Methods and compositions for antibody-drug conjugates

Abstract

The invention relates to compounds and methods that include a lysosomal-targeting antibody linked to a lysosomal enzyme inhibitor for the treatment or prevention of conditions or disorders, such as cancer.

Description

WSGR Docket No.63634-701.601 METHODS AND COMPOSITIONS FOR ANTIBODY-DRUG CONJUGATES CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No.63 / 494,414 filed on April 5, 2023, and U.S. Provisional Application No.63 / 501,756 filed on May 12, 2023, which are incorporated herein by reference in their entirety. BACKGROUND

[0002] Antibody–drug conjugates or ADCs are a class of biopharmaceutical drugs designed as a targeted therapy for treating cancer. ADCs consist of three different components, antibody, linker, and drug / payload, that are responsible for the delivery of payload specifically to the targeted cells, releasing the drug / payload exclusively inside the target cells and blocking the biological activity of the target cells, respectively. INCORPORATION BY REFERENCE

[0003] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. SUMMARY

[0004] Disclosed herein is a compound comprising a lysosomal-targeting antibody linked to a lysosomal enzyme inhibitor.

[0005] Disclosed herein is a method of treating a condition, the method comprising administering to a subject in need thereof a therapeutically-effective amount of a compound disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0006] FIG.1 shows relative cell number in head and neck cancer cell line, FaDu cells, non- treated, or treated with Carmofur (100nM), alone and in combination with cisplatin (12uM) recorded by real-time cell growth monitoring.

[0007] FIG.2 shows relative cell number in FaDu cells following treatment with cisplatin (12uM, 72 hours) and recover with or without Carmofur (100nM) or recombinant acid ceramidase (rAC; 0.2 µg / mL) measured by MTT assay.

[0008] FIG.3 shows hydrogen-1 NMR (1H NMR) analysis of an example Carmofur-linker compound purified by HPLC.WSGR Docket No.63634-701.601

[0009] FIG.4 shows liquid chromatography-mass spectrometry (LC-MS) analysis of an example Carmofur-linker compound purified by HPLC.

[0010] FIG.5 shows1H NMR analysis of an example Carmofur-linker compound using mass spectrometry coupled to liquid chromatography diode array.

[0011] FIG.6 shows purity analysis of an example Carmofur-linker compound and its stability in different solvents: DMSO, Methanol, 10% buffer PBS (pH 7.4) and 90% CH3CN, 90% Histidine buffer and 10% CH3CN, DMF, CH3CN, 10% Succinate buffer and 90% CH3CN, DMA, CH3CN:Water (50:50) (0.05%), or 10% Histidine buffer and 90% CH3CN.

[0012] FIG.7 shows representative immunostaining of insulin-like growth factor 2 receptor (IGF2R) expression in FaDu cells localized to the membrane.

[0013] FIG.8 shows representative immunostaining of IGF2R expression in FaDu cells localized to the ER / lysosomes.

[0014] FIG.9 shows representative immunostaining of acid ceramidase (AC) expression in FaDu cells showed lysosomal distribution.

[0015] FIG.10 shows flow cytometry analysis of expression of and localization of insulin like growth factor 1 receptor (IGF1R) and IGF2R on the surface of FaDu cells, assessed by flowcytometry.

[0016] FIG.11 shows representative immunostaining of uptake of anti-IGF2R antibody in live FaDu cells incubated in the presence of anti-IFG2R antibody for 2h, followed by fixation and permeabilization, and incubation with secondary Ab.

[0017] FIG.12 shows representative immunostaining of fixed and permeabilized FaDu cells incubated with secondary Ab only.

[0018] FIG.13 shows representative immunostaining of fixed and permeabilized FaDu cells incubated with primary and secondary antibodies.

[0019] FIG.14 shows representative images of FaDu cells following treatment with or without anti-IGF2R-MMAE conjugate (10nM and 100nM) for 48 hours illustrating decreased in viability of the treated cells, captured bright field microscopy.

[0020] FIG.15 shows cell viability in FaDu cells incubated with anti IGF2R-MMAE ADC (0- 100nM) for 48 hours.

[0021] FIG.16 shows co-expression analysis of exemplary genes coding for target receptors of ADCs and Ceramide Synthases in tumor (T) versus normal (N) tissue.

[0022] FIG.17 shows human epidermal growth factor receptor 2 (HER2) expression in breast cancer (BRCA) compared to normal breast tissue, as an example of the differential expression of a receptor utilized by a clinically validated ADC (e.g. KADCYLA® (ado-trastuzumab emtansine),WSGR Docket No.63634-701.601 evaluated using RNAseq data of patients’ samples from The Cancer Genome Atlas (TCGA) dataset on GEPIA.

[0023] FIG.18 shows ceramide synthase 2 (CERS2) expression in BRCA compared to normal breast tissue, evaluated using RNAseq data of patients’ samples from The Cancer Genome Atlas (TCGA) dataset on GEPIA, showing higher differentiation and lower variation.

[0024] FIG.19 shows positive and significant correlation of HER2 and CERS2 expression in breast cancer (p=0.043).

[0025] FIG.20 shows correlation of ceramide synthase 4 (CERS4) and overall survival (OS) in breast cancer (p=0.023).

[0026] FIG.21 shows Nectin 4 expression in bladder cancer (BLCA) compared to normal tissue (N.S.).

[0027] FIG.22 shows CERS2 expression in BLCA compared to normal tissue (N.S.).

[0028] FIG.23 shows correlation of Nectin 4 and CERS2 in bladder cancer (p=7.9e-08).

[0029] FIG.24 shows correlation of CERS2 and OS in bladder cancer (N.S).

[0030] FIG.25 shows Trop 2 expression in BLCA compared to normal tissue (N.S.).

[0031] FIG.26 shows CERS2 expression in BLCA compared to normal tissue (N.S.).

[0032] FIG.27 shows correlation of Trop-2 and CERS2 in bladder cancer (p=8.8e-06).

[0033] FIG.28 shows correlation of CERS2 and OS in bladder cancer (N.S.).

[0034] FIG.29 shows EGFR expression in head and neck squamous cell carcinoma (HNSC) compared to normal tissue (N.S.).

[0035] FIG.30 shows CERS2 expression in HNSC compared to normal tissue (p<0.01).

[0036] FIG.31 shows correlation of EGFR and CERS2 in HNSC (p=0.0014).

[0037] FIG.32 shows correlation of CERS2 and OS in HNSC (p=0.05).

[0038] FIG.33 shows tissue factor (TF) expression in endocervical adenocarcinoma (CESC) (N.S.).

[0039] FIG.34 shows CERS2 expression in CESC compared to normal tissue (N.S.).

[0040] FIG.35 shows correlation of TF and CERS2 in CESC (N.S.).

[0041] FIG.36 shows correlation of CERS2 and OS in in cervical cancer (p=0.015).

[0042] FIG.37 shows folate receptor alpha (FRa) expression in ovarian cancer (OV) compared to normal tissue (p<0.01).

[0043] FIG.38 shows CERS2 expression in OV compared to normal tissue (N.S.).

[0044] FIG.39 shows correlation of FRa and CERS2 in OV (N.S.).

[0045] FIG.40 shows correlation of CERS2 and OS in in ovarian cancer (N.S.).

[0046] FIG.41 shows CERS2 expression in adrenocortical carcinoma (ACC), bladder urothelialWSGR Docket No.63634-701.601 carcinoma (BLCA), breast cancer (BRCA), esophageal cancer (ESCA), brain cancer (GBM), head and neck cancer (HNSC), kidney renal clear cell carcinoma (KIRCH), compared to normal tissue.

[0047] FIG.42 shows CERS2 expression in brain cancer (LGG). adrenocortical pancreatic cancer (PAAD), rectal cancer (READ, N.S.), prostate cancer (PRAD, N.S.), and skin cancer (SKCM), compared to normal tissue.

[0048] FIG.43 shows CERS4 expression in ACC, BRCA, KICH, PRAD and READ), compared to normal tissue.

[0049] FIG.44 shows CERS6 expression in ACC, BRCA, cholangiocarcinoma (CHOL), colon cancer (COAD), and ESCA, compared to normal tissue.

[0050] FIG.45 shows CERS6 expression in leukemia (LAML), PAAD, READ, stomach cancer (STAD), and thymoma (THYM), compared to normal tissue.

[0051] FIG.46 shows co-expression analysis of example target genes in tumor (T) versus normal (N) tissue, illustrating increased expression of HER2 in BRCA, CHOL, COAD, PAAD, STAD and THYM.

[0052] FIG.47 shows co-expression analysis of example target genes in tumor (T) versus normal (N) tissue, illustrating increased expression of Nectin-4 in BRCA, CHOL, COAD, PAAD, READ and STAD.

[0053] FIG.48 shows co-expression analysis of example target genes in tumor (T) versus normal (N) tissue, illustrating increased expression of TROP-2 in CHOL, COAD, PAAD, PRAD, READ, STAD and THYM.

[0054] FIG.49 shows co-expression analysis of example target genes in tumor (T) versus normal (N) tissue, illustrating increased expression of EGFR in ESCA, HNSC, LGG, PAAD, STAD and THYM.

[0055] FIG.50 shows CERS2 expression in CHOL, COAD, EDVA, and HNSC, compared to normal tissue.

[0056] FIG.51 shows CERS2 expression in PAAD, READ, SKCM, and STAD, compared to normal tissue.

[0057] FIG.52 shows co-expression analysis of IGF2R and CERS2 in ESCA (R=0.3, p=5e-05).

[0058] FIG.53 shows co-expression analysis of IGF2R and CERS2 in CAAD (R=0.35, p=2.3e- 09).

[0059] FIG.54 shows co-expression analysis of IGF2R and CERS2 in HNSC (R=0.5, p=0).

[0060] FIG.55 shows co-expression analysis of IGF2R and CERS2 in PAAD (R=0.47, p=5.2e- 11).

[0061] FIG.56 shows co-expression analysis of IGF2R and CERS2 in READ (R=0.43, p=2.4e-WSGR Docket No.63634-701.601

[0062] FIG.57 shows co-expression analysis of IGF2R and CERS2 in SKCM (R=0.16, p=0.0042).

[0063] FIG.58 shows co-expression analysis of IGF2R and CERS2 in STAD (R=0.35, p=2.3e-13).

[0064] FIG.59 shows IGF2R expression in tumor compared to normal samples in various types of cancer.

[0065] FIG.60 shows co-expression of IGF2R and EGFR in HNSCC cell lines (DepMap portal; (R=0.51, p=2.2e-36).

[0066] FIG.61 shows IGF2R expression in HNSCC cells compared to normal cells.

[0067] FIG.62 shows correlation of high CERS2 expression and poor OS in HNSCC (p=3.4e-08; DepMap).

[0068] FIG.63 shows correlation of EGFR and OS in HNSCC (p=0.052; DepMap).

[0069] FIG.64 shows acid ceramidase expression in tumor compared to normal samples in various types of cancer.

[0070] FIG.65 shows correlation of high acid ceramidase expression and poor OS in cancer.

[0071] FIG.66 shows alteration frequency of ceramide synthases 1-6 gene expression in various types of cancer.

[0072] FIG.67 shows alteration frequency of ceramide synthases 1-6 gene expression in cell lines derived from various types of cancer.

[0073] FIG.68 shows correlation of differential expression of IGF2R and ASAH1 genes in tumorous cells of various types of cancer compared to healthy cells.

[0074] FIG.69 shows CerS1 expression in various types of cancer compared to normal tissue.

[0075] FIG.70 shows CerS2 expression in various types of cancer compared to normal tissue.

[0076] FIG.71 shows CerS3 expression in various types of cancer compared to normal tissue.

[0077] FIG.72 shows CerS4 expression in various types of cancer compared to normal tissue.

[0078] FIG.73 shows CerS5 expression in various types of cancer compared to normal tissue.

[0079] FIG.74 shows CerS6 expression in various types of cancer compared to normal tissue.

[0080] FIG.75 shows IGF2R expression in various types of cancer compared to normal tissue.

[0081] FIG.76 shows gene expression of IGF2R, AC gene (ASAH1), ceramide synthases (CerS1- 6), and EGFR in 10 randomly selected patient samples.

[0082] FIG.77 shows co-expression of IGF2R and EGFR in HNSCC cell lines (DepMap portal).

[0083] FIG.78 shows a chemical structure of an example ADC-FZ-01 antibody-drug conjugate

[0084] FIG.79 shows an illustrative flow chart depicting example associations between acid ceramidase and cancer.WSGR Docket No.63634-701.601

[0085] FIG.80 shows an example workflow to test effects of AC inhibition and cisplatin treatment in HNSCC cells. DETAILED DESCRIPTION

[0086] Disclosed herein are compounds comprising a lysosomal-targeting antibody linked to a lysosomal enzyme inhibitor. In some embodiments, a lysosomal-targeting antibody linked to a lysosomal enzyme inhibitor via a linker designed to be released in the lysosome. In some embodiments, the release in the lysosome may be through activity of lysosomal protease. Also provided herein are methods for treating a condition (e.g., cancer) comprising administering to a subject in need thereof a compound comprising a lysosomal-targeting antibody linked to a lysosomal enzyme inhibitor.

[0087] In some embodiments, a compound disclosed herein inhibits acid ceramidase (AC). The role of AC in oncogenesis is associated with ceramide synthesis. Various cancer cells have significantly higher expression of ceramide synthases (CerS). AC maintains the hydrolysis of ceramide and creates Sphingosine-1-phosphate, a secreted signaling molecule, that supports oncogenesis, angiogenesis, and immunosuppression. Thus, in some embodiments, the present disclosure provides a compound capable of inhibiting AC, inhibiting ceramide metabolism, and / or inducing lysosomal membrane permeability. In some embodiments, treatment with a compound disclosed herein (e.g., a compound capable of inhibiting AC) in cancer cells with ceramide synthesis enhances ceramide buildup and / or cytotoxic effects faster than on target toxicity would affect normal cells.

[0088] In some embodiments, the present disclosure provides an antibody-drug conjugate (ADC). In some embodiments, the antibody-drug conjugate is used for treating cancer. In some embodiments, the cancer is advanced stage cancer. In some embodiments, the cancer is recurrent cancer. In some embodiments, the cancer is drug-resistant cancer cells. In some embodiments, the cancer is dormant cancer cells. In some embodiments, the cancer is drug-resistant dormant cancer cells. Non-limiting examples of cancers include: stomach adenocarcinoma, pancreatic adenocarcinoma, brain lower grade glioma, glioblastoma multiforme, prostate adenocarcinoma, skin cutaneous melanoma, liver hepatocellular carcinoma, head and neck squamous cell carcinoma, esophageal carcinoma, rectum adenocarcinoma, colon adenocarcinoma, sarcoma, and thyroid carcinoma.

[0089] In some embodiments, the cancer is Head and Neck Cancer. In some embodiments, the antibody-drug conjugate is used for treating head and neck squamous cell carcinoma (HNSCC) (e.g., Head and Neck Cancer (HNC)). HNSCC is a type of cancer that originates in the squamousWSGR Docket No.63634-701.601 cells that line the moist mucosal surfaces of the head and neck, including the pharynx, larynx, and nasal cavity and oral cavity. HNC can have a high rate or recurrence (e.g., greater than about 50%) and HNC can have a poor response to therapy (e.g., about 30% response rate to first line therapies). HNC is also associated with poor overall survival of subjects diagnosed with HNC (e.g., median overall survival of about 6 months to about 10 months). In some embodiments, the HNSCC is advanced stage and recurrent HNSCC.

[0090] In some embodiments, the ADC is a targeted inhibitor of lysosomal enzymes. In some embodiments, the lysosomal enzymes are associated with cancer progression, drug resistance, and / or therapy-induced senescence. In some embodiments, the ADC inhibits the lysosomal enzyme in the lysosome. In some embodiments, the ADC comprises a surface receptor. In some embodiments, the antibody of the ADC targets a surface receptor. In some embodiments, the surface receptor is involved in lysosomal trafficking. In some embodiments, the ADC is delivered via biological lysosomal trafficking. In some embodiments, the lysosomal trafficking receptor is IGF2R. In some embodiments, the ADC delivers targeted inhibitors of lysosomal enzymes.

[0091] In some embodiments, the surface receptor is insulin-like growth factor 2 receptor (IGF2R). In some embodiments, the IGF2R targets the ADC to the lysosome of a target cell (e.g., cancer cell). In some embodiments, the drug of the ADC is Carmofur. In some embodiments, Carmofur is an inhibitor of Acid Ceramidase. In some embodiments, the ADC targets drug-resistant dormant cancer cells. In some embodiments, the drug-resistant cancer cells are generated via therapy- induced senescence (TIS) and serve as a source of recurrent cancer.

[0092] The etiology of HNSCC is complex and involves multiple known and unknown factors. In some embodiments, HNSCC is caused by HPV. In HPV-negative cases, factors include smoking, spicy food, and other causes. HPV-positive and -negative status represent clinically and molecularly distinct cancers with unique tumor microenvironment and patients with unique prognostic characteristics. Overall, HPV-positive patients show substantially better survival and clinical outcomes after standard-of-care treatment compared to HPV-negative. The significant gap remains with regards to identification of new targets, based on the distinct biomarkers, characteristic to the molecular patients’ cohorts in HNSCC.

[0093] Mechanisms of drug resistance in HNSCC provide an obstacle against therapy. Mechanisms of resistance include upregulation of pro-survival pathways (e.g., PI3K / AKT / mTOR), drug efflux (MRD), DNA repair mechanisms, and epithelial to mesenchymal transition. Resistance to chemotherapy due to therapy-induced senescence (TIS) is another mechanism of resistance. TIS is a process by which cancer cells stop dividing and become dormant in response to chemotherapy or radiation therapy. Chemotherapy-resistant senescent cancer cells can recover and give rise toWSGR Docket No.63634-701.601 recurrent and / or more aggressive cancer cells. Senescent cells are metabolically inactive and can be invisible during PET / CT, thus initially giving an impression of effectiveness of cancer treatment. The dormant cells recover, giving rise to recurrent cancer. TIS involves inhibition of apoptosis, including engagement of inhibitor of apoptosis family of proteins (IAP).

[0094] In some embodiments, the ADC is targeting TIS-associated drug-resistant HNSCC. In some embodiments, the ADC is targeting other cancers (e.g., lung cancer, non-small cell lung cancer, liver cancer, ovarian cancer, cervical cancer, lymph node carcinoma, osteosarcoma, mucosal melanoma, hepatocellular carcinoma, urothelial cancer, pancreatic cancer, gastric cancer, prostate cancer, stomach cancer, intestinal cancer, head and neck cancer, ovarian cancer, breast cancer, or bladder cancer). In some embodiments, the ADC targets drug-resistant cancer cells. Non-limiting examples of drugs used to treat cancer (e.g., cancer drugs) include: chemotherapy, immunotherapy, radiation, and hormonal therapy. In some embodiments, the ADC targets dormant cancer cells. In some embodiments, the ADC targets senescent cancer cells. In some embodiments, the cancer cell stops dividing upon treatment with a cancer drug (e.g., chemotherapy). In some embodiments, the cancer cell becomes dormant following treatment with a cancer drug. In some embodiments, the dormant cancer cell begins to replicate / divide upon clearance of the cancer drug. In some embodiments, the ADC of the present disclosure lessens a likelihood of occurrence of the cancer cells from becoming dormant. In some embodiments, the ADC of the present disclosure lessens a likelihood of occurrence of reactivation of dormant cancer cells. In some embodiments, the ADC of the present disclosure induces programmed cell death of the cancer cells.

[0095] In some embodiments, the ADC is used as an adjuvant for subjects with HNSCC. In some embodiments, the ADC comprises an antibody. In some embodiments, the antibody targets the ADC to a target cell (e.g., cancer cell). In some embodiments, the antibody targets a surface cancer target. Non-limiting examples of surface cancer targets include: IGF2R, PSA, PAP, CA125, CEA, AFP, HCG, CA19-9, CA15-3, CA27-29, LDH, NSE, HER2, Nectin-4, EGFR, Trop-2, Nectin-4, CEACAM5, c-MET, FOLATE RECEPTOR ALPHA, HER3, LIV-1, MESOTHELIN, and TISSUE FACTOR.

[0096] Additional non-limiting examples of antibody targets include: IGF2R (aka CIM6PR / CD222), Acid Ceramidase (AC), IGF2, IGF1R, CD44, HER2, FOLH1, cMET, CD19, CD22, CD70, Nectin4, HER3, CD33, HER1, TROP2, CLDN182, ROR1, MUC1, PSMA, AXL, ADAM9, CD123, Claudin18.2, KAAG1, PRLR, TF, CD37, MSLN, CD99, CD117, CA125, CD56, CSP1, BCMA, B7H4, CD74, AGS16ENPP3, HER4, CD71, LIV1, TPBG, CLDN, CanAg, HER, CD30, CTLA4, TACSTD2, CXCR4, TNFRSF17, 5T4, CD248, FGFR2, ETB, CD166, LY6E, CD79B, CFC1B, MTX5, CD20, SLC34A2, CD205, CDH6, CA6DS6, MMP14, CLDN6, Notch3, CD228,WSGR Docket No.63634-701.601 SC16, CA9, STEAP1, CD25, TNBC, CD269, CEACAM5, DLK1, LYPD3, DLL3, AREG, DNA, MUC16, DPEP3, PDL1, EDB, ROR2, EFNA4, SEZ6, EGFR, SORT1, EGFREGFR27, TAA,EphA2, TIM1, ER36, aVintegrin, CD276, VEGFA, CD28, LRRC15, FLT3, LY75, AG72, MICAB, FXYD6, MS4A1, FZD11, MTX3, GCC, CD7, GD4, CD138, GIPR, NSCLC, GPC3, CD163, GPI, CD79, GPNMB, SAIL, GPRC5D, SCLC, GUCY2C, SLAMF7, CCR7, SLTRK6, CD352, SSTR2, AMHR2, STn,CD38, TACSTD, CD3E, CD98, HLADR, TM4SF1, HSP90, TNF, IGFR1, TNFRSF8, IL2RA, CEACAM4, CD4, VTCN1, LAMP1, and CD446.

[0097] In some embodiments, the antibody comprises an anti-IGF2R antibody. In some embodiments, the anti-IGF2R antibody is conjugated to a targeted inhibitor. In some embodiments, the targeted inhibitor inhibits lysosomal enzymes.

[0098] Lysosomal Enzyme Inhibitors

[0099] In some embodiments, the lysosomal enzyme inhibitor induces cell death via lysosomal function destabilization and / or lysosomal membrane permeabilization.

[0100] Non-limiting examples of lysosomal enzymes include: alpha-galactosidase A, alpha-N- acetylgalactosaminidase, alpha-galactosidase, AMSH / STAMBP, cathepsin 3, cathepsin 6, arylsulfatase A, arylsulfatase, arylsulfatase G, BAD-LAMP, CD63, and CD-M6PR. In some embodiments, the targeted inhibitor is an inhibitor of lysosomal enzymes. In some embodiments, the targeted inhibitor inhibits acid ceramidase. In some embodiments, the targeted inhibitor is an inhibitor of acid ceramidase. In some embodiments, the inhibitor of acid ceramidase is N-oleoyl- ethanol amine (NOA). In some embodiments, the inhibitor of acid ceramidase is LCL204. In some embodiments, the inhibitor of acid ceramidase is B13. In some embodiments, the inhibitor of acid ceramidase is Carmofur.

[0101] Additional non-limiting examples of lysosomal enzyme inhibitors include: Maytansine, Calicheamicin, Auristatin, Camptothecin, Taxol, SN-38, Carmofur, Vinblastine, Daunomycin, Doxorubicin, Methotrexate, N-[(1R,2R)-2-hydroxy-1-(hydroxymethyl)-2-(4-mitrophenyl)ethyl]- tetradecanamide (D-NMAPPD), N-Oleoyethanolamine, N-((2S,3R)-1,3-dihydroxy-5-((2-oxo-2H- chromen-7-yl)oxy)pentan-2-yl)dodecanamide (RBM14C12), a Auristatin, a Maytansinoid, a Tubulysin, a Calicheamicins, a Duocarmycin, an Exatecan, a Pyrrolobenzodiazepine, a TLR agonist, and a STING agonist.

[0102] In some embodiments, the lysosomal enzyme inhibitor is an inhibitor of acid ceramidase. Non-limiting examples of inhibitors of acid ceramidase include: D-NMAPPD, N- Oleoylethanolamide, RBM14C12, D-e- MAPP, and Carmofur.

[0103] In some embodiments, the lysosomal enzyme inhibitor is Carmofur. In some embodiments, the lysosomal enzyme inhibitor is N-[(1R,2R)-2-hydroxy-1-(hydroxymethyl)-2-(4-WSGR Docket No.63634-701.601 mitrophenyl)ethyl]-tetradecanamide. In some embodiments, the lysosomal enzyme inhibitor is N- Oleoylethanolamine. In some embodiments, the lysosomal enzyme inhibitor is N-((2S,3R)-1,3- dihydroxy-5-((2-oxo-2H-chromen-7-yl)oxy)pentan-2-yl)dodecanamide.

[0104] Additional non-limiting examples of lysosomal enzyme inhibitors include: inhibitors of acid ceramidase, inhibitors of ceramide metabolism: Inhibitors of Ceramide Synthases, Inhibitors of Acid Sphingomyelinase (FIASMAs), Inhibitors of Sphingosine Kinase, inhibitors of glycosylcermaide synthase.

[0105] In some embodiments, the lysosomal enzyme inhibitor is an inhibitor of sphingolipid metabolism. Non-limiting examples of inhibitors of sphingolipid metabolism include: (R)-FTY720- OMe, THI, GT-11, N-C12-deoxysphingosine, N-C16-deoxysphingosine, N-C24:1- deoxysphingosine, N-C12-desoxymethylsphinganine, N-C16-deoxysphinganine, N-C16- desoxymethylsphinganine, N-C24:1-deoxysphinganine, N-C24:1-desoxymethylsphinganine, N- C12-desoxymethylsphingosine, N-C16-desoxymethylsphingosine, N-C24:1- desoxymethylsphingosine, 1-deoxysphingosine, 1-desoxymethylsphinganine, 1- desoxymethylsphingosine, Adamantanyl Galactosyl(ß) Ceramide, C10 bisphosphonate, Safingol, 1- deoxysphinganine, Enigmol(2S,3S,5S), N-C6-Enigmol(2S,3S,5S), and D-threo-PPMP.

[0106] In some embodiments, the lysosomal enzyme inhibitor alters lysosomal membrane permeability (LMP). Non-limiting examples of compounds that alter lysosomal membrane permeability (LMP) include: antipsychotics, antidepressants, immunosuppressive drugs, antihistamine drugs, antimalarial drugs, photosensitizers, plant compounds, thiosemicarbazone analogs, tyrosine kinase inhibitors, detergents, reactive oxygen species, antibiotics, DNA-damaging drugs, and antimitotic agents.WSGR Docket No.63634-701.601

[0107] Linkers

[0108] In some embodiments, the antibody is conjugated to the inhibitor via a linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker is an enzyme labile cleavable linker. In some embodiments, the enzyme labile cleavable linker is a valine-citrulline linker. In some embodiments. In some embodiments, the linker is a chemically labile linker. In some embodiments, the chemically labile linker is a disulfide linker. In some embodiments, the linker is an acid-labile linker. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the non-cleavable linker is a maleimidocaproyl (MC) linker. In some embodiments, the linker is any combination of any linkers of the present disclosure.

[0109] In some embodiments, the lysosomal-targeting antibody and the lysosomal enzyme inhibitor are linked by a linker. Non-limiting examples of a linker include: a cleavable linker, a non-cleavable linker, a self-immolative linker, a click chemistry linker, and a hybrid linker.

[0110] In some embodiments, the lysosomal-targeting antibody and the lysosomal enzyme inhibitor are linked by a linker, wherein the linker is a cleavable linker. Cleavable Linkers: These linkers can be cleaved under specific conditions typically found within target cells, leading to the release of the cytotoxic drug. Cleavable linkers can be further classified into Acid-Cleavable Linkers, Reduction-Cleavable Linkers, and Protease-Cleavable Linkers.

[0111] Acid-Cleavable Linkers are sensitive to acidic conditions found within lysosomes or endosomes of target cells. Non-limiting examples of acid-cleavable linkers include hydrazone, acetals, and ortho esters.

[0112] Reduction-Cleavable Linkers are sensitive to reductive conditions found within the cytoplasm of cells. Non-limiting examples of reduction-cleavable linkers include disulfide bonds and thioethers.

[0113] Protease-Cleavable Linkers are sensitive to specific proteases like cathepsin B or matrix metalloproteinases (MMPs). Non-limiting examples of protease-cleavable linkers include: glycyl glycyl phenylalanyl glycine (GGFG), Val-Cit, Val-Ala, Valine-Citrulline, Valine-Alanine.

[0114] Additional non-limiting examples of a cleavable linker include: hydrozone, acetal, ortho- ester, GGFG, Val-Ala, Valine-Citrulline, and mc-vc-PABC, Valine-Alanine.

[0115] In some embodiments, the cleavable linker is a tetrapeptide liner. Non-limiting examples of tetrapeptide linkers include: GGFG, DXd, GFLG, and ALAL.

[0116] In some embodiments, the lysosomal-targeting antibody and the lysosomal enzyme inhibitor are linked by a linker, wherein the linker is a non-cleavable linker. In contrast to cleavable linkers, non-cleavable linkers can remain intact after internalization into target cells. The cytotoxic drug is released from the linker-antibody complex through lysosomal degradation or proteolysis ofWSGR Docket No.63634-701.601 the entire complex. Non-cleavable linkers are often composed of non-hydrolyzable bonds such as thioether or amide bonds, for example, thioethers, maleimide linkage, amide bonds, and carbamate linkage).

[0117] In some embodiments, the linker is a non-cleavable linker. Non-limiting examples of a non- cleavable linker include: a maleimide linkage, a thioether bond, an amide linkage, and a carbamate linkage. Non-limiting examples of a self-immolative linker include: a p-aminobenzyl alcohol (PAB) linker and a 1,6-elimination-based linker. Non-limiting examples of a non-cleavable linker include: a maleimide linkage, a thioether bond, an amide linkage, and a carbamate linkage.

[0118] In some embodiments, the lysosomal-targeting antibody and the lysosomal enzyme inhibitor are linked by a linker, wherein the linker is a self-immolative linker. Self-immolative linkers are cleavable linkers that contain a triggering mechanism causing the linker to undergo spontaneous intramolecular rearrangement or fragmentation upon reaching the target site, leading to release of the cytotoxic drug. Non-limiting examples of a self-immolative linker include: a p- aminobenzyl alcohol (PAB) linker and a 1,6-elimination-based linker.

[0119] In some embodiments, the lysosomal-targeting antibody and the lysosomal enzyme inhibitor are linked by a linker, wherein the linker is a click chemistry linker. Click chemistry refers to a group of reactions that are highly selective and occur rapidly under mild conditions. Click chemistry linkers allow the conjugation of cytotoxic drugs to antibodies with high efficiency, for example, Azide-alkyne cycloaddition (Azides, Alkynes).

[0120] In some embodiments, the lysosomal-targeting antibody and the lysosomal enzyme inhibitor are linked by a linker, wherein the linker is a hybrid linker. Hybrid linkers combine characteristics of different linker types, offering advantages such as improved stability, controlled release, or enhanced selectivity.

[0121] In some embodiments, the lysosomal-targeting antibody and the lysosomal enzyme inhibitor are linked by a linker, wherein the linker comprises a GGFG linkage.

[0122] In some embodiments, the lysosomal-targeting antibody and the lysosomal enzyme inhibitor are linked by a linker, wherein the linker comprises a maleimide linkage.

[0123] In some embodiments, the lysosomal-targeting antibody and the lysosomal enzyme inhibitor are linked by a linker, wherein the linker comprises an amide linkage.

[0124] In some embodiments, the linker comprises a maleimidocaproyl linkage.

[0125] In some embodiments, the lysosomal-targeting antibody and the lysosomal enzyme inhibitor are linked by a linker, wherein the linker comprises a L-valine-L-citrulline-p-aminobenzyl linkage.

[0126] In some embodiments, the linker comprises maleimidocaproyl-L-valine-L-citrulline-p-WSGR Docket No.63634-701.601 aminobenzyl.

[0127] In some embodiments, the antibody is an anti-IGF2R antibody. In some embodiments, the linker is an enzyme labile cleavable linker. In some embodiments, the enzyme labile cleavable linker is a cathepsin-B cleavable linker. In some embodiments, the anti-IGF2R antibody is conjugated to the inhibitor via cathepsin-B cleavable linker. In some embodiments, the cathepsin- B cleavable linker is MC-valine-citrulline. In some embodiments, the targeted inhibitor is a lysosomotrophic agent. Non-limiting examples of lysosomotrophic agents include: chloroquine, ammonium chloride, azithromycin, and hydroxychloroquine.

[0128] In some embodiments, the ADC is a treatment for cancer. In some embodiments, the cancer is a solid tumor. Non-limiting examples of solid tumors include lung cancer, non-small cell lung cancer, liver cancer, ovarian cancer, cervical cancer, lymph node carcinoma, osteosarcoma, mucosal melanoma, hepatocellular carcinoma, urothelial cancer, pancreatic cancer, gastric cancer, prostate cancer, stomach cancer, intestinal cancer, head and neck cancer, ovarian cancer, breast cancer, and bladder cancer.

[0129] In some embodiments, the ADC delivers an inhibitor of a lysosomal enzyme. Lysosomal activity can be oncogenic. In some embodiments, lysosomal activity results in cancer cell proliferation, angiogenesis of the cancer cell, and / or immunosuppression. In some embodiments, the ADC of the present disclosure lessens a likelihood of occurrence of the cancer cell proliferation, angiogenesis of the cancer cell, and / or immunosuppression that occurs as a result of lysosomal activity. In some embodiments, the lysosomal activity in cancer cells leads to the cancer cell becoming drug-resistant. In some embodiments, lysosomal activity in cancer cells leads to drug- resistance induced replicative arrest of the cancer cells. In some embodiments, the ADC of the present disclosure lessens a likelihood of occurrence of drug-resistance induced replication arrest of the cancer cells. In some embodiments, the lysosomal enzyme is acid ceramidase. In some embodiments, acid ceramidase is necessary for survival of cancer cells. In some embodiments, acid ceramidase is necessary for reactivation of dormant cancer cells. In some embodiments, the inhibition of acid ceramidase results in apoptosis of dormant cancer cells. In some embodiments, the inhibitor of acid ceramidase results in dormant cancer cells not being reactivated. In some embodiments, the drug of the ADC is a cytotoxic drug. In some embodiments, the ADC is delivered to target cells via biological lysosomal trafficking. In some embodiments, the ADC delivered to target cells via biological lysosomal trafficking is less toxic than ADC delivery via non-biological lysosomal trafficking. In some embodiments, the surface receptor of the ADC is recycled to the target cell’s surface. In some embodiments, the ADC is administered to a subject subsequent to chemotherapy. One of the mechanisms of drug resistance can be therapy-inducedWSGR Docket No.63634-701.601 senescence (TIS). Chemotherapy-resistant senescent cancer cells can recover and give rise to recurrent, frequently more aggressive cancer. In some embodiments, the ADC is administered subsequently to chemotherapy and induces apoptosis in senescent cancer cells. In some embodiments, the senescent cancer cells are cancer cells that escaped chemotherapy. In some embodiments, the ADC of the present disclosure is a senolytic drug. In some embodiments, a senolytic drug is a drug capable of killing senescent cells, such as senescent cancer cells.

[0130] Non-limiting examples of an ADC of the present disclosure include: Anti- HER2-mc-vc- PABC-Carmofur, Anti- IGF2R-mc-vc-PABC-Carmofur, Anti- HER2-MC-GGFG-Carmofur, Anti- IGF2R-mc-GGFG -Carmofur, Anti- EGFR-mc-GGFG -Carmofur, Anti- IGF2R-mc-GGFG -D- NMAPPD, Anti- IGF2R-mc-GGFG -N-Oleoylethanolamine, Anti- IGF2R-mc-GGFG RBM14C12, Anti- HER2-mc-GGFG -D-NMAPPD, Anti- HER2-mc-GGFG -N- Oleoylethanolamine, Anti- HER2-mc-GGFG RBM14C12, Anti- EGFR -mc-GGFG -D-NMAPPD, Anti- EGFR -mc-GGFG -N-Oleoylethanolamine, Anti- EGFR -mc-GGFG RBM14C12, Anti- IGF1R -mc-GGFG -D-NMAPPD, Anti- IGF1R -mc-GGFG -N-Oleoylethanolamine, Anti- IGF1R - mc-GGFG RBM14C12.

[0131] One example of an ADC of the present disclosure is shown in FIG.78.WSGR Docket No.63634-701.601

[0132] In some embodiments, a compound disclosed herein is:or a pharmaceutically-acceptable ionized form thereof.

[0133] Lysosomal-Targeting Antibody

[0134] In some embodiments, the target expression of the lysosomal-targeting antibody is elevated and / or homogenously expressed in a target tissue of a subject in need thereof. In some embodiments, the target expression of the lysosomal-targeting antibody co-expresses with one or more ceramide synthases.

[0135] Non-limiting examples of lysosomal-targeting antibody include: anti-HER2, anti-IGF2R, anti-EGFR, anti-acid ceramidase, anti-IGF1R, anti-Cd44, anti-FOLH1, anti-cMET, anti-IGF1R, anti-CD19, anti-CD22, anti-CD70, anti-Nectin4, anti-HER3, anti-HER1, anti-CD33, anti-TROP2, anti-CLDN182, anti-ROR1, anti-MUC1, and anti-PSM.

[0136] In some embodiments, the lysosomal-targeting antibody is anti-HER2. A non-limiting example of an anti-HER2 antibody is Trastuzumab or Enhertu.

[0137] IGF2R is a natural trafficking receptor of glycosylated proteins to the lysosome. In some embodiments, the IGF2R is effective in trafficking a compound disclosed herein to the lysosome. In some embodiments, the lysosomal-targeting antibody is anti-IGF2R. A non-limiting example of an anti-IGF2R antibody is Hum-Anti-IGF2R.

[0138] The IGF2R, also known as CIM6PR (Cation-Independent Mannose 6-Phosphate Receptor) can be a transmembrane glycoprotein essential for homeostasis and regulation of cell growth,WSGR Docket No.63634-701.601 differentiation, and survival. In some embodiments, IGF2R is ubiquitously expressed in human tissues. In some embodiments, the IGF2R protein is localized to the ER-Golgi secretory pathway to transport newly synthesized glycosylated proteins from the ER to the lysosome. In some embodiments, the IGF2R is localized on the plasma membrane. In some embodiments, the IGF2R protein binds to M6P, IGF2, and or M6P-tagged ligands. In some embodiments, the IGF2R protein delivers the tagged protein to the lysosome. In some embodiments, the ADC does not neutralize IGF2R. In some embodiments, the IGF2R targets the ADC to a target cell (e.g., cancer cell).

[0139] In some embodiments, the lysosomal-targeting antibody is anti-IGF1R. A non-limiting example of an IGF1R antibody is hz208F2-4. A non-limiting example of an IGF-1R–targeted antibody–drug conjugate is W0101.

[0140] In some embodiments, the lysosomal-targeting antibody is anti-EGFR. A non-limiting example of an anti-EGFR antibody is Cetuximab.

[0141] In some embodiments, the lysosomal-targeting antibody is anti-Nectin 4. A non-limiting example of an anti-Nectin 4 antibody is Enfortumab.

[0142] In some embodiments, the lysosomal-targeting antibody is anti-Trop-2. A non-limiting example of an anti-Trop-2 antibody is Sacituzumab.

[0143] In some embodiments, the lysosomal-targeting antibody is anti-TF. A non-limiting example of an anti- TF antibody is Tisotumab.

[0144] In some embodiments, the lysosomal-targeting antibody is anti-FOLRa. A non-limiting example of an anti- FOLRa antibody is Mirvetuximab.

[0145] Acid Ceramidase

[0146] Acid ceramidase (AC, N-acylsphingosine deacylase) can be a lysosomal enzyme that plays an important role in the metabolism of sphingolipids, which are a class of lipids found in cell membranes. AC is encoded by the ASAH1 gene, which cleaves ceramides into sphingosine (SPH) and free fatty acids within lysosomes. In some embodiments, the substrate for AC is ceramide. In some embodiments, ceramide is a bioactive lipid. In some embodiments, ceramide is involved in apoptosis, inflammation and / or signal transduction. Ceramide can be synthesized in response to a variety of cellular stresses, such as oxidative stress, DNA damage, and ER stress. In some embodiments, the AC can convert ceramide into sphingosine, and a pro-survival molecule, Sphingosine-1-Phosphate. In some embodiments, the AC can convert ceramide into sphingosine. In some embodiments, the AC can convert ceramide into Sphingosine-1-Phosphate. In some embodiments, AC can be an important rheostat of ceramide levels and can play a major role in regulation of cell death and survival. FIG.79 depicts illustrative reasoning for targeting acid ceramidase in cancer cells.WSGR Docket No.63634-701.601

[0147] Ceramide is a key intermediate in sphingolipid metabolism and an important signaling molecule, is synthesized by a family of six ceramide synthases (CerS), Ceramide Synthases 1-6, each of which synthesizes ceramides with distinct acyl chain lengths. CerS1 synthesizes C18 ceramide. CerS2 synthesizes C20, C22, C24, C26 ceramides. CerS3 synthesizes C22, C24 and C26 ceramides. CerS4 synthesizes C18 and C20 ceramide2. CerS5 synthesizes C16 ceramide. CerS6 synthesizes C14 and C16 ceramides.

[0148] Down regulation of C18 ceramide can be observed in, for example, HNSCC (and not in non-squamous cancers). CerS1 sensitizes human embryonic kidney cells to a wide range of drugs including cisplatin, carboplatin, doxorubicin and vincristine, whereas CerS5 only sensitized cells to doxorubicin and vincristine and CerS4 did not affect sensitivity to any of the drugs. C16- and C18- ceramides were demonstrated to play two opposing roles in human head and neck squamous cell carcinomas, pro-survival and proapoptotic, respectively. C18:0 ceramides in HNSCC suppressed the proliferation of cancer cells, whereas short-chain C16:0 ceramides induced proliferation.

[0149] Short and long chains C16&C18

[0150] Among all types of ceramides, certain have different roles in the sphingolipids pathway. Depending on the length of the acyl chain, each either promotes cell death or cell proliferation. For example, C16-ceramide engenders cell proliferation and C18-ceramide mediates apoptosis. C16- ceramide (resp. C18-ceramide) expression is mainly determined by CerS1 or CerS4 (resp. CerS5 or CerS6). This latter distinction sorts cancer types according to ceramide synthases expressions.

[0151] Cancer types for which the tumoral expression of CerS1 or CerS4 is higher than the normal tissue expression can be better candidates for cancer therapies due to the inhibition of Acid Ceramidase specifically in tumor cells leading to more cell death in this case. Cancer types for which the tumoral expression of CerS5 or CerS6 is lower than in the normal tissue expression can be better candidates for cancer therapies due to the inhibition of Acid Ceramidase specifically in tumor cells leading to less cell proliferation.

[0152] Focus on comparison between different levels of C16- and C18-ceramides -products of ceramide synthases 1, 4, 5, and 6. In some embodiments, CerS 5 / 6 overexpressing cancer is not suitable for a therapy comprising a compound disclosed herein if inhibiting AC increases C16- ceramide and increases proliferation. In some embodiments, CerS1 / 4 low expressing cancers (C18- ceramide low) are not suitable for a therapy comprising a compound disclosed herein, if inhibiting AC to induce apoptosis requires high synthesis and accumulation of C18-ceramide.

[0153] In some embodiments, a compound disclosed herein is more effective in cancer types having low CerS5 / 6 expression (tumor and normal cells) and high CerS1 / 4 expression (tumor cells) as the balance between C16-ceramide and C18-ceramide is shifted towards apoptosis.WSGR Docket No.63634-701.601

[0154] In some embodiments, a compound disclosed herein is less effective in cancer types having high CerS5 / 6 expression (tumor cells) and low CerS1 / 4 expression (tumor and cancers cells), as balance between C16-ceramide and C18-ceramide is shifted towards pro-survival.

[0155] In some embodiments, AC is overexpressed in target cells. In some embodiments, the target cells are cancer cells. In some embodiments, the cancer cells are HNSCC cancer cells. In some embodiments, overexpression of AC is associated with tumor progression and resistance to chemo- and radiation therapy. In some embodiments, AC downregulation by pharmacological inhibitors or genetic manipulation suppresses tumorigenesis and sensitizes cancer cells to chemo- and radiation therapies. Ceramide can be involved in cellular senescence in normal cells in response to various stressors such as DNA damage, oxidative stress, and / or telomere shortening. In some embodiments, ceramide is involved in therapy-induced senescence (TIS) in cancer cells. Ceramide can be generated by various pathways, including de novo synthesis, hydrolysis of sphingomyelin, and salvage of sphingolipids, and can act as a signaling molecule to regulate cell growth, differentiation, and apoptosis. In some embodiments, ceramide is broken down through AC activity. Carmofur is a chemotherapy drug that can be used in the treatment of various types of cancer, including colon, breast, and pancreatic cancers. In some embodiments, Carmofur inhibits the enzyme dihydropyrimidine dehydrogenase (DPD), which leads to accumulation of uracil in cancer cells and leads to cell death. In some embodiments, Carmofur inhibits AC activity. In some embodiments, Carmofur is administered as an anti-cancer treatment. In some embodiments, the compound is an inhibitor of acid ceramidase.

[0156] Disclosed herein is a method of treating a condition, the method comprising administering to a subject in need thereof a therapeutically-effective amount of a compound, the compound comprising a lysosomal-targeting antibody linked to a lysosomal enzyme inhibitor.

[0157] In some embodiments, the condition is a cancer. In some embodiments, the condition is head and neck cancer. In some embodiments, the compound inhibits acid ceramidase.

[0158] Dosing and Administration

[0159] In practicing the methods or use provided herein, therapeutically-effective amounts of the compounds described herein are administered to a subject having a disease or condition to be treated. A therapeutically-effective amount can vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the compounds used, and other factors. Subjects can be, for example, humans, elderly adults, adults, adolescents, pre-adolescents, children, toddlers, infants, or neonates. A subject can be a patient. In some embodiments, the subject is human.

[0160] The particular dosage of a compound required to treat the condition can depend on theWSGR Docket No.63634-701.601 severity of the condition, the route of administration, and related factors that can be decided by the attending physician. The particular dosage of a compound required to treat the cancer can depend on the severity of the cancer, the route of administration, and related factors that can be decided by the attending physician.

[0161] A therapeutically-effective amount of a compound of the present disclosure can be expressed as mg of the compound per kg of subject body mass. In some embodiments, a therapeutically-effective amount is 1-1,000 mg / kg, 1-500 mg / kg, 1-250 mg / kg, 1-100 mg / kg, 1-50 mg / kg, 1-25 mg / kg, or 1-10 mg / kg.

[0162] In some embodiments, the therapeutically-effective amount is from about 50 mg to about 1,000 mg.

[0163] In some embodiments, a therapeutically-effective amount is 5 mg / kg, 10 mg / kg, 25 mg / kg, 50 mg / kg, 75 mg / kg, 100 mg / kg, 150 mg / kg, 200 mg / kg, 250 mg / kg, 300 mg / kg, 400 mg / kg, 500 mg / kg, 600 mg / kg, 700 mg / kg, 800 mg / kg, 900 mg / kg, 1,000 mg / kg, about 5 mg / kg, about 10 mg / kg, about 25 mg / kg, about 50 mg / kg, about 75 mg / kg, about 100 mg / kg, about 150 mg / kg, about 200 mg / kg, about 250 mg / kg, about 300 mg / kg, about 400 mg / kg, about 500 mg / kg, about 600 mg / kg, about 700 mg / kg, about 800 mg / kg, about 900 mg / kg, or about 1,000 mg / kg.

[0164] A compound described herein can be present in a composition in a range of from about 1 mg to about 5 mg, from about 5 mg to about 10 mg, from about 10 mg to about 15 mg, from about 15 mg to about 20 mg, from about 20 mg to about 25 mg, from about 25 mg to about 30 mg, from about 30 mg to about 35 mg, from about 35 mg to about 40 mg, from about 40 mg to about 45 mg, from about 45 mg to about 50 mg, from about 50 mg to about 55 mg, from about 55 mg to about 60 mg, from about 60 mg to about 65 mg, from about 65 mg to about 70 mg, from about 70 mg to about 75 mg, from about 75 mg to about 80 mg, from about 80 mg to about 85 mg, from about 85 mg to about 90 mg, from about 90 mg to about 95 mg, from about 95 mg to about 100 mg, from about 100 mg to about 125 mg, from about 125 mg to about 150 mg, from about 150 mg to about 175 mg, from about 175 mg to about 200 mg, from about 200 mg to about 225 mg, from about 225 mg to about 250 mg, or from about 250 mg to about 300 mg.

[0165] A compound described herein can be present in a composition in an amount of about 1 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, or about 300 mg.

[0166] In some embodiments, a therapeutically-effective amount can be administered 1-35 times per week, 1-14 times per week, or 1-7 times per week. In some embodiments, a therapeutically-WSGR Docket No.63634-701.601 effective amount can be administered 1-10 times per day, 1-5 times per day, 1 time, 2 times, or 3 times per day.

[0167] In some embodiments, a compound disclosed herein can be administered in therapeutically- effective amounts by various forms and routes including, for example, by intravenous, intravitreal, subcutaneous, intramuscular, oral, rectal, aerosol, parenteral, ophthalmic, pulmonary, transdermal, vaginal, otic, nasal, intraocular, and topical administration. Non-limiting examples of parenteral or systemic administration includes subcutaneous, intravenous, intraperitoneal, and intramuscular injections.

[0168] In some embodiments, the administering is oral. In some embodiments, the administering is by infusion.

[0169] In some embodiments, a method disclosed herein further comprises administering a therapeutically-effective amount of a second therapy. EXAMPLES EXAMPLE 1: Assays for assessing cytotoxic activity

[0170] A preliminary study in an HNSCC cell line (FaDu) was performed to obtain supporting data for choosing IGF2R as an ADC antigen and to validate strategy for using Carmofur as a payload. First, expression of IGF2R and AC proteins by FaDu cells was confirmed (FIGs.7-9). This experiment showed that IGF2R protein was localized to the surface of HNC cells (e.g., FaDu cells) (FIG.7) and to the ER / lysosomes (FIG.8), and showed lysosomal distribution of AC expression (FIG.9), and therefore the HNC cells were suitable for update of IGF2R antibodies. Expression of and localization of IGF1R and IGF2R was also assessed by flow cytometry and confirmed on the surface of cancer cells (FIG.10).

[0171] Next, several anti-IGF2R antibodies were screened and a monoclonal IgG1 antibody was identified as suitable for uptake by live cells. Uptake of the antibody by live cells were incubated with an anti-IGF2R antibody for 2h, then fixation and permeabilization, and secondary Ab was applied and visualized by fluorescent microscopy (FIG.11). Controls for non-specific signal was confirm by incubation with secondary only (FIG. 12) and fixed and permeabilized cells were incubated with primary and secondary antibodies (FIG.13). The experiment showed that HNC cells up-took the IGF2R antibodies, and therefore would be able to deliver the ADC to the HNC cells (FIGs.11-13).

[0172] Once the antibody uptake was confirmed (FIGs.11-13), the anti-IGF2R Ab was conjugated with anti-IGF2R-Monomethyl auristatin E (MMAE) using oYo-link technology (AlphaThera), to test the ability of IGF2R to internalize ADC. Treating FaDu cells with anti-IGF2R-MMAEWSGR Docket No.63634-701.601 conjugate induced cytotoxic effects, based on cell morphology and cell viability assay, CellTiterGlo (FIGs.14-15). Specifically, IGF2R mediated update of the anti-IGF2R-MMAE ADC induced cell death in HNC cells at a nanomolar range. The effective range was 10-100 nM and suggested an acceptable rate of internalization via IGF2R. Evidence was collected for use of Carmofur as an effective payload that achieves the desired effect following chemotherapy. The workflow for this experiment is depicted in FIG.80. Briefly, FaDu cells were treated with cisplatin for 72 hours and then underwent a 24 hour recovery period. During that recovery period, the FaDu cells either: recovered without any additional treatment, recovered with the additional of a recombinant acid ceramidase (rAC), or recovered the acid ceramidase inhibitor Carmofur. The experiment additionally included a negative control of FaDu cells not treated with any treatment. Cell viability in FaDu cells treated with the various recoveries was measured and live-cell growth of the FaDu cells was monitored. Carmofur significantly inhibited cell proliferation (FIG.1) and the recovery from replicative arrest, induced by cisplatin, while rAC reverted this effect (FIG.2). FIG.1 depicts the cell viability of the FaDu cells from this experiment and FIG.2 depicts the live- cell growth monitoring of the FaDu cells of this experiment. Overall, this experiment showed that AC inhibition by Carmofur blocked the recovery of the HNC cells from cisplatin treatment.

[0173] The heterogeneity of tumor cells is a risk in the development of ADCs for solid tumors. To mitigate this risk, analyses were conducted of transcriptomics data from the public domain and a hypothesis was developed for patient segment selection for LT-CAR. The proposed molecular signature was IGF2R+ Ceramide Synthase +, AC +. Analysis of RNAseq data from 9,736 tumors and 8,587 paired normal samples from the TCGA and the GTEx (GEPIA) showed increased expression of IGF2R in multiple types of cancer, including colon, esophageal, HNSCC, sarcoma, etc. (FIG.75). IGF2R gene expression in HNSCC was 33.09 transcripts per million (red bars in FIG.75) compared to 17.39 transcripts per million in paired normal tissues (black bars in FIG.75). The role of AC in oncogenesis is tightly associated with ceramide synthesis. Cancer cells have significantly higher expression of ceramide synthases (CerS). AC maintains the hydrolysis of ceramide and creates Sphingosine-1-phosphate, a secreted signaling molecule, that supports oncogenesis, angiogenesis, and immunosuppression. Gene expression analysis from patient-derived tumor xenografts (Crown Bio) showed positive expression for IGF2R, AC gene (ASAH1) and ceramide synthases (CerS2, CerS4 and CerS5), within the range comparable to EGFR (FIG.76). Going back to GEPIA, the overall survival associated with high expression of ceramide synthase 2 was analyzed and found that patient overall survival with high CerS2 was significantly worse compared to low (p=3.4e-08, FIG.76). Overall, this analysis showed that increased ceramide turnover in HNC patients was associated with poor survival. These data supported the hypothesisWSGR Docket No.63634-701.601 that in cancer cells with ceramide synthesis, AC inhibition leads to ceramide buildup and cytotoxic effect faster than on-target toxicity would affect normal cells. Confirming the expression of IGF2R, CerS genes and AC gene (ASAH1) in HNSCC xenograft increases the feasibility of LT-CAR delivery to the tumor and efficacy of AC inhibition and tumor suppression. EXAMPLE 2: Synthesis and characterization of an anti-IGF2R-Carmofur ADC

[0174] An anti-IGF2R-Carmofur ADC (e.g., Lysosome-Targeted CARmofur, LT-CAR) is generated. The structure of an ADC of the present disclosure is depicted in FIG.78. ADC synthesis, purification and characterization is performed. The first step is to synthesize, purify and characterize the Carmofur-linker compound. A clinically validated linker Maleimidocaproyl-L- valine-L-citrulline-p-aminobenzyl alcohol p-nitrophenyl carbonate (MC-Val-Cit-PABC) is selected for the linker. This linker is hydrolyzed by cathepsin B, a lysosomal protease overexpressed in multiple cancers. The Carmofur-linker compound is purified by HPLC and characterized by1H conjugatable impurities. In step two, a stochastic maleimide conjugation is performed to interchain cysteine residues of the anti-IGF2R IgG1 antibody with the Carmofur-linker compound. Monomeric ADC is purified by size exclusion chromatography (SEC). The Drug-to-Antibody ratio (DAR) is assessed by hydrophobic interaction chromatography (HIC), RP-LC / MS, RP-LC, and UV / V. The ADC is further purified to remove free drug and the purity is confirmed by LC / MS. The expected purity is 90%. To characterize LT-CAR, the affinity to the surface IGF2R receptor is evaluated and is updated and tracked in a panel of HNSCC cell lines (A-253, SCC-15, SCC-25, SCC-9, FaDu and Detroit 562). Surface IGF2R expression in HNSCC cell lines and the binding of LT-CAR to the HNSCC cells is assessed by flow cytometry, using rabbit anti-IGF2R and anti- mouse IgG antibodies, respectively.

[0175] To assess the internalization of the LT-CAR by the cells, cells are incubated with LT-CAR labeled with Zenon reagent (Life Technologies) for 1-6h and evaluated by FACS. The Zenon reagent binds specifically to antibodies, generates a bright signal, and is an easy labeling method, that can be completed within minutes. To track the lysosomal delivery of LT-CAR, the pH- sensitive dye, pHrodo to label LT-CAR is used and the increase and clearance of the signal is tracked over time (1- 12 h) in cells incubated with pHrodo -LT-CAR using flow cytometry.

[0176] The release of Carmofur in the lysosome is determined, by measuring AC activity and ceramide levels. AC activity in cell extract is measured, using a substrate mixture of C12-ceramide. After incubation, the reaction is quenched with ice-cold methanol with C18-ceramide as a reference and the lipids are extracted. The ceramide species is then quantified using an LC / MS system andWSGR Docket No.63634-701.601 the peak area ratio of the ceramide to an internal standard is used to determine the amount of each ceramide species. The secondary functional validation is the ability of LT-CAR to induce cell cycle arrest and inhibit cell growth. Cell growth / viability in the presence of LT-CAR is assessed using CellTiterGlo assay in cells treated with 8 concentrations of LT-CAR (0-100 nM) for 72 h. Cell cycle arrest and apoptosis are assessed using annexin V / propidium iodide staining, by flow cytometry, in cells treated with LT-CAR (at IC50 and 10x IC50) for 2 h, 12 h, 24 h, and 72 h. The Acceptance criteria of the ADC is: IC50 of LT-CAR in the <100 nM range, >50% increase in ceramide with high IGF2R expression. EXAMPLE 3: Assays for assessing ceramidase expression

[0177] Cisplatin induces cell cycle arrest and senescence in cancer cells, including HNSCC. Acid ceramidase expression in HNC is associated with sensitivity of the cancer cells to cisplatin and that AC inhibition by N-oleoyl-ethanolamine (NOE) sensitized HNC cells to cisplatin treatment via ceramide-induced apoptosis. P53 mutations are common in HNSCC patients. The cell cycle arrest at the G2 / M phase is associated with the activation of the checkpoint kinase 1 (Chk1) and Chk2 pathways, and subsequent induction of senescence via p53-p21 and p16-Rb. Ceramide activates a pathway involving the protein kinase JNK, which can phosphorylate p53 and promote stabilization and activation in senescent cells. Senescent cells can accumulate ceramide and thus contribute to the anti-proliferative effects of senescence and promote resistance to the anti-proliferative drugs such as cisplatin. Some gain-of-function mutations in p53 (e.g., R175H, R248Q, R273H, and R282W) are associated with resistance to cisplatin in HNSCC and other cancers. Recurrent cancer, arising from re-activation of the dormant senescent cells, requires hydrolysis of ceramide by AC and resumption of cell cycle. To confirm that LT-CAR can inhibit ceramide hydrolysis and reduces the recovery of HNSCC cells from cisplatin-induced senescence, cell viability assays and colony formation assays in HNSCC cells, treated with cisplatin or doxorubicin for 72 h and allowed to recover for 10 days, with or without LT-CAR, Carmofur or Venetoclax (positive control) are performed. To track induction of and recovery from senescence, Beta-Glo® Assay (Promega) is used.

[0178] Interaction between tumor cells and tumor microenvironment (TME) plays a role in oncogenesis, including in the recovery of tumor cells from chemotherapy. Tumor organoids (tumoroids) are three-dimensional structures grown from tumor cells that closely mimic the architecture of the tumor and TME. Tumoroids are used to evaluate the effect of LT-CAR in the context of TME. PDX-derived tumoroids are purchased from Crown Bio. Crown Bio provides complete data package with the tumoroids, including WES, RNAseq, growth curves and drugWSGR Docket No.63634-701.601 response. To confirm that LT-CAR can penetrate a 3D culture and to determine the dose and the frequency of treatment, tumoroids cultured in matrigel are treated with pHrodo -LT-CAR for 24, 48 and 72h after which cells are fixed and evaluated by high-resolution laser scanning microscopy. Next, tumoroids cultured in matrigel are treated with cisplatin (5uM) for 3 days, followed by LT- CAR with additional 5 days. Tumoroids growth are evaluated by tracking the average diameter of the colonies and cell death is monitored continuously by plate-based RealTime-Glo Annexin V Apoptosis and Necrosis Assay (Promega). Selected tumoroids are fixed and whole mounted and analyzed by multiplex IHC for markers of senescence and apoptosis (beta-galactosidase, BCL2, p53, p16, p21, cleaved caspase-3).

[0179] To demonstrate POC in vivo, the CDX (FaDu) and PDX (IGF2R+ / AC+) are utilized. To establish a HNC xenograft model, 5 x 106FaDu cells in matrigel are injected subcutaneously into the hind flank of Nude BALB / c mice. When the tumor size reaches 5 mm, animals are treated with 2 rounds of cisplatin, followed by LT-CAR, Carmofur alone or vehicle. Tumor size and animal weight are measured twice weekly. Animals are sacrificed 5 days after the last treatment or at the humane endpoint, whichever happens first. Tumors are excised and evaluated for ceramide content, using LC / MS and for markers of senescence, cell cycle and apoptosis, using IHC, qPCR and immunoblot. To establish PDX model of HNSCC, the PDX colony is expanded to 3 mice with 3 different PDX models at the ASRC comparative medicine unit. Mice are treated and evaluated similarly to CDX model. The Acceptance criteria for the ADC are: >50% decrease in growth with LT-CAR, during cancer cells / tumors recovery from chemotherapy. EXAMPLE 4: Synthesis and characterization of a carmofur-linker compound

[0180] To synthesize Carmofur-linker compound, Maleimidocaproyl-L-valine-L-citrulline-p- aminobenzyl alcohol pnitrophenyl carbonate32 (MC-Val-Cit-PABC), was conjugated to Carmofur.WSGR Docket No.63634-701.601

[0181] The Carmofur-linker compound was purified by HPLC and characterized by1H NMR (FIG.3) and LC-MS (FIG.4).

[0182] Stability evaluation of the Carmofur-linker compound showed a presence of the product and its derivative, and their relative abundance, and was used to determine the purity of the product (FIG.5). This assay was repeated after the lyophilized product was exposed to difference solvents, and then used to calculate purity over time.

[0183] Based on the stability evaluation of the Carmofur-linker compound, 50% of the product was destabilized after conjugation (FIG.5). The product was missing a part of molecule that was essential for AC inhibition.

[0184] Stability of the payload-linker was evaluated in different solvents DMSO, Methanol, 10% buffer PBS (pH 7.4) and 90 % CH3CN,DMF, CH3CN, 10% Succinate buffer and 90% CH3CN, DMA, CH3CN:Water (50:50)(0.05%FA), 10% Histidine buffer and 90 %CH3CN (FIG.6).

[0185] Over 30 different linkers and conditions were examined which did not result in the desired product. The hexylamine urea moiety was easily hydrolyzed under base conditions. The reaction transform to 77% completion but after separation and lyophilization, this was reduced to 50%:50%.WSGR Docket No.63634-701.601EXAMPLE 5: Gene expression analysis

[0186] RNA sequencing expression data of 9,736 tumors and 8,587 normal samples from the TCGA and the GTEx projects was analyzed on the web server GEPIA using a standard processing pipeline. Gene expression analysis for illustrative target receptors (e.g., HER2, Nectin 4, Trop-2, EGFR, TF, FOLRa, and IFG2R) was performed as shown and described in FIGs.16-40 and TABLE 1.WSGR Docket No.63634-701.601

[0187] TABLE 1

[0188] Co-Expression was analyzed for target receptor genes and Ceramide Synthases in various tumors compared to normal tissue (FIG.16).

[0189] Anti-HER2 (e.g., Trastuzumab) - Breast Cancer

[0190] Expression of HER2 and CERS2 was assessed in breast cancer and compared to normal tissue (FIGs.17-18). This experiment showed that HER2 and CERS2 expression was elevated in breast cancer compared to normal breast tissue (FIGs.17-18). Analysis of co-expression of HER2 and Ceramide Synthases showed that HER2 and CERS2 expression was significantly correlated in breast cancer (p=0.043) (FIG.19). Both CerS2 and CerS4 produce long chain ceramides. Analysis of co-expression of Ceramide synthase 4 (CERS4) and overall survival (OS) in breast cancer samples showed that CERS2 expression did not correlate with poor survival, instead, high expression of CERS4 was associated with poor survival (p=0.023) (FIG.20). In these experiments.

[0191] Anti-Nectin 4 (e.g., Enfortumab or Padcev) - Bladder Cancer

[0192] Expression of Nectin 4 and CERS2 was assessed in bladder cancer and compared to normal tissue (FIGs.21-22). This experiment showed that Nectin 4 and CERS2 expression was elevated inWSGR Docket No.63634-701.601 bladder cancer compared to normal tissue (N.S.) (FIGs.21-22). Analysis of co-expression of Nectin 4 and Ceramide Synthases in bladder cancer showed that Nectin 4 and CERS2 expression was positively correlated (N.S.) (FIG.23). Analysis of co-expression of CERS2 and overall survival (OS) in bladder cancer showed that CERS2 expression was not significantly correlated with poor survival (FIG.24).

[0193] Anti-Trop 2 (e.g., Sacituzumab or Trodelvy) - Bladder Cancer and Breast Cancer

[0194] Expression of Trop 2 and CERS2 was assessed in bladder cancer and compared to normal tissue (FIGs.25-26). This experiment showed that Trop 2 and CERS2 expression was elevated in bladder cancer compared to normal tissue (N.S.) (FIGs.25-26). Analysis of co-expression of Nectin 4 and Ceramide Synthases in bladder cancer showed that Trop 2 and CERS2 expression was positively correlated (NS) (FIG.27). Analysis of co-expression of CERS2 and overall survival (OS) in bladder cancer showed that CERS2 expression did not significantly correlate with poor survival (FIG.28).

[0195] Anti-EGFR (e.g., Cetuximab or RM-1929 ADC) - Head and Neck Cancer

[0196] Expression of EGFR and CERS2 was assessed in head and neck cancer and compared to normal tissue (FIGs.29-30). This experiment showed that EGFR and CERS2 expression was elevated in HNSC compared to normal tissue (N.S., significant, respectively) (FIGs.29-30). Analysis of co-expression of Nectin 4 and Ceramide Synthases in HNSC cancer showed that EGFR and CERS2 expression positively correlated (significantly) (FIG.31). Analysis of co-expression of CERS2 and overall survival (OS) in HNSC cancer showed that CERS2 expression significantly correlated with poor survival (p=0.05) (FIG.32).

[0197] Anti-TF (e.g., Tisotumab or Tivdak) – Cervical cancer

[0198] Expression of TF and CERS2 was assessed in cervical cancer and compared to that of normal tissue (FIGs.33-34). This experiment showed that CERS2 expression is lower in cervical cancer than in normal tissue (FIG.34). Analysis of co-expression of TF and Ceramide Synthases in cervical cancer showed that TF expression was low and did not correlate with CERS2 (FIG.35). Co-expression of CERS2 and overall survival (OS) was also assessed in cervical cancer samples (FIG.36).

[0199] Anti-FRa (e.g., Mirvetuximab or Elahere) – Ovarian cancer

[0200] Expression of FRa and CERS2 was assessed in ovarian cancer and compared to normal tissue (FIGs.37-38). This experiment showed that FRa expression in cervical cancer was significantly higher compared to normal tissue (FIG.37) and CERS2 expression in ovarian cancer was not significantly different compared to normal tissue (FIG.38). Co-expression of FRa and Ceramide Synthases was assessed in cervical cancer samples (FIG.39). Analysis of co-expressionWSGR Docket No.63634-701.601 of CERS2 and overall survival (OS) in ovarian cancer showed that CERS2 does not correlate with overall survival (FIG.40). EXAMPLE 6: Analysis of expression of ceramide synthases in various cancers

[0201] Assessment of ceramide synthases expression in various cancer types was performed as shown and described in FIGs.41-45.

[0202] CERS2 expression was elevated in breast cancer (BRCA), esophageal cancer (ESCA), brain (GBM and LLG) cancer, head and neck cancer (HNSC), kidney renal clear cell carcinoma (KIRCH), pancreatic cancer (PAAD), rectal cancer ((READ, N.S.), prostate cancer (PRAD, N.S.) and skin cancer (SKCM) compared to normal tissue (FIGs.41-42). CERS4 expression was elevated in breast cancer (BRCA), kidney renal clear cell carcinoma (KIRCH), and prostate cancer (PRAD) compared to normal tissue (FIG.43). CERS6 expression was elevated in breast cancer (BRCA), cholangiocarcinoma (CHOL), colon cancer (COAD), esophageal cancer (ESCA), Leukemia (LAML), pancreatic cancer (PAAD), rectal cancer (READ), stomach cancer (PRAD), and thymoma (THYM) compared to normal tissue (FIGs.44-45).

[0203] Co-expression was analyzed for exemplary target receptors in the cancers identified to exhibit evaluated ceramide synthases expression (FIGs.46-49).

[0204] Anti-HER2 (e.g., Trastuzumab)

[0205] Co-expression was analyzed for HER2 genes and showed potential therapeutic effects of administering a combination of an ADC comprising an anti-HER2 with inhibitors of ceramide metabolism for breast cancer (BRCA), colon (COAD), cholangiocarcinoma (CHOL), pancreatic adenocarcinoma (PAAD), rectum adenocarcinoma (READ), stomach adenocarcinoma (STAD), and thymoma (THYM) (FIG.46).

[0206] Anti-Nectin 4 (e.g., Enfortumab or Padcev)

[0207] Co-expression was analyzed for HER2 genes and showed potential therapeutic effects of administering a combination of an ADC comprising an anti-HER2 with inhibitors of ceramide metabolism for bladder cancer, BRCA, CHOL, COAD, PAAD, READ, and STAD (FIG.47).

[0208] Anti-Trop 2 (e.g., Sacituzumab or Trodelvy)

[0209] Co-expression was analyzed for HER2 genes and showed potential therapeutic effects of administering a combination of an ADC comprising an anti-HER2 with inhibitors of ceramide metabolism for bladder cancer, CHOL, COAD, PAAD, Prostate adenocarcinoma (PRAD), READ, STAD, and THYM (FIG.48).

[0210] Anti-EGFR (e.g., Cetuximab or RM-1929 ADC)

[0211] Co-expression was analyzed for HER2 genes and showed potential therapeutic effects ofWSGR Docket No.63634-701.601 administering a combination of an ADC comprising an anti-HER2 with inhibitors of ceramide metabolism for HNSC, ESCA, PAAD, STAD, and THYM (FIG.49). EXAMPLE 7: Analysis of transcriptomics data

[0212] ASAH1 and IGF2R (the mAb target) expression in various cancers were analyzed as shown and described in FIGs.50-65 and TABLES 2-12.

[0213] Analysis of the co-expression of AC, IGF2R and Ceramide synthases

[0214] The datasets used for the comparisons between tumorous and normal gene expressions are TCGA (The Cancer Genome Atlas; www.cancer.gov / ccg / research / genome-sequencing / tcga) and GTEx (Genome-Tissue expression; www.gtexportal.Dorg / home / ). Both datasets are available through the GEPIA2 browser (Genome Expression Profiling Interactive Analysis 2; gepia2.cancer- pku.cn / #index).

[0215] Data of 31 cancer types were analyzed, including Adrenocortical Carcinoma (ACC), Bladder Urothelial Carcinoma (BLCA), Breast Invasive Carcinoma (BRCA), Cervical Squamous Cell Carcinoma and Endocervical Adenocarcinoma (CESC), Cholangio Carcinoma (CHOL), Colon Adenocarcinoma (COAD), Lymphoid Neoplasm Diffuse Large B-Cell Lymphoma (DLBC), Esophageal Carcinoma (ESCA), Glioblastoma Multiforme (GBM), Head and Neck Squamous Cell Carcinoma (HNSC), Kidney Chromophobe (KICH), Kidney Renal Clear Cell Carcinoma (KIRC), Kidney Renal Papillary Cell Carcinoma (KIRP), Acute Myeloid Leukemia (LAML), Brain Lower Grade Glioma (LGG), Liver Hepatocellular Carcinoma (LIHC), Lung Adenocarcinoma (LUAD), Lung Squamous Cell Carcinoma (LUSC), Ovarian Serous Cystadenocarcinoma (OV), Pancreatic Adenocarcinoma (PAAD), Pheochromocytoma and Paraganglioma (PCPG), Prostate Adenocarcinoma (PRAD), Rectum Adenocarcinoma (READ), Sarcoma (SARC), Skin Cutaneous Melanoma (SKCM), Stomach Adenocarcinoma (STAD), Testicular Germ Cell Tumors (TGCT), Thyroid Carcinoma (THCA), Thymoma (THYM), Uterine Corpus Endometrial Carcinoma (UCEC), and Uterine Carcinosarcoma (UCS).

[0216] In particular, the gene expression tool (using the ANOVA differential method and the standards parameters) was used to compare gene expression levels between tumorous cells and healthy cells for all 31 cancer types. All IGF2R, ASAH1, CerS1, CerS2, CerS3, CerS4, CerS5 and CerS6 genes were analyzed individually. For each gene analysis, the result is a table containing 31 rows (for each cancer type) and 4 columns (Cancer type ID, tumorous expression of the gene (transcript per million), Normal expression of the gene (transcript per million) and the p-value).

[0217] IGF2R

[0218] Expression of IGF2R was increased in multiple types of cancer compared to normal tissueWSGR Docket No.63634-701.601 (FIGs. 50-51 and 59). IGFR2 expression was elevated CHOL, COAD, Esophageal carcinoma (ESCA), HNSC, PAAD, READ, SKCM, and STAD compared to normal tissue (FIGs.50-51). Analysis of co-expression of IGFR2 and Ceramide Synthases showed that IGFR2 and CERS2 expression was significantly correlated in ESCA, CAAD, HNSC, PAAD, READ, SKCM, and STAD (FIGs.52-58).

[0219] Expression level of IGF2R on the surface of cancer cells was determined to be significantly elevated compared to normal cells.

[0220] Co-expression of IGF2R and EGFR was assessed in HNSC cell lines (DepMap portal) (FIG.60). IGF2R expression was elevated in HNSC compared to normal cells (FIG. 61). Overall survival was associated with high expression of CerS2 or high EEGFR (GEPIA). Patient overall survival with high CerS2 was significantly worse compared to patient overall survival with low CerS2 (p=3.4e-08) (FIG. 62). EGFR overexpression is not significantly correlated with overall survival (FIG.63).

[0221] Cancer types for which IGF2R expression is greater in tumorous tissues were considered better candidates for the mAb, for a higher specificity. According to IGF2R expression experiments, a compound disclosed herein may be effective in the cancer types: ESCA, SKCM, SARC, HNSC, PAAD, THCA, READ, STAD, PRAD, COAD, GBM, and / or LGG (Table 2).

[0222] ASAH1

[0223] Analysis of RNAseq data from 9,736 tumors and 8,587 paired normal samples from the TCGA and the GTEx (GEPIA) showed increased expression of acid ceramidase in multiple types of cancer compared to normal tissue (FIG.64). Acid Ceramidase gene expression in patient samples was adversely correlated with disease free survival (FIG.65).

[0224] Cancer types for which ASAH1 is largely overexpressed in tumorous tissues can be considered good candidates because the cancers can react more intensely to a Carmofur treatment. On the other hand, cancer types for which ASAH1 expression is greater in normal tissues but still high in tumorous tissues can also be considered good candidates because of this high baseline expression. According to the ASAH1 (AC) expression experiments, a compound disclosed herein can be effective, for example, in the cancer types: SKCM, GBM, LGG, PAAD, PCPG, CHOL, STAD, LAML, PRAD, BRCA, TGCT, and / or COAD (Table 2).

[0225] TABLE 2. AC and IgF2R expression in various cancersWSGR Docket No.63634-701.601WSGR Docket No.63634-701.601

[0226] Correlation of differential expression of IGF2R and ASAH1 genes in tumor compared to normal cells showed significant differentiation in both genes or in ASAH1 alone in the following cancer types: Skin Cutaneous Melanoma (SKCM), Glioblastoma multiforme (GBM), Brain Lower Grade Glioma (LGG), Pancreatic adenocarcinoma (PAAD), Pheochromocytoma and Paraganglioma (PCPG), Cholangio carcinoma (CHOL), Stomach adenocarcinoma (STAD), Acute Myeloid Leukemia (AML), and Prostate adenocarcinoma (PRAD) (FIG.68).

[0227] Based on types of cancer that showed high ASAH1 or high ASAH1 / IGF2R expression, cancers suitable for treatment with an ADC as a first line therapy (top right and bottom right quarters of FIG.68) include STAD, PAAD, LGG, GBM, PRAD, SKCM, AML, and PCPG (Table 3):

[0228] TABLE 3. First-line group:WSGR Docket No.63634-701.601

[0229] Based on types of cancer that showed elevated IGF2R but not elevated ASAH1, examples of cancers suitable for treatment with an ADC as a combination therapy (top left quarter of FIG. 68) include LIHC, HNSC, ESCA, READ, COAD, SARC, and THCA (Table 4):

[0230] . TABLE 4. Combination therapy group:

[0231] Ceramide synthase 1-6

[0232] Expression of Ceramide Synthase (CerS) was also assessed to evaluate the potential effect of treatment on the different cancer types. Expression of each CerS (CerS1-6) was individually assessed in various cancer types was performed as shown and described in FIGs.69-74.

[0233] Based on the ASAH1 and IGF2R expression analysis, the expression of ceramide synthases was applied to indications that were chosen for first line and combination treatments. Ceramide synthases expression and correlation between CerS5 / 6 vs CerS1 / 4 were assess as shown in Tables 5-11.WSGR Docket No.63634-701.601

[0234] TABLE 5. Baseline Ceramide Synthases expression in normal tissue:WSGR Docket No.63634-701.601

[0235] TABLE 6. Ceramide Synthase 1: Differential expression of Ceramide Synthases in tumor tissue compared to normal:WSGR Docket No.63634-701.601

[0236] TABLE 7. Ceramide Synthase 2: Differential expression of Ceramide Synthases in tumor tissue compared to normal:WSGR Docket No.63634-701.601

[0237] TABLE 8. Ceramide Synthase 3: Differential expression of Ceramide Synthases in tumor tissue compared to normal:WSGR Docket No.63634-701.601

[0238] TABLE 9. Ceramide Synthase 4: Differential expression of Ceramide Synthases in tumor tissue compared to normal:WSGR Docket No.63634-701.601

[0239] TABLE 10. Ceramide Synthase 5: Differential expression of Ceramide Synthases in tumor tissue compared to normal:WSGR Docket No.63634-701.601

[0240] TABLE 11. Ceramide Synthase 6: Differential expression of Ceramide Synthases in tumor tissue compared to normal:

[0241] Indications were selected for evaluation based on baseline expression and relationship of CerS1+CerS4 to CerS5+CerS6 to exclude high CerS5 / 6 expression and low CerS1 / 4 expression and include low CerS5 / 6 expression and high CerS1 / 4 expression, as shown in Table 12.WSGR Docket No.63634-701.601sulliniSreCfon oisserpxee nilesaB.21E LBATWSGR Docket No.63634-701.601

[0242] Based on the above, the following indications were selected: Acute Myeloid Leukemia, Adrenocortical carcinoma, Cholangio carcinoma, Head and Neck squamous cell carcinoma, Kidney Chromophobe, Lung adenocarcinoma, Ovarian serous cystadenocarcinoma, Ovarian serous cystadenocarcinoma, Pancreatic adenocarcinoma, Pheochromocytoma and Paraganglioma, Prostate adenocarcinoma, Skin Cutaneous Melanoma, Thymoma, Uterine Carcinosarcoma, and Uterine Corpus Endometrial Carcinoma.

[0243] Other indications can be included based, for example, on dynamics of ceramide synthases expression following various types of drug therapy.

[0244] EXAMPLE 8: Evaluation of the anti-cancer effect of ADC (e.g. aIGF2R-Carmofur) on cancer cells

[0245] The effect of aIGF2R-Carmofur is assessed on cell cycle arrest and apoptosis in cells treated with aIGF2R-Carmofur alone or with cisplatin.

[0246] arrest and apoptosis are assessed using annexin V / propidium iodide staining, by flow cytometry, in

[0247] TIS is stimulated in the HNSCC cell panel using cisplatin or docetaxel. The efficacy of aIGF2R-Carmofur as an adjuvant is assessed by measuring apoptosis, cell cycle arrest, and colony formation. Cisplatin can induce cell cycle arrest and senescence in cancer cells, including HNSCC. Senescent cells can accumulate ceramide, which can contribute to the anti-proliferative effects of senescence and promote resistance to the anti-proliferative drugs such as cisplatin or docetaxel. Recurrent cancer, arising from re-activation of the dormant senescent cells, can require hydrolysis from cisplatin-induced senescence, a cell viability assay and colony formation assay is performed in HNSCC cells, treated with cisplatin or docetaxel and allowed to recover with or without Assay (Promega) is used.

[0248] Evaluating aIGF2R-Carmofur ability to penetrate 3D tumoroids and to induce senolytic effect in the context of tumor microenvironment, using multiplex-immunofluorescence on FFPE tumoroids treated with consecutively with cisplatin and aIGF2R-Carmofur.

[0249] microenvironment (TME). Tumor lines are selected with high expression of IGF2R, AC andWSGR Docket No.63634-701.601 penetration is evaluated by confocal microscopy. Next, tumoroids is treated with cisplatin, followed diameter. Senescence and cell death is assessed on FFPE section by IHC (beta-Gal, BCL2, p53, p16, p21, caspase-3).

[0250] Evaluating the effect of aIGF2R-Carmofur in vivo, by following tumor growth and recovery from TIS in a CDX (FaDu) and a PDX mouse models, treated consecutively with cisplatin followed by aIGF2R-Carmofur.

[0251] To establish CDX xenograft model, 5 x 106 FaDu cells in a volume of 100 µL (Matrigel- FADU cell suspension) is injected subcutaneously into the hind flank of each mouse. When the tumor size reaches 100 mm3, mice are randomized into 6 groups (N=8 / group), five groups are Carmofur (5mg / kg), Xevinapant (Positive control, IAP inhibitor, works efficiently in HNSCC and in xenograft (50mg / kg)) or vehicle.

[0252] The PDX model is used because PDX better preserve tumor stromal cells which play a role in onset of and recovery from therapy induced senescence. The PDX model is established in BALB / c Nude.8 w.o. mice are randomized into 4 groups (N=8 / group, vehicle, ADC, payload, and positive control). For both, CDX and PDX studies, tumor growth and animals’ weight are tracked. Carcasses are used to collect samples from major organs (kidney, liver, heart, lung and brain tissues) and perform H&E staining for obtaining the initial evidence related to toxicity of the investigational compound, to establish the reference for the future safety and dosing studies. Serum is tested for toxicity and immunogenicity.

[0253] Potential drug combination and indications based on the expression of IGF2R, ASAH1 and CerS1-6 follow drug treatment.

[0254] Inclusion IGF2R high, AC high, CerS1 / CERS4 high, CerS5 / CerS6 low

[0255] Drug combination analysis

[0256] The impact of existing therapies over gene expression is also analyzed. The impact of commonly used medications affecting the expression of some genes either immediately or after some time is studied. Through a browser, drug or small molecule induced gene expression signatures is assessed. For a given drug, a signature corresponds to impact over the expression of different genes in multiple cells. Datasets are downloaded and sorted according to a signature for a given gene of interest. For example, searches for all medications that affect (either positively or negatively) the expression of a given gene are conducted. As for the cancer type study, the genes of interests are IGF2R, ASAH1, CerS1, CerS2, CerS3, CerS4, CerS5 and CerS6. To analyze the data further, the signatures are sorted by cell lines (i.e., cancer types), onset times, and operate crossWSGR Docket No.63634-701.601 analysis to find drugs that have complementary effects on different genes.

[0257] Combination therapies comprising an AC inhibitor

[0258] If following drug treatment CerS6 goes up and AC also goes up, then short chain ceramides (C14 and C16) are low, and cells do not proliferate. Inhibition of AC in this case leads to proliferation.

[0259] If following drug treatment CerS4 goes up and AC also goes up, then long ceramides (C18) are low and cells proliferate. Inhibition of AC in this case blocks proliferation. IGF2R stays unchanged to allow ADC uptake.WSGR Docket No.63634-701.601 EMBODIMENTS

[0260] The following non-limiting embodiments provide illustrative examples of the invention, but do not limit the scope of the invention.

[0261] Embodiment 1. A compound comprising a lysosomal-targeting antibody linked to a lysosomal enzyme inhibitor.

[0262] Embodiment 2. The compound of embodiment 1, wherein the lysosomal-targeting antibody is anti-HER2.

[0263] Embodiment 3. The compound of embodiment 1, wherein the lysosomal-targeting antibody is anti-IGF2R.

[0264] Embodiment 4. The compound of embodiment 1, wherein the lysosomal-targeting antibody is anti-IGF1R.

[0265] Embodiment 5. The compound of embodiment 1, wherein the lysosomal-targeting antibody is anti-EGFR.

[0266] Embodiment 6. The compound of any one of embodiments 1-5, wherein the lysosomal enzyme inhibitor is Carmofur.

[0267] Embodiment 7. The compound of any one of embodiments 1-5, wherein the lysosomal enzyme inhibitor is N-[(1R,2R)-2-hydroxy-1-(hydroxymethyl)-2-(4-mitrophenyl)ethyl]- tetradecanamide.

[0268] Embodiment 8. The compound of any one of embodiments 1-5, wherein the lysosomal enzyme inhibitor is N-Oleoylethanolamine.

[0269] Embodiment 9. The compound of any one of embodiments 1-5, wherein the lysosomal enzyme inhibitor is N-((2S,3R)-1,3-dihydroxy-5-((2-oxo-2H-chromen-7-yl)oxy)pentan- 2-yl)dodecanamide.

[0270] Embodiment 10. The compound of any one of embodiments 1-9, wherein the lysosomal-targeting antibody and the lysosomal enzyme inhibitor are linked by a linker.

[0271] Embodiment 11. The compound of any one of embodiments 1-10, wherein the lysosomal-targeting antibody and the lysosomal enzyme inhibitor are linked by a linker, wherein the linker is a cleavable linker.

[0272] Embodiment 12. The compound of one of embodiments 1-10, wherein the lysosomal- targeting antibody and the lysosomal enzyme inhibitor are linked by a linker, wherein the linker is a non-cleavable linker.

[0273] Embodiment 13. The compound of one of embodiments 1-10, wherein the lysosomal- targeting antibody and the lysosomal enzyme inhibitor are linked by a linker, wherein the linker is aWSGR Docket No.63634-701.601 self-immolative linker.

[0274] Embodiment 14. The compound of one of embodiments 1-10, wherein the lysosomal- targeting antibody and the lysosomal enzyme inhibitor are linked by a linker, wherein the linker is a click chemistry linker.

[0275] Embodiment 15. The compound of one of embodiments 1-10, wherein the lysosomal- targeting antibody and the lysosomal enzyme inhibitor are linked by a linker, wherein the linker comprises a GGFG linkage.

[0276] Embodiment 16. The compound of one of embodiments 1-10, wherein the lysosomal- targeting antibody and the lysosomal enzyme inhibitor are linked by a linker, wherein the linker comprises a maleimide linkage.

[0277] Embodiment 17. The compound of one of embodiments 1-10, wherein the lysosomal- targeting antibody and the lysosomal enzyme inhibitor are linked by a linker, wherein the linker comprises an amide linkage.

[0278] Embodiment 18. The compound of one of embodiments 1-10, wherein the lysosomal- targeting antibody and the lysosomal enzyme inhibitor are linked by a linker, wherein the linker comprises a maleimidocaproyl linkage.

[0279] Embodiment 19. The compound of one of embodiments 1-10, wherein the lysosomal- targeting antibody and the lysosomal enzyme inhibitor are linked by a linker, wherein the linker comprises a L-valine-L-citrulline-p-aminobenzyl linkage.

[0280] Embodiment 20. The compound of one of embodiments 1-10, wherein the linker comprises maleimidocaproyl-L-valine-L-citrulline-p-aminobenzyl.WSGR Docket No.63634-701.601

[0281] Embodiment 21. The compound of embodiment 1, wherein the compound is:

[0282] or an ionized form thereof.

[0283] Embodiment 22. The compound of one of embodiments 1-21, wherein the compound is an inhibitor of acid ceramidase.

[0284] Embodiment 23. A method of treating a condition, the method comprising administering to a subject in need thereof a therapeutically-effective amount of a compound of any one of embodiments 1-22.

[0285] Embodiment 24. The method of embodiment 23, wherein the condition is a cancer

[0286] Embodiment 25. The method of embodiment 23, wherein the condition is head and neck cancer.

[0287] Embodiment 26. The method of one of any one of embodiments 23-25, wherein the compound inhibits acid ceramidase.

[0288] Embodiment 27. The method of any one of embodiments 23-26, wherein the administering is oral.

[0289] Embodiment 28. The method of any one of embodiments 23-26, wherein the administering is by infusion.

[0290] Embodiment 29. The method of any one of embodiments 23-28, wherein the therapeutically-effective amount is from about 50 mg to about 1,000 mg.

[0291] Embodiment 30. The method of any one of embodiments 23-29, wherein the subject is human.

Claims

WSGR Docket No.63634-701.601 CLAIMS WHAT IS CLAIMED IS:

1. A compound comprising a lysosomal-targeting antibody linked to a lysosomal enzyme inhibitor.

2. The compound of claim 1, wherein the lysosomal-targeting antibody is anti-HER2.

3. The compound of claim 1, wherein the lysosomal-targeting antibody is anti-IGF2R.

4. The compound of claim 1, wherein the lysosomal-targeting antibody is anti-IGF1R.

5. The compound of claim 1, wherein the lysosomal-targeting antibody is anti-EGFR.

6. The compound of claim 1, wherein the lysosomal enzyme inhibitor is Carmofur.

7. The compound of claim 1, wherein the lysosomal enzyme inhibitor is N-[(1R,2R)-2- hydroxy-1-(hydroxymethyl)-2-(4-mitrophenyl)ethyl]-tetradecanamide.

8. The compound of claim 1, wherein the lysosomal enzyme inhibitor is N- Oleoylethanolamine.

9. The compound of claim 1, wherein the lysosomal enzyme inhibitor is N-((2S,3R)-1,3- dihydroxy-5-((2-oxo-2H-chromen-7-yl)oxy)pentan-2-yl)dodecanamide.

10. The compound of claim 1, wherein the lysosomal-targeting antibody and the lysosomal enzyme inhibitor are linked by a linker.

11. The compound of claim 1, wherein the lysosomal-targeting antibody and the lysosomal enzyme inhibitor are linked by a linker, wherein the linker is a cleavable linker.

12. The compound of claim 1, wherein the lysosomal-targeting antibody and the lysosomal enzyme inhibitor are linked by a linker, wherein the linker is a non-cleavable linker.

13. The compound of claim 1, wherein the lysosomal-targeting antibody and the lysosomal enzyme inhibitor are linked by a linker, wherein the linker is a self-immolative linker.

14. The compound of claim 1, wherein the lysosomal-targeting antibody and the lysosomal enzyme inhibitor are linked by a linker, wherein the linker is a click chemistry linker.

15. The compound of claim 1, wherein the lysosomal-targeting antibody and the lysosomal enzyme inhibitor are linked by a linker, wherein the linker comprises a GGFG linkage.

16. The compound of claim 1, wherein the lysosomal-targeting antibody and the lysosomal enzyme inhibitor are linked by a linker, wherein the linker comprises a maleimide linkage.

17. The compound of claim 1, wherein the lysosomal-targeting antibody and the lysosomal enzyme inhibitor are linked by a linker, wherein the linker comprises an amide linkage.WSGR Docket No.63634-701.601 18. The compound of claim 1, wherein the lysosomal-targeting antibody and the lysosomal enzyme inhibitor are linked by a linker, wherein the linker comprises a maleimidocaproyl linkage.

19. The compound of claim 1, wherein the lysosomal-targeting antibody and the lysosomal enzyme inhibitor are linked by a linker, wherein the linker comprises a L-valine-L-citrulline-p- aminobenzyl linkage.

20. The compound of claim 1, wherein the linker comprises maleimidocaproyl-L-valine-L- citrulline-p-aminobenzyl.

21. The compound of claim 1, wherein the compound is:or an ionized form thereof.

22. The compound of claim 1, wherein the compound is an inhibitor of acid ceramidase.

23. A method of treating a condition, the method comprising administering to a subject in need thereof a therapeutically-effective amount of a compound, the compound comprising a lysosomal- targeting antibody linked to a lysosomal enzyme inhibitor.

24. The method of claim 23, wherein the condition is a cancer.

25. The method of claim 23, wherein the condition is head and neck cancer.

26. The method of claim 23, wherein the compound inhibits acid ceramidase.

27. The method of claim 23, wherein the administering is oral.

28. The method of claim 23, wherein the administering is by infusion.WSGR Docket No.63634-701.601 29. The method of claim 23, wherein the therapeutically-effective amount is from about 50 mg to about 1,000 mg.

30. The method of claim 23, wherein the subject is human.

Images