels of the anti-MUCl-C epitope (Fig. 13d) and responsiveness to the anti-MUCl-C ADC (Fig. 13e). These results collectively indicated that BC cells resistant to ET and CDK4 / 6 inhibitors are targets for the anti-MUCl-C ADC. Eliminating the CSC population is arguably of importance for achieving long-term responses and potentially cures. We therefore evaluated effectiveness of the anti-MUCl-C ADC on self-renewal capacity as evidenced by tumorsphere formation (Yamashita, N. et al., iScience, 26, 2023). In this way, we found that (i) MCF-7, MCF- 7 / ER(Y537S) and MCF-7 / ER(D538G) (Fig. 6b), (ii) MCF-7-AR (Fig. 13f), and (iii) T47D and T47D-AR (Fig. 13g) CSCs are all sensitive to targeting with the anti-MUCl-C ADC, indicating that drug-resistant HR+ / HER2- BC cells are potential candidates for treatment with this agent. To extend these results, we studied ZR-75-1 cells, which unlike MCF-7 and T47D cells, are null for PTEN, which confers resistance to CDK4 / 6 inhibitors in HR+ BC cells (Costa, C. et al., Cancer Discov, 10, 2020). As shown for MCF-7 / AR and T47D / AR cells, we found that ZR-75-1 cells are sensitive to the anti-MUCl-C ADC as evidenced by loss of self-renewal capacity (Fig. 6c). We therefore treated established ZR-75-1 tumors in the 4th mammary fat pad with the anti-MUCl-C ADC and found complete and durable responses, which were significantly different as compared to control tumors (Fig. 6d). Moreover, as shown in MUCl.Tg mice and other mouse tumor xenograft models (Panchamoorthy, G. etal., JCI Insight, 3, 2018), treatment with the anti-MUCl- C ADC was not associated with significant weight loss or other overt toxicities (Fig. 6e). To extend these results, we studied effects of the anti-MUCl-C ADC against the patient-derived xenograft ER-WT PDX1415 model derived from a patient with a HR+ / HER2- BC refractory to treatment with aromatase inhibitor, fulvestrant, capecitabine, taxol, eribulin, and carboplatin-gemcitabine (Jeselsohn, R. et al., Cancer Cell, 33, 2018). PDX1415 tumor samples were implanted subcutaneously in ovari ectomi zed NOD-SCID-IL2Rgc- / - mice with E2 supplementation. Treatment of established PDX1415 tumors with the anti-MUCl-C ADC resulted in suppression of growth that was significantly different vs that in the control mice (Fig. 6f). As found for the ZR- 75-1 tumor-bearing nude mice, the anti-MUCl-C ADC had no significant effect on body weight

[0483]

[0484] of N0D-SCTD-IL2Rgc- / - mice (Fig. 6g). These results demonstrate that treatment of HR+ / HER2- BC cells with the anti-MUCl-C ADC inhibits self-renewal capacity and tumorigenicity in concert with targeting of the CSC population.

[0485] Expression of MUC1-C in treatment-resistant HR+ / HER2- BCs. Given the findings that MUC1-C is a target for treating drug-resistant HR+ / HER2- BCs, we asked if MUC1 expression is associated with clinical outcome in this patient population. Approximately half of the luminal B patients in the TCGA database lack treatment information. Among the remaining half with treatment data, -90% (or -45% of all patients with Luminal B BCs) received ER targeted therapy. Analysis of MUCI-high vs MUCl-low expression in this treatment-resistant cohort demonstrated that MUC1 associates with a significant decrease in progression-free survival (PFS) (Fig. 7a). We therefore next analyzed 29 HR+ / HER2- BCs for which the associated clinical characteristics and treatment information are detailed in Supplemental Tables S3a and S3b. MUC1-C was detectable in different patterns of apical, diffuse membrane and cytoplasmic expression (Fig. 7b). Assessment of HR+ / HER2- BCs based on MUC1-C staining found that over 90% of tumors express 2+ and 3+ levels (Fig. 7c). Further analysis of HR+ / HER2- BCs with pathogenic ESRI mutations identified expression of MXJC1-C at 1+ to 3+ levels (Fig. 7d, Supplemental Table S3b). We also evaluated a cohort of patients with HR+ / HER2- BCs refractory to ET in combination with palbociclib or abemaciclib (Supplemental Table S3c). Among these, 3 patients had MUC I -C 3+ positive tumors before and after treatment (Fig. 7e). In an additional two patients, MUC1-C levels were undetectable or 2+ in metastatic tumor tissue pre-treatment and were increased to 3+ posttreatment in the setting of progressive disease (Figs. 7e and 7f). These findings indicate that MUC1-C is expressed in a high proportion of treatment-resistant HR+ / HER2- tumors.

[0486] Materials and Methods

[0487] Cell culture. MCF-7 and T47D cells (ATCC) were cultured in MEM (#10-010-CV, Corning, Corning, NY, USA) and RPMI medium (#11875-119, Thermo Fisher Scientific, Waltham, MA, USA), respectively, supplemented with 10% fetal bovine serum (FBS; #100-106, GEMINI Bio-Products, West Sacramento, CA, USA) and 10 ug / mL human recombinant insulin (#12585-014, Thermo Fisher Scientific). ZR-75-1 cells (ATCC) were cultured in RPMI medium with 10% FBS. MCF-7 cells with TALEN knock-in Y537S and D538G ER mutations were

[0488]

[0489] cultured as described in Jeselsohn, R etal., Cancer Ce / / (2018) 33, 173-186. For hormone deprived conditions, cells were cultured in phenol -red-free DMEM (#17-205-CV, Coming) supplemented with 10% charcoal stripped FBS (#F6765, Sigma-Aldrich, Burlington, MA, USA). Cells harboring knock-in ER mutations were cultured under HD conditions for a minimum of two days prior to sample collection, with the media refreshed daily. Cells were treated with abemaciclib (cat# S7158; Selleck Chemicals, Houston, TX, USA), samuraciclib (ICEC09402, cat# S8722; Selleck Chemicals), PF-3644022 (cat# PZ0188, Sigma), 0-Estradiol (cat# E2758-250MG, Sigma) and GO-203 (cat# S8674, Selleck Chemicals). Authentication of the cells was performed every 3-4 months by short tandem repeat (STR) analysis. Cells were monitored for mycoplasma contamination every 3-4 months using the MycoAlert Mycoplasma Detection Kit (#LT07-218, Lonza, Rockland, MA, USA). Cells were maintained for 3-4 months when performing experiments.

[0490] Gene silencing. MUClshRNA (MISSION shRNA TRCN0000122938; Sigma, St. Louis, MO, USA) or a control scrambled shRNA (CshRNA; Sigma) was inserted into the pLKO-tet-puro vector (Plasmid #21915; Addgene, Cambridge, MA, USA) as described in Haratake, N. et al., J Thorac Oncol, (2024). The MUClshRNA#2 (MISSION shRNA TRCN0000430218) SRC- 3shRNA#l (TRCNTRCN0000365196), SRC-3shRNA#2 (TRCN0000370321), MEDlshRNA#! (TRCN0000019800), and MEDlshRNA#2 (TRCN0000019800) were produced in HEK293T cells as described in Haratake, N. et al., J Thorac Oncol, (2024). Vector-transduced cells were selected for growth in 1-2 pg / ml puromycin. Cells were treated with 0.1% DMSO as the vehicle control or 500 ng / ml doxycycline (DOX; Millipore Sigma).

[0491] Quantitative reverse-transcription PCR (qRT-PCR). Total cellular RNA was isolated using Trizol reagent (#15596018, Thermo Fisher Scientific). cDNAs were synthesized using the High Capacity cDNA Reverse Transcription Kit (#4387406, Thermo Fisher Scientific) as described in Haratake, N. et al., J Thorac Oncol, (2024). The cDNA samples were amplified using the Power SYBR Green PCR Master Mix (#4367659, Thermo Fisher Scientific) and the CFX96 Real-Time PCR System (BIO-RAD, Hercules, CA, USA) as described Haratake, N. et al., J Thorac Oncol, (2024). Primers used for qRT-PCR are listed in Supplementary Table S2.

[0492] Immunoblot analysis. Total lysates and chromatin-bound protein prepared from nonconfluent cells were subjected to immunoblot analysis using anti-MUCl-C (#MA5-11202, 1:1000

[0493]

[0494] dilution; Thermo Fisher Scientific, Waltham, MA, USA), anti-NaKATPasel (301 OS, 1 : 1000, Cell Signaling Technology, Danvers, MA, USA), anti-LaminBl (66095-1-Ig, 1:2000, Proteintech, Rosemont, IL, USA), anti-a-tubulin (2144S, 1:1000, CST), anti-H3 (9715S, 1:1000, CST), antiER (8644S, 1:1000, Cell Signaling Technology), anti-b-actin (A5441, 1:10000 dilution, Sigma- Aldrich), anti-SRC-3 (2126S, 1:1000, CST), anti-pSRC3(S857) (PA5-106189, Thermo Fischer Scientific), anti-MK2 (12155S, 1:1000, CST), anti-MEDl (51613S, 1:1000, CST), anti- pMEDl(T1457) (ab60950, 1:500, abeam), anti-CDK7 (2916S, 1:1000, CST), anti— pCDK7(T170) (abl55976, 1:1000, abeam), anti-pPol II(pRpbl)(S7) (13780S, 1:500, CST), antiPol II(Rpbl) (14958T, 1:1000, CST), anti-pRB(S780) (9307S, 1:1000, CST), anti-RB (9309S, 1:1000, CST) and anti-E2Fl (3742, 1:1000, CST) as described in Haratake, N. et al., J Thorac Oncol, (2024).

[0495] Co-immunoprecipitation studies. Nuclear lysates were isolated as described in Yamamoto, M. et al., Cancer Res (2019) 79, 2031-2041. Nuclear proteins were incubated with anti-MUCl-C (#MA5-11202; Thermo Fisher Scientific), anti-SRC-3 (2126S, CST), anti-MEDl (51613S, CST), Armenian Hamster IgG (abl8479, abeam) or Rabbit IgG (#NI01-100UG, Sigma), precipitated with Dynabeads Protein G (#10004D; Thermo Fisher Scientific) and analyzed as described in Yamamoto, M. et al., Cancer Res (2019) 79, 2031-2041.

[0496] Colony formation assays. Cells were seeded in 24-well plates for 24 hours and then treated with (i) 0.1% DMSO or 500 ng / mL DOX, and (ii) PBS or GO-203. For cells harboring knock-in ER mutations were continuously maintained under HD conditions. After 7—14 days, cells were fixed with methanol and stained with 1% crystal violet ( LC135417, LabChem, Zelienople, PA, USA). Growth was quantified at 590 nm using a spectrophotometer and normalized to vehicle treatment.

[0497] Cell proliferation and drug sensitivity assays. Cells were seeded at a density of 1500 to 3500 cells per well in 96-well plates. The next day, the cells were treated with different concentrations of the drug. Cell viability and proliferation were assessed using the Alamar Blue Reagent (cat# DALI 100, Thermo Fisher Scientific) following the company protocol. The IC50 values were determined by nonlinear regression of the dose-response data using Prism 10.0 (SCR_002798, GraphPad Software). Fluorescence intensity (560 nm excitation / 590 nm emission) was measured in at least triplicate wells.

[0498]

[0499] Subcellular protein extraction. Subcellular Protein Fractionation Kit (cat# 78840, Thermo Fisher Scientific) was used to isolate subcellular proteins.

[0500] Flow cytometry. Cells (1 x 106) were blocked by incubation with 1% BSA / PBS for 20 minutes on ice. Cells were then incubated with 40 pg / ml mAb 3D1 or 40 pg / ml IgGl kappa isotype control antibody (cat# 60070.1 ; STEMCELL Technologies, Vancouver, BC, Canada) for 1 hour on ice. Goat Anti-Mouse IgG (Alexa Fluor 488) (abl50113, 1:100 dilution, Abeam) was used as the secondary reagent. Antibody binding to the cell surface was measured by MACSQuant Analyzer 10 Flow Cytometer (Miltenyi Biotec, Bergisch Gladbach, NRW, Germany) and analyzed by FlowJo vlO.6.2 (BD Biosciences, Franklin Lakes, NJ, USA) software.

[0501] Tumorsphere formation assays. Cells (5 x 103) were seeded per well in 6-well ultra-low attachment culture plates (cat# 3471, Corning Life Sciences) in DMEM / F12 50 / 50 medium (cat# 10565-018, Corning Life Sciences) with 20 ng / mL EGF (cat# E9644, Millipore Sigma), 20 ng / mL bFGF (#100-18B, Thermo Fisher Scientific), and 2%B27 supplement (#17504044, Thermo Fisher Scientific). Cells were treated with PBS or anti-MUCl-C ADC. After 7 days, tumorspheres over 100 pm were counted under an inverted microscope in triplicate wells.

[0502] RNA sequence analysis. Total RNA from cells cultured in triplicates was isolated using RNeasy Plus Mini Kit (#74134, QIAGEN, Hilden, Germany) as described in Nakashoji, A. et al., Cancer Res Commnn (2024) 11 (7): 1736-1753. TruSeq Stranded mRNA (Illumina, San Diego, CA, USA) was used for library preparation. Raw sequencing reads were aligned to the human genome (GRCh38) using STAR. Raw feature counts were normalized and differential expression analysis using DESeq2 as described 58. Differential expression rank order for subsequent GSEA was performed using the packages tidyverse (v2.0.0), dbplyr (v2.4.0), and enrichplot (vl.22.0) in R (v4.3.2). Hallmark Gene Sets were queried through the Molecular Signatures Database.

[0503] HR+ / HER2- ZR-75-1 and PDX1415 tumor xenograft studies. ZR-75-1 cells were suspended in 50% RPMI-1640:50% Matrigel® at 1 x 108 / ml. Athymic nude (nu / nu NCr bred inhouse) 7-week-old female mice were implanted subcutaneously (SC) with an 0.18 mg estradiol pellet (Innovative Research, FL, USA). After 24 hours, the mice were implanted with 100 pl of ZR-75-1 cells in the 4th mammary fat pad. Tumors were staged to 150 mm3 (range 80-195 mm3) and mice were randomized into groups using StudyLog®. Treatment was performed using once weekly x 5 intravenous (IV) dosing of (1) 10% DMSO in 90% D5W vehicle control (n=20) and

[0504]

[0505] (2) 5 mg / kg huMAb 3D1-MMAE ADC (n=10). Tumor volumes were collected as bidirectional caliper measurements using StudyDirector® (StudyLog, San Franscisco, CA). Individual body weights were recorded at each tumor volume assessment. The estradiol pellet was replaced in all surviving mice at day 103 to ensure adequate maintenance of estradiol. Mice were not dosed if they had body weight loss of 15% or greater. Mice were humanely sacrificed when their tumor reached approximately 1500 mm3 in volume or if clinical signs suggested estrogen toxicity. For patient-derived xenograft (PDX) studies, patient consent for tumor tissue implantation in nude mice was obtained under an Institutional Review Board (IRB)-approved protocol (Dana- Farber / Harvard Cancer Center IRB protocol 93-085). Tumor samples from PDX1415 were dipped in 50% matrigel and implanted subcutaneously in ovariectomized NOD-SCfD-IL2Rgc- / - mice (Jackson Laboratory, ME, USA), supplemented with 0.18 mg E2 pellets. When tumors reached 150-200 mm3, mice were randomized into two arms of once weekly x 5 intravenous (IV) dosing of (1) vehicle control (n=6) and (2) 7.5 mg / kg anti-MUCl-C ADC (n=6).

[0506] Immunohistochemistry (IHC). HR+ / HER2- breast cancer specimens from patients who underwent surgery or biopsy at the Department of Surgery, Keio University School of Medicine were subjected to IHC with an anti-MUCl-C rabbit monoclonal antibody (16564, 1 : 1000 dilution; CST, heat-induced epitope retrieval, pH 6.0) as previously described in Ozawa, H. et al. Biomedicines (2024) 12, 1509. The determinations were performed independently by two investigators. IHC score of MUC1-C on membranous, cytoplasmic or apical membranous region were scored as 0, 1+, 2+ or 3+ based on the highest intensity occupying > 10% of the evaluated area. The representative IHC score for each specimen was determined by adopting the maximum score of each region. The apical membrane of mammary duct epithelium was used as internal controls for MUC1-C staining. The ethics committee of Keio University School of Medicine approved this study under the approval number 20190057. Informed consent or a suitable substitute in accordance with the Declaration of Helsinki was obtained from the patients in the study.

[0507] Detection of ESRI mutations in HR+ / HER2- BCs. DNA extraction from patient tumor samples, target amplification and sequencing were performed as described in Takeshima, K. et al., Oncol Lett (2020) 20, 1231-1238. under the protocol 20190057 approved by the ethics committee of Keio University School of Medicine.

[0508]

[0509] Statistical analysis. Each experiment was performed at least three times. Unpaired two- tailed Student’s t-tests were used to assess differences between the mean±SD of two groups. P- values were considered significant at p<0.05. GraphPad Prism9 was used for all statistical analyses. Asterisks represent *P < 0.05, **P < 0.01, ***P < 0.001, ****p < 0.0001 with CI = 95%.

[0510] Data availability. The accession number for the RNA-seq data is GEO Submission GSE272989.

[0511] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Exemplary methods and materials are described herein, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention.

[0512] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are considered to be within the scope of this invention.

[0513] All publications, patents, and patent applications, Genbank sequences, websites and other published materials referred to throughout the disclosure herein are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application, Genbank sequences, websites and other published materials was specifically and individually indicated to be incorporated by reference. In the event that the definition of a term incorporated by reference conflicts with a term defined herein, this specification shall control.

Claims

WHAT TS CLAIMED IS:

1. A method to treat cancer comprising administering to a subject in need thereof at least one endocrine therapy, at least one CDK4 / 6 inhibitor, and at least one MUC1-C inhibitor.

2. The method of claim 1, wherein the cancer is HR+ / HER- breast cancer.

3. The method of claim 1 or 2, wherein the MUC1-C inhibitor is an antibody drug conjugate (ADC).

4. The method of claim 3, wherein the ADC is 3D1-MMAE.

5. A method to treat cancer comprising administering to a subject in need thereof at least one MUC1-C inhibitor, wherein the cancer is breast cancer presenting a mutation in the ESR-1 gene.

6. The method of claim 5, wherein the mutation in the ESR-1 gene is independently selected from Y537S and D538G.

7. A method to treat breast cancer comprising administering to a subject in need thereof at least one MUC1-C inhibitor, wherein the breast cancer is resistant to at least one endocrine therapy treatment.

8. A method to treat breast cancer comprising administering to a subject in need thereof at least one MUC1-C inhibitor, wherein the breast cancer is resistant to at least one CDK4 / 6 inhibitor treatment.

9. A method to treat breast cancer comprising administering to a subject in need thereof at least one MUC1-C inhibitor, wherein the breast cancer is resistant to both endocrine therapy and CDK4 / 6 inhibitor treatment.

10. The method of any one of claims 7 -9, wherein the breast cancer is HR+ / HER- breast cancer.

11. The method of any one of claims 1-10, wherein the MUC1-C inhibitor is an ADC.

12. The method of claim 11, wherein the ADC is 3D1-MMAE.

13. The method of any one of claims 1-10, wherein the MUC1-C inhibitor is a peptide 14. The method of claim 13, wherein the peptide is GO-203.

15. The method of any one of claims 1-10, wherein the MUC1-C inhibitor is a small RNA (sRNA) molecule.

16. The method of claim 15, wherein the sRNA molecule is independently selected from a siRNA, shRNA, and sgRNA.

17. The method of any one of claim 1-10, wherein the MUC1-C inhibitor is a small molecule.

18. The method of claim 17, wherein the small molecule is salinomycin.

19. A means for treating HR+ / HER2- breast cancer comprising a means for inhibiting MUC1-C, wherein the HR+ / HER2- breast cancer is resistant to 1 or more of a means for inhibiting CDK4 / 6 and a means for modulating hormone receptors.

20. A pharmaceutical composition comprising:a) an endocrine therapy;b) a CDK 4 / 6 inhibitor; andc) a MUC 1 -C inhibitor.

21. The pharmaceutical composition of claim 20 wherein, the MUC1-C inhibitor is independently selected from an ADC, a small molecule, a peptide, and an sRNA molecule.

22. The pharmaceutical composition of claim 21, wherein the ADC is 3D1-MMAE.

23. The pharmaceutical composition of claim 21, wherein the peptide is GO-203.

24. The pharmaceutical composition of claim 21, wherein the sRNA molecule is independently selected from siRNA, shRNA, and sgRNA.

25. The pharmaceutical composition of claim 21, wherein the small molecule is salinomycin.

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