[0012] In some embodiments, the anti-Trop-2 ADC comprises sacituzumab (hRS7; described, e.g., in W02003074566, Figs 3 and 4). [0013] In some embodiments, the anti-Trop-2 ADC is selected from sacituzumab govitecan, datopotamab deruxtecan (DS- 1062), ESG-401, SKB-264, DAC-02 and BAT-8003.

[0014] In some embodiments, the ami-Trop-2 ADC is sacituzumab govitecan.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIGs. 1A and IB show data from The Cancer Genome Atlas (TCGA) to illustrate relative RNA expression levels of Trop-2 (FIG. 1A) and Topi (FIG. IB) in various human tumor types, and including TCGA tumor (left columns; patterned open circles), tumor-adjacent normal (middle columns; solid circles), and Genotype Tissue Expression normal (GTEx; right columns; open circles) expression levels. NS = not significant. GTEx, Genotype Tissue Expression; ns, not significant; TCGA, The Cancer Genome Atlas. ns=P>0.05 *=P<0.05 **=P<0.01 ***=P<0.001 ****=P<0.0001

[0016] FIGs. 2A-2C show immunohistochemistry images, illustrating TROP2 expression in bladder epithelium obtained from an exemplary' bladder cancer patient (FIG. 2A), a normal human volunteer (FIG. 2B), and a healthy cynomolgus monkey (cyno; FIG. 2C), respectively.

[0017] FIGs. 3A-3D show results of a flow cytometry analysis of Trop-2 expression in a parental MDA-MB436 human breast adenocarcinoma (TNBC) cell line (FIG. 3 A), a Trop2+ transduced MDA-MB436 cell line (FIG. 3B), a UM-UC3 human NMIBC cell line (FIG. 3C), and human primary bladder epithelial cells (FIG. 3D). Numeric values reflect geometric mean fluorescence intensity (gMFI) of Trop2 expression. Values of 692 in FIG. 3A, 665 in FIG. 3B, 245 in FIG. 3C, and 855 in FIG. 3D reflect geometric mean fluorescence intensity (gMFI) of Trop- 2 expression. Higher values indicate higher surface Trop-2 expression.

[0018] FIGs. 4A-4C illustrate SG-mediated cytotoxic effects as determined by cell viability (FIG. 4A), apoptosis (FIG. 4B), or DNA damage (FIG. 4C) on MDA-MB-436 Trop2+ (transduced) and UM-UC3 Trop2+ tumor cell lines and human normal primary' bladder epithelial cells.

[0019] FIG. 5 illustrates the design of a non-human primate study of SG intravesical administration in non-human primates (NHPs).

[0020] FIG. 6 illustrates concentrations of total SN-38 (top panel) and free SN-38 (bottom panel) present in the dosing solution prior to instillation (circles) compared with contents from drained bladders (squares) following ih of NHP bladder instillation with SG. [0021] FIG. 7 illustrates a pharmacokinetic (PK) modeling exercise to predict an effication human dose of SG administered by intravesical instillation. Eq. = equivalent; HEffD = human efficacious dose; MFD = maximum feasible dose; NHP = nonhuman primate.

[0022] FIGs. 8A and 8B illustrate results of a flow cytometry analysis of TROP-2 expression in endogenous (FIG. 8A) and engineered (FIG. 8B) NMIBC cell lines and tumors, respectively.

[0023] FIGs. 9A-9D illustrate rapid internalization of SG in TROP-2 high expressor cells. FIG. 9A shows fluorescence microscope images illustrating SG (top panel) and a control ADC (bottom panel) internalization in hTrop2 expressing live cells. FIG. 9B shows fluorescence intensity traces illustrating relative SG internalization in UM-UC3-TROP-2 transduced cells (

) compared to its control ADC ( ~‘V - ) FIG. 9C shows fluorescence intensity traces illustrating SG internalization in TROP-2 transduced UM-UC3 compared to other tested cell lines expressing endogenous Trop-2. FIG. 9D shows a bar graph comparing relative SG internalization levels at the I2-hr time point of FIG. 9C. TROP-2 transduced UM-UC3, TROP-2high RT112, TROP-2int J82 and TROP-21ow UM-UM3. One-way ANOVA: * p< 0.0313; **** p< 0.0001.

[0024] FIGs. 10A--10C show fluorescence microscope images depicting a confocal analysis of Trop-2 dependent SG internalization via endocytosis. FIG. 10A illustrates SG colocalization with Trop-2. FIG. 10B illustrates SG colocalization with LAMP1, a marker of lysosome. FIG. 10C shows a 3-D visualization of internalized SG. FIG. 10D shows quantification of immunofluorescence emitted by SG-bound secondary antibody against human IgG, before and after 1 hour incubation at 37°C.

[0025] FIG. 11 illustrates results of an in vitro SG stability test at low pH and in urine.

[0026] FIGs. 12A-12C illustrate results of an UMUC3 Trop2+ orthotopic xenograft model of NMIBC involving intravesicular SG administration. FIG. 12A shows in vivo images of mice treated with intravesical injection of saline. FIG. 12B shows in vivo images of mice treated with intravesical injection of control ADC. FIG. 12C shows in vivo images of mice treated with intravesical injection of SG.

[0027] FIGs. 13A-13C illustrate the designs in vivo mouse NMIBC studies involving subcutaneous UMUC3 Trop2+ xenografts with intraperitoneal injection of SG (FIG. 13 A) or orthotopic UMUC3 Trop2+ xenografts with intravesicular administration of SG (FIG. 13B). FIG. 13C illustrates a representative bioluminescent image obtained in the study of FIG. 13B. DETAILED DESCRIPTION

[0028] Provided herein are therapies for treating bladder cancer comprising intravesical administration of an effective amount of an anti-Trop-2 antibody-drug conjugate (ADC) to a subject.

Definitions

[0029] As used herein, the term “antibody” refers to a polypeptide that includes canonical immunoglobulin sequence elements sufficient to confer specific binding to a particular target antigen (e.g., a heavy chain variable domain, a light chain variable domain, and/or one or more CDRs sufficient to confer specific binding to a particular target antigen). Thus, the term antibody includes, for example, and without limitation, human antibodies, non-human antibodies, antibody fragments, and antigen-binding agents that include antibody fragments, inclusive of synthetic, engineered, and modified forms thereof. The term antibody includes, by way of example, both naturally occurring and non-naturally occurring antibodies. In general, an antibody may comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding molecule thereof. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains, CHI, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one constant domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the Abs may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. Naturally-produced antibodies are glycosylated, typically on the CH2 domain. Examples of antibodies include monoclonal antibodies, monospecific antibodies, polyclonal antibodies, multispecific antibodies (including bispecific antibodies), engineered antibodies, recombinantly produced antibodies, wholly synthetic antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, tetrameric antibodies comprising two heavy chain and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chainantibody heavy chain pairs, intrabodies, antibody fusions (sometimes referred to herein as “antibody conjugates”), heteroconjugate antibodies, single domain antibodies, monovalent antibodies, single chain antibodies or single-chain Fvs (scFv), camelized antibodies, affybodies. Fab fragments, Fab' fragments, F(ab’)2 fragments, Fd' fragments, Fd fragments, isolated CDRs, single chain Fvs, polypeptide-Fc fusions, single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof); cameloid antibodies; disul fide-l inked Fvs (sdFv), anti-idiotypic (anti-Id) antibodies (including, e.g., anti-anti-Id antibodies), minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as “antibody mimetics”), single chain or Tandem diabodies (TandAb®), Anticalins®, Nanobodies®, minibodies, BiTE®s, ankyrin repeat proteins or DARPINs®, Avimers®, DARTs, TCR-like antibodies, Adnectins®, Affilins®, Trans-bodies®, Affibodies®, TrimerX®, MicroProteins, m Fynomers®, Centyrins®, KALBITOR®s, and antigen-binding fragments of any of the above.

[0030] As used herein, the term “antibody-drug conjugate” generally refers to a compound comprising an antibody targeting a tumor antigen and an anticancer agent payload, optionally- connected by a linker. In some embodiments the tumor antigen is tumor-associated calcium signal transducer 2 (Trop-2; NCBI Gene ID: 4070). In some embodiments the tumor antigen targeted antibody is an anti-Trop-2 antibody (e.g., sacituzumab or datopotamab) In some embodiments the payload is a topoisomerase I inhibitor (e.g., SN38 or Dxd). Many ADC linker chemistries are known to a skilled artisan and referenced herein (e.g., CL2A).

[0031] Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. In certain embodiments, the term “about” includes the indicated amount ± 10%. In other embodiments, the term “about” includes the indicated amount ± 5%. In certain other embodiments, the term “about” includes the indicated amount ± 1%. Also, to the term “about X” includes description of “X”. Also, the singular forms “a” and “the” include plural references unless the context clearly dictates otherwise. Thus, e.g., reference to “the compound” includes a plurality' of such compounds and reference to “the assay” includes reference to one or more assays and equivalents thereof known to those skilled in the art.

[0032] As used herein, the term “intravesical administration” refers to administering a drug right into the bladder. In some embodiments, the drug is administered into the bladder via a catheter. The term “intravesical administration” and the term “intravesical instillation” are used interchangeably herein.

[0033] As used herein, the term “topoisomerase I inhibitor” refers to small molecule compounds capable of inhibiting the activity of a DNA topoisomerase type I enzyme (Topi). Type I topoisomerases can catalyze changes in DNA topology' via transient single-stranded breaks in DNA. Type I topoisomerases can be further classified as Type 1A and a Type IB subtypes. A description of type I topoisomerases can be found, for example, in Baker el al. (2009) Nucleic Acids Res 37(3), 693-703. Topoisomerase inhibitors that can be used as payloads in the ADCs described herein include camptothecin (CPT) and non-camptothecin based inhibitors. Useful camptothecins include, for example, topotecan, irinotecan, belotecan, exatecan, and derivatives thereof. Useful non-camptothecins include, for example, indenosinoquinolines (e.g., indeno[ 1,2- c] isoquinoline, NSC314622, indotecan (LMP-400), indimitecan (LMP-776)), phenanthridines (e.g., topovale (ARC-111), and indolocarbazoles (e.g., BE-13793C). In some embodiments the topoisomerase I inhibitor is a camptothecin (e.g., an irinotecan, topotecan, belotecan, or exatecan derivative, such as SN38 or Dxd). In some embodiment the topoisomerase I inhibitor is SN38. In some embodiments the topoisomerase I inhibitor is Dxd.

[0034] As used herein, the terms “effective amount” or "therapeutically effective amount" refer to that amount of a therapeutic agent administered in the methods provided herein (e.g., ADC, adenosine pathway inhibitor, checkpoint inhibitor) that, when administered alone or in combination with another therapeutic agent to a cell, tissue, or subject is sufficient to effect treatment or a beneficial result in the subject. The therapeutically effective amount may vary depending on the subject, and disease or condition being treated, the weight and age of the subject, the severity of the disease or condition, and the manner of administering, which can readily be determined by one of ordinary skill in the art. An effective amount further refers to that amount of the therapeutic agent, which when used in the context of the combination therapies provided herein, is sufficient to treat, prevent, alleviate, ameliorate or mitigate a disease condition, or delayer slow the progression of a disease, and that amount sufficient to affect an increase in rate of treatment, healing, prevention or amelioration of such conditions. When applied to an individual therapeutic agent administered alone, an effective amount refers to that active ingredient alone. When applied to a combination, a therapeutically effective amount refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously. In some embodiments an effective amount or therapeutically effective amount of a therapeutic agent (e.g., ADC, adenosine pathway inhibitor, checkpoint inhibitor) administered to a subject in the methods provided herein with one or more additional therapeutic agents, as described herein, can (i) reduce the number of diseased cells; (ii) reduce tumor size; (hi) inhibit, retard, slow to some extent, and preferably stop the diseased cell infiltration into peripheral organs; (iv) inhibit (e.g., slow to some extent and preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay occurrence and/or recurrence of a tumor; and/or (vii) relieve to some extent one or more of the symptoms associated with cancer. In various embodiments, the amount is sufficient to ameliorate, palliate, lessen, and/or delay one or more of symptoms of cancer.

[0035] As used herein, the terms “treatment,” “treat,” and “treating” refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease or disorder, or one or more symptoms thereof, as described herein. In some embodiments, treatment may be administered after one or more symptoms have developed. In other embodiments, treatment may be administered in the absence of symptoms. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g. , in light of a history of symptoms and/or in light of genetic or other susceptibility factors ). Treatment may also be continued after symptoms have resolved, for example to prevent or delay their recurrence. In some embodiments the methods provided herein refer to the treatment of a subject having cancer (e.g., a human cancer patient). In some embodiments treating a subject having cancer (e.g., a human cancer patient) comprises inhibiting cancer or cancer cell proliferation in the treated subject. In some embodiments treating a human cancer patient using the methods provided herein results in the observation of anti-tumor effects or anti-cancer effects in the treated patient.

[0036] As used herein, the terms “inhibition of cancer” and “inhibition of cancer cell proliferation” refer to the inhibition of the growth, division, maturation or viability of cancer cells, and/or causing the death of cancer cells, individually or in aggregate with other cancer cells, by cytotoxicity, nutrient depletion, or the induction of apoptosis.

[0037] As used herein, the terms “anti-tumor effect,” “anti-cancer effect,” or “anti-cancer efficacy” refer to a biological effect that can present as a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in tumor cell proliferation, a decrease in the number of metastases, an increase in overall or progression-free survival, an increase in life expectancy, or amelioration of various physiological symptoms associated with the tumor. In some embodiments anti-cancer effects are measured using one or more of the endpoint criteria applied in the clinical studies described herein (e.g., primary, secondary, or exploratory endpoints). Exemplary clinical endpoint criteria that can be used to measure anti-cancer effects in connection with the methods provided herein include objective response rate (ORR), complete response (CR) rate, partial response (PR) rate, disease control rate (DCR), progression-free survival (PFS), overall survival (OS), biomarker-based signals, e.g., of intratumoral immune activation or induction of cancer cell death (e.g.. tumor tissue or blood based biomarkers), patient quality of life (QoL) indicators (e.g., based on patient surveys), and others. List of Abbreviations and Acronyms

Anti-TROP-2 ADCs

[0038] The treatment methods provided herein comprise intravesical administration of an anti- Trop-2 antibody-drug conjugate (ADC) to a subject, such as a human NMIBC patient. In some embodiments, the anti-Trop-2 antibody-drug conjugate (ADC) comprises an anti-Trop-2 antibody, an anti-cancer agent payload, and an optional linker connecting the anti-Trop-2 antibody and payload. In some embodiments the anti-cancer agent payload in the anti-Trop-2 ADC is a topoisomerase I inhibitor (e.g., SN38, Dxd). In some embodiments the anti-cancer agent payload in the anti-Trop-2 ADC does not include a topoisomerase I inhibitor.

[0039] Examples of anti-Trop-2 antibodies that can be used in anti-Trop-2 ADCs to perform the methods provided herein include, but are not limited to, those described in W02020016662 (Abmart), W02020249063 (Bio-Thera Solutions), US20190048095 (Bio-Thera Solutions), WO2013077458 (LivTech/Chiome), EP20110783675 (Chiome), W02015098099 (Daiichi Sankyo), W02017002776 (Daiichi Sankyo), W02020I30125 (Daiichi Sankyo), W02020240467

(Daiichi Sankyo), US2021093730 (Daiichi Sankyo), US9850312 (Daiichi Sankyo),

CN112321715 (Biosion), US2006I93865 (hnmunomedics/Gilead), WO2011068845

(Immunomedics/Gilead), US2016296633 (hnmunomedics/Gilead), US2017021017

(Immunomedics/Gilead), US2017209594 (Immunomedics/Gilead), US2017274093

(Immunomedics/Gilead), US20181 10772 (Immunomedics/Gilead), US2018185351

(Immunomedics/Gilead), US2018271992 (Immunomedics/Gilead), WO2018217227

(Immunomedics/Gilead), US2019248917 (Immunomedics/Gilead), CN 1 11534585

(Immunomedics/Gilead), US2021093730 (Immunomedics/Gilead), US2021069343

(Immunomedics/Gilead), US8435539 (Immunomedics/Gilead), US8435529

(Immunomedics/Gilead), US9492566 (hnmunomedics/Gilead), W02003074566 (Gilead),

WO2020257648 (Gilead), US20I3039861 (Gilead), WO2014163684 (Gilead), US9427464 (LivTech/Chiome), US10501555 (Abruzzo Theranostic/Oncoxx), WO2018036428 (Sichuan Kelun Pharma), WO2013068946 (Pfizer), W02007095749 (Roche), and W02020094670 (SynAffix).

[0040] In some embodiments of the methods provided herein, the anti-Trop-2 ADC comprises an antibody selected from sacituzumab (hRS7), datopotamab (hTINA HIL I ), or a Trop-2 binding fragment thereof. In some embodiments the anti-Trop-2 ADC is sacituzumab (hRS7). In some embodiments the anti-Trop-2 antibody is datopotamab (hTINA HILI).

[0041] In some embodiments of the methods provided herein, the anti-Trop-2 ADC comprises a VH-CDR1 , a VH-CDR2, a VH-CDR3, a VL-CDRI, a VL-CDR2 and a VL-CDR3 selected from one of Tables 1 to 4. In some embodiments the anti-Trop-2 ADC comprises the following VH- CDR1, a VH-CDR2, a VH-CDR3, a VL-CDRI, a VL-CDR2 and a VL-CDR3 amino acid sequences (according to Kabat), respectively:

• SEQ ID NOs: 1, 2, 3, 4, 5, and 6, or

• SEQ ID NOs: 7, 8, 9, 10, I I, and 12.

[0042] In some embodiments of the methods provided herein, the anti-Trop-2 ADC comprise variable domains (VH and VL) selected from Table 5. In some embodiments the anti-Trop-2 ADC comprises the following VH and VL amino acid sequences, respectively:

• SEQ ID NOs: 49 and 50, or

• SEQ ID NOs: 51 and 52.

[0043] In some embodiments of the methods provided herein the anti-Trop-2 ADC comprises an anti-Trop-2 antibody, an anti-cancer agent payload, and an optional linker connecting antibody and payload. In some embodiments the linker is non-cleavable (e.g., a maleimidocaproyl or maleimidomethyl cyclohexane- 1 -carboxylate linker). In some embodiments the linker is cleavable. In some embodiments the linker is acid cleavable (e.g., a hydrazone linker). In some embodiments the cleavable linker is reducible (e.g., a disulphide linker). In some embodiments the linker is protease cleavable (e.g., a dipeptide or tetrapeptide linker). In some embodiments, the linker is selected from linkers disclosed in USPN 7,999,083 (e.g., CL2A, CL6, CL7, CLX, or CLY). In some embodiments, the linker is CL2A. Additional linker chemistries usefill for anti- Trop-2 ADCs are described, for example in WO21225892 (Shanghai Escugen Biotechnology; ESG-401, STI-3258), W022010797 (BiOneCure Therapeutics; BIO-106), CN112237634 (Shanghai Fudan-Zhangjiang Biopharmaceutical; FDA018-ADC), WO191 14666 (Sichuan Kelun Pharmaceutical; KLA264), WO22078524 (Hangzhou DAC Biotech; DAC-002), WO 15098099 (Daiichi Sankyo; datopotamab deruxtecan), WO21147993 (Jiangsu Hengrui Medicine; SHR- A1921), and W021052402 (Sichuan Baili Pharmaceutical; BL-M02D1).

[0044] Exemplaty anti-cancer agent payloads that can be used in anti-Trop-2 ADCs in the methods provided herein include, for example, microtubule inhibitors, DNA cleavage agents, and topoisomerase I inhibitors. In some embodiments the microtubule inhibitor is an auristatin (e.g., monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF), a taxane, a vinca alkaloid, an epothilone) or maytansinoid (e.g., mertansine (DM1) or ravtansine (DM4)). In some embodiments the DNA cleavage agent is a calicheamicin (e.g., ozogamicin). In some embodiments the topoisomerase I inhibitor is a camptothecin (e.g., an irinotecan, topotecan, belotecan, or exatecan derivative, such as SN38 or Dxd). In some embodiment the topoisomerase I inhibitor is SN38. In some embodiments the topoisomerase I inhibitor is is Dxd. In some embodiments the topoisomerase I inhibitor is not a camptothecin. In some embodiments the non-camptothecin topoisomerase I inhibitor is selected from an indenosinoquinoline (e.g., indeno[l,2-c]isoquinoline, NSC314622, indotecan (LMP-400), indimitecan (LMP-776)), a phenanthridine (e.g., topovale (ARC-111), and a indolocarbazole (e.g., BE-13793C).

[0045] Additional illustrative anti-cancer agent payloads that can be conjugated to anti-Trop-2 ADCs include without limitation anthracyline (e.g., doxorubicin, daunorubicin, epirubicin, idarubicin), pyrrolobenzodiazepine (PBD), or dimer thereof, DNA cross-linking agent SC-DR002 (D6.5), duocarmycin, a duocarmycin (A, Bl, B2, Cl, C2, D, SA, CC-1065), tubulysin B and analogs thereof (e.g., Tub 196), and other anti-cancer agents described herein.

[0046] Exemplary anti-Trop-2 ADCs that can be used in the methods provided herein are described in WO21225892 (Shanghai Escugen Biotechnology; ESG-401, STI-3258), W022010797 (BiOneCure Therapeutics; BIO-106), CN112237634 (Shanghai Fudan-Zhangjiang Biopharmaceutical; FDA018-ADC), WO19114666 (Sichuan Kelun Pharmaceutical; KLA264), WO22078524 (Hangzhou DAC Biotech; D AC-002), WO 15098099 (Daiichi Sankyo; datopotamab deruxtecan), WO21147993 (Jiangsu Hengrui Medicine; SHR-A1921), and WO2 1052402 (Sichuan Baili Pharmaceutical; BL-M02D1).

[0047] In some embodiments of the methods provided herein the anti-Trop-2 ADC is selected from sacituzumab govitecan (Immunomedics/Gilead), datopotamab deruxtecan (DS-1062, Dato- Dxd; Daiichi Snakyo/ AstraZeneca), SKB-264 (KL-A264; Klus Pharma, Sichuan Kelun Pharma), ESG-401 (Shanghai Escugen Biotechnology/Levena Biopharma), JS-108 (DAC-002; Junshi Bio/Hangzhou DAC), FDA018-ADC (Shanghai Fudan Zhangjiang Bio Pharma), STI-3258 (Sorrento), OXG-64 (Oncoxx), BDI-4702 (OBI Pharma), BL-M02D1 (Systimmune), Anti-Trop- 2 Ab (Mediterrania Theranostic/Legochem), KD-065 (Nanjing KAED1 Biotech), Anti-Trop2 sdAb (Kisoli Biotech), Anti-Trop-2 ADC (Shandong Fontacea), LIV-2008 (LivTech/Yakult Honsha), TROP2-TRACTr (BiTE; Janux), TROP-2-IR700 (Chiome, photosensitizer), TROP2- XPAT (Amunix), BAT-8003 (Bio-Thera Solutions), GQ-1003 (Genequantum Healthcare, Samsung BioLogics), DAC-002 (Hangzhou DAC Biotech, Shanghai Junshi Biosciences), El -3s (hnmunomedics/Gilead, IBC Pharmaceuticals), s BioTech), humanized anti-Trop2-SN38 antibody conjugate (Shanghai Escugen Biotechnology, TOT Biopharma), anti-Trop2 antibody- CLB-SN-38 conjugate (Shanghai Fudan-Zhangjiang Bio-Pharmaceutical), TROP2-Ab8 (Abmart), Trop2-IgG (Nanjing Medical University (NMUJ), 90Y-DTPA-AF650 (Peking University First Hospital), and hRS7-CM (SynAffix), 89Zr-DFO-AF650 (University of Wisconsin-Madison). In some embodiments the anti-Trop-2 ADC is sacituzumab govitecan (Immunomedics/Gilead). In some embodiments, the anti-Trop-2 ADC is selected from sacituzumab govitecan, datopotamab deruxtecan (DS-1062), ESG-401, SKB-264, DAC-02 and BAT-8003. In some embodiments, the anti-Trop-2 ADC is sacituzumab govitecan. In some embodiments the anti-Trop-2 ADC is datopotamab deruxtecan (DS-1062, Dato-Dxd; Daiichi Snakyo/ AstraZeneca). Further examples of usefol anti-Trop-2 therapeutics include, but are not limited to, those described in W02016201300 (Gilead), and CN108440674 (Hangzhou Lonzyme Biological Technology).

[0048] Exemplary anti-Trop-2 ADCs that can be used in the methods provided herein are described, for example, in USPN 7,999,083 and USPN 9,028,833, which are hereby incorporated herein by reference in their entireties. In some embodiments, the anti-Trop-2 ADC comprises a topoisomerase I inhibitor. In some embodiments, the topoisomerase I inhibitor is selected from irinotecan, topotecan, and SN-38. In some embodiments, the anti-Trop-2 ADC has a structural formula of mAb-CL2A-SN-38, with a structure represented by:

(described, e.g., in USPN 7,999,083). In some embodiments, the drug-antibody ratio (DAR) of CL2A-SN38 to anti-Trop-2 antibody in the anti-Trop2 ADC is >7.0 (e.g., DAR = 7.6). In some embodiments, the drug-antibody ratio (DAR) of CL2A-SN38 to anti-Trop-2 antibody in the anti- Trop2 ADC is between 7.0 and 8.0 (e.g., DAR = 7.6). In some embodiments, the anti-Trop-2 ADC comprises sacituzumab (hRS7; described, e.g., in W02003074566, Figs 3 and 4). In some embodiments the anti-Trop-2 ADC is sacituzumab govitecan (IMMU-132). Sacituzumab govitecan (SG) is an antibody-drug conjugate (ADC) composed of the following 3 components:

• The humanized monoclonal antibody hRS7 IgGlK, which binds to trophoblast cell-surface antigen 2 (Trop-2; TACSTD2; EGP-1; NCBI Gene ID: 4070), a transmembrane calcium signal transducer that is overexpressed in many epithelial cancers, including triple-negative breast cancer (TNBC).

• The camptothecin-derived agent SN-38, a topoisomerase I inhibitor.

• A hydrolyzable linker (CL2A) that links the humanized monoclonal antibody to SN-38. [0049] Additional exemplary anti-Trop-2 ADCs that can be used in the methods provided herein are described in WO21225892 (Shanghai Escugen Biotechnology). In some embodiments the anti- Trop-2 ADC comprises a linker-payload conjugate having a structure represented by:

attached to an anti-Trop-2 antibody (e.g., hRS7). In some embodiments the anti-Trop-2 ADC has a DAR of between 1 and 8. In some embodiments the anti-Trop-2 ADC has a DAR of between 7.0 and 8.0. In some embodiments the anti-Trop-2 ADC is ESG-401 (STI-3258).

[0050] Additional exemplary anti-Trop-2 ADCs that can be used in the methods provided herein are described in US20210101906 (Sichuan Kelun Pharmaceutical). In some embodiments the anti- Trop-2 ADC comprises a linker-payload conjugate (TL035) having a structure represented by:

attached to an anti-Trop-2 antibody (e.g., hRS7). In some embodiments the anti-Trop-2 ADC has a DAR of between 1 and 8. In some embodiments the anti-Trop-2 ADC has a DAR of between 7.0 and 8.0. In some embodiments the anti-Trop-2 ADC has a DAR of about 7.0. In some embodiments the anti-Trop-2 ADC is KL-A264.

[0051] Additional exemplary anti-Trop-2 ADCs that can be used in the methods provided herein are described in US2016297890 (Daiichi Sankyo). In some embodiments the anti-Trop-2 ADC comprises a linker-payload conjugate having a structure represented by:

attached to an anti-Trop-2 antibody (e.g., hTINAl-HlLl). In some embodiments the anti-Trop-2 ADC has a DAR of between 1 and 8. In some embodiments the anti-Trop-2 ADC has a DAR of <7.0. In some embodiments the anti-Trop-2 ADC has a DAR of about 4. In some embodiments the anti-Trop-2 ADC is datopotamab deruxtecan.

Treatment Methods

[0052] In one aspect, provided herein are methods of treating bladder cancer in a subject comprising intravesical administration of an effective amount of an anti-Trop-2 antibody-drug conjugate (anti-Trop-2 ADC) to the subject.

[0053] In some embodiments of the methods provided herein, the anti-Trop-2 ADC comprises a topoisomerase I inhibitor. In some embodiments, the topoisomerase I inhibitor is a camptothecin (CPT). In some embodiments, the topoisomerase I inhibitor is a topotecan, irinotecan, belotecan or exatecan. In some embodiments, the topoisomerase I inhibitor is SN38 or Dxd. In some embodiments, the topoisomerase I inhibitor is selected from irinotecan, topetecan, and SN-38. In some embodiments, tire topoisomerase I inhibitor is SN38.

[0054] In some embodiments of the methods provided herein, the anti-Trop-2 ADC has a structural formula of mAh -CL2A- SN-38, with a structure represented by:

described, e.g., in U.S. Patent No. 7,999,083).

[0055] In some embodiments of the methods provided herein, the anti-Trop-2 ADC comprises sacituzumab (hRS7; described, e.g., in W02003074566, Figs. 3 and 4). In some embodiments, the anti-Trop-2 ADC is selected from sacituzumab govitecan, datopotamab deruxtecan (DS- 1062), ESG-401, SKB-264, DAC-02 and BAT-8003. In some embodiments, the anti-Trop-2 ADC is sacituzumab govitecan.

[0056] In some embodiments of the methods provided herein, the bladder cancer is non-muscle invasive bladder cancer (NMIBC). In some embodiments, the NMIBC is classified as Tis (carcinoma in site, CIS: flat, high-grade, non-papillary carcinomas confined to the urothelium), Ta (confinement to the epithelium or mucosa), or T1 (invasion of the subepithelial connective tissue or lamina propria).

[0057] In some embodiments of the methods provided herein, the method further comprises coadministering an additional therapeutic agent or therapeutic modality. In some embodiments, the additional therapeutic agent or therapeutic modality comprises BCG, mitomycin, germcitabine, a taxane, interferon, vairubicin or a combination thereof. In some embodiments, the additional therapeutic agent or therapeutic modality’ comprises one, two, three, or four additional therapeutic agents and/or therapeutic modalities. In some embodiments, the additional therapeutic agent or therapeutic modality is administered via intravesical route. In some embodiments, the additional therapeutic agent or therapeutic modality is administered intravenously.

[0058] In some embodiments of the methods provided herein, the subject is treatment naive. In some embodiments, the bladder cancer has progressed following at least one prior anti-cancer therapy. In some embodiments, the bladder cancer is resistant or refractive to at least one anticancer therapy. In some embodiments, the subject has been previously treated with BCG, interferon, or a combination thereof. In some embodiments, the subject has previously been treated additional intravesical therapy such as mitomycin, germcitabine, a taxane, interferon, valrubicm or a combination thereof. In some embodiments, the subject failed to respond to previous BCG with or without interferon therapy. In other embodiments, the subject responded to previous BCG with or without interferon therapy but disease subsequently recurred. In some embodiments, the subject has been treated mitomycin, germcitabine, a taxane, interferon, vairubicin or a combination thereof after failure to respond to BCG with or without interferon therapy. In some embodiments, the subject has been treated mitomycin, germcitabine, a taxane, interferon, vairubicin or a combination thereof after a relapse following BCG with or without interferon therapy. In some embodiments, the subject received at least 6, at least 7, at least 8, or at least 9 administration of BCG. In further embodiments, the at least 6, at least 7, at least 9, or at least 9 instillations of BCG were over 2 cycles of BCG.

[0059] In some embodiments of the methods provided herein, the bladder is emptied via a catheter prior to administration of the anti-Trop-2 ADC. In some embodiments, the bladder is emptied by the subject by voiding the bladder. In some embodiments, the bladder is emptied less than about 5 minutes, less than about 10 minutes, less than about 15 minutes, less than about 20 minutes, less than about 25 minutes, less than about 30 minutes, less than about 35 minutes, less than about 40 minutes, less than about 45 minutes, less than about 55 minutes, or less than about 60 minutes prior to administration of the anti-Trop-2 ADC. In some embodiments, the bladder is emptied about 0 to 15 minutes prior to administration of the anti-Trop-2 ADC. In some embodiments, the bladder is emptied about 15 to 30 minutes prior to administration of the anti- Trop-2 ADC. In some embodiments, the bladder is emptied about 30 to 45 minutes prior to administration of the anti-Trop-2 ADC. In some embodiments, the bladder is emptied about 5 minutes prior to administration of the anti-Trop-2 ADC. In some embodiments, the bladder is emptied about 10 minutes prior to administration of the anti-Trop-2 ADC. In some embodiments, the bladder is emptied about 15 minutes prior to administration of the anti-Trop-2 ADC. In some embodiments, the bladder is emptied about 20 minutes prior to administration of the anti-Trop-2 ADC. In some embodiments, the bladder is emptied about 25 minutes prior to administration of the anti-Trop-2 ADC. In some embodiments, the bladder is emptied about 30 minutes prior to administration of the anti-Trop-2 ADC. In some embodiments, the bladder is emptied about 35 minutes prior to administration of the anti-Trop-2 ADC. In some embodiments, the bladder is emptied about 40 minutes prior to administration of the anti-Trop-2 ADC. In some embodiments, the bladder is emptied about 45 minutes prior to administration of the anti-Trop-2 ADC. In some embodiments, the bladder is emptied about 50 minutes prior to administration of the anti-Trop-2 ADC. In some embodiments, the bladder is emptied about 55 minutes prior to administration of the anti-Trop-2 ADC. In some embodiments, the bladder is emptied about 60 minutes prior to administration of the anti-Trop-2 ADC. In some embodiments, the anti-Trop-2 ADC is held in the bladder is held in the bladder for at least about 15 minutes, at least about 30 minutes, at least about 45 minutes, at least about 1 hour, at least about 1.5 hours, at least about 2 hours, at least about 2.5 hours, at least about 3 hours, or longer. In some embodiments, the anti-Trop-2 ADC is held in the bladder is held in the bladder for about 15 minutes or less, about 30 minutes or less, about 45 minutes or less, about 1 hour or less, about 1.5 hours or less, about 2 hours or less, about 2.5 hours or less, or about 3 hours or less. In some embodiments, the anti-Trop-2 ADC is held in the bladder is held in the bladder for about 30 minutes to about 3 hours. In some embodiments, the anti-Trop- 2 ADC is held in the bladder is held in the bladder for about 1 hour to about 3 hours. In some embodiments, the anti-Trop-2 ADC is held in the bladder is held in the bladder for about 1.5 hour to about 2.5 hours. In some embodiments, the anti-Trop-2 ADC is held in the bladder is held in the bladder for about 1 hour to about 2 hours. In some embodiments, the anti-Trop-2 ADC is held in the bladder for about 2 hours. In some embodiments, the subject is positioned upright, prone, supine, and in the left and right lateral decubitus positions during the time that the anti-Trop-2 ADC is in the bladder.

[0060] In some embodiments of the methods provided herein, the subject is human.

[0061] In some embodiments of the methods provided herein, the anti-Trop-2 ADC is administered to the subject at a dose level of about 200 mg to about 500 mg per dose. In some embodiments, the anti-Trop-2 ADC is administered to the subject at a dose level of about 250 mg to about 500 mg per dose. In some embodiments, the anti-Trop-2 ADC is administered to the subject at a dose level of about 300 mg to about 500 mg per dose. In some embodiments, the anti- Trop-2 ADC is administered to the subject at a dose level of about 350 mg to about 500 mg per dose, hi some embodiments, the anti-Trop-2 ADC is administered to the subject at a dose level of about 400 mg to about 500 mg per dose. In some embodiments, the anti-Trop-2 ADC is administered to the subject at a dose level of about 450 mg to about 500 mg per dose. In some embodiments, the anti-Trop-2 ADC is administered to the subject at a dose level of about 200 mg per dose. In some embodiments, the anti-Trop-2 ADC is administered to the subject at a dose level of about 250 mg per dose. In some embodiments, the anti-Trop-2 ADC is administered to the subject at a dose level of about 300 mg per dose. In some embodiments, the anti-Trop-2 ADC is administered to the subject at a dose level of about 350 mg per dose. In some embodiments, the anti-Trop-2 ADC is administered to the subject at a dose level of about 400 mg per dose. In some embodiments, the anti-Trop-2 ADC is administered to the subject at a dose level of about 450 mg per dose. In some embodiments, the anti-Trop-2 ADC is administered to the subject at a dose level of about 500 mg per dose.

[0062] In some embodiments, an anti-cancer effect is observed as determined by objective response rate (ORR), disease control rate (DCR), progression free survival (PFS), overall survival (OS), complete response (CR), partial response (PR), radiographic response rate, or change from baseline in blood and tumor tissue microenvironment pharmacodynamic (PD) biomarkers.

EXAMPLES

[0063] The following examples are offered to illustrate, but not to limit the claimed invention.

EXAMPLE 1. Safety analysis of SG for the treatment of NMIBC

Example 1.1 Trop-2 and TOPI RNA expression profiles in human and NHP bladder tissue

[0064] The Cancer Genome Atlas (TCGA) dataset were combined and normalized with RNA expression data from healthy tissue samples in the Genotype Tissue Expression (GTEx) project dataset to compare gene expression across tissue types (A) Trop-2 and (B) TOPI RNA expression were found to be broadly upregulated in tumor samples relative to tumor-adjacent-nonnal and healthy normal tissue samples (FIGs. 1A and IB).

Abbreviations of studies in FIGs 1A and IB:

ACC Adrenocortical carcinoma

BLCA Bladder Urothelial Carcinoma

BRCA Breast invasive carcinoma

CESC Cervical squamous cell carcinoma and endocervical adenocarcinoma CHOL Cholangiocarcinoma

LCML Chronic Myelogenous Leukemia

COAD Colon adenocarcinoma

DLBC Lymphoid Neoplasm Diffuse Large B-cell Lymphoma

ESCA Esophageal carcinoma

GBM Glioblastoma multiforme

HNSC Head and Neck squamous cell carcinoma

KICH Kidney Chromophobe

KIRC Kidney renal clear cell carcinoma

KIRP Kidney renal papillary cell carcinoma

LAML Acute Myeloid Leukemia

EGG Brain Lower Grade Glioma

LIHC Liver hepatocellular carcinoma

LUAD Lung adenocarcinoma

LUSC Lung squamous cell carcinoma

MESO Mesothelioma

OV Ovarian serous cystadenocarcinoma

PAAD Pancreatic adenocarcinoma

PCPG Pheochromocytoma and Paraganglioma

PRAD Prostate adenocarcinoma

READ Rectum adenocarcinoma

SARC Sarcoma

SKCM Skin Cutaneous Melanoma

ST AD Stomach adenocarcinoma

TGCT Testicular Germ Cell Tumors

THYMThymoma

THCA Thyroid carcinoma

UCS Uterine Carcinosarcoma UCEC Uterine Corpus Endometrial Carcinoma

UVM Uveal Melanoma

[0065] RNA expression data from primary’ tumors and tumor- adjacent-normal samples were compared across numerous cancer indications. Trop-2 and TOPI expressions are significantly upregulated in bladder cancer and most cancer types relative to normal tissues (FIGs. 1A and IB).

Example 1.2 Trop-2 Protein expression profiles in human and NHP bladder tissue

[0066] Trop-2 protein expression was analyzed by immunohistochemistry in 20 commercially procured paired human bladder cancer tissues, 20 normal adjacent human tissues and 3 commercially procured healthy cynomolgus monkey bladder tissues. Tissues were sectioned at 5 pm. Slides were stained with Trop-2 antibody (SP295, Abeam, Cat No. ab227691) on the Leica Bond Rxm using routine immunohistochemistry’ (IHC) method A. Trop-2 protein expression was found to be high in both UC (left) and normal bladder epithelium (right). It was concluded that high UC expression supports the use of SG as intravesical treatment if safety studies can establish that exposure of normal bladder epithelium to SG does not have major deleterious effects. FIG. 2B and 2C show that Trop-2 expression is comparable in normal human and normal cyno bladder epithelium. This expression pattern is believed to support use of cynomolgus monkey as safety testing species to evaluate effect of intravesical SG administration.

[0067] Trop-2 expression upon IHC was shown to be highly prevalent across normal and bladder cancer tissues in humans (FIGs. 2A and 2B)

Example 1.3 Cytotoxicity on normal epithelial bladder cells

[0068] A fluorescence-activated cell sorting (FACS) assessment of Trop2+ expression was performed in transduced breast cancer and bladder cancer cell lines along with with an evaluation of Trop2+ expression in human primary cells (MDA-MB-436 human breast adenocarcinoma cell line (TNBC); lentiviral transduced (Trop2+) MDA-MB-436 cells, UM-UC3 human NMIBC cell line, and normal primary bladder epithelial cells (ATCC; PCS-420-010; Lot 70036782); FACS Ab: RS7 anti-human Trop-2.). Results are shown in FIGs. 3A-3D. It was observed that human primary bladder epithelial cells and tumor cells both expressed high level of TROP-2.

[0069] Trop-2-positive cell lines (MDA-MB-436 or UM-UC3) and human primary' bladder epithelial cells were incubated for 30 minutes with control ADC (cADC; non-Trop-2-binding antibody), or SG (1 or 10 pg/mL), washed, and cultured for 24 or 48 h in fresh medium. Free SN- 38 from SG and cADC preparations were comparable, as determined by liquid chromatography/mass spectrometry (LC/MS) (cADC: h679-CL2A-SN38 (drug-to-antibody ratio [DAR] 7.12); SG: hRS7-CL2A-SN38 (DAR 7.18)). Cell viability was measured with CellGlow titer; Apoptosis was measured by flow cytometry staining with annexin V and propidium iodine (Pl). DNA damage was measured by flow cytometry staining with gH2AX; C- Error bars are showing standard error of the mean.

[0070] It was found that Trop-2+ tumor cell lines MDA-MB-436 or UM-UC3 were more sensitive to cytotoxic effects of SG than normal epithelial cells, consistent with the broad upregulation of the SN-38 target TOPI by tumor cells to compare with normal cells. SG demonstrated a notable impact on cell viability (FIG. 4A) including apoptosis (FIG. 4B) and a DNA damage (FIG. 4C) in Trop 2-expressing bladder cancer cell lines compared with normal primary¹ human bladder tissues.

Example 1.4 Tolerability in NHP

[0071] FIG. 5 illustrates the design of an NHP study to evaluate a possible SG dosing schedule and to perform tolerability and PK assessments. A repeat dose NHP toxicity study was conducted in a cynomolgus monkey model system, and tolerability was assessed (clinical observations, body weights, food consumption, urine cytology, clinical pathology) during the in-life phase post-life prior to and following intravesical instillation of SG once weekly for two weeks at 10 mg/mL (FIG. 5). Pharmacokinetic (PK) exposure assessments for free SN-38 were conducted out to 120 h postdose following each dosing event. Formulations were administered as either vehicle control article (20 mM MES [2-(N-morpholino) ethanesulfonic acid], 25 mM trehalose, 0.01% w/vc polysorbate 80, 0.9% NaCl, in sterile water for inj., USP, pH adjusted with NaOH to 6.5 ± 0.2; suspended in diluent as 0.9% sodium chloride for inj., USP) or SG formulated in vehicle control article at 10 mg/mL.

[0072] No abnormal findings occurred in the NHP toxicity study (n=4 treated naive animals) following 2 once weekly instillations of SG for 1 h each. DI or D7 concentration of (top panel) total SN-38 (free + ADC bound, top panel) and free SN-38 detected in the dosing solution (dots, bottom panel) prior to instillation was compared with contents from the drained bladder (squares) following the 1 h bladder instillation. Results from the NHP toxicity study showed no abnormal in-life, urine cytology, clinical pathology, or histopathological findings following two once- weekly dosing events at 10 mg/mL. No systemic or local toxicity was observed, which correlates with a lack of free SN-38 detection in serum and only small changes in free SN-38 pre-procedure (dosing solution) versus postprocedure (drained bladder contents) in tested NHPs (FIG. 6).

[0073] No measurable systemic exposure of total SN-38 (conjugated and unconjugated), free SN-38, or SN-38 glucuronide was detected in serum up to 120 h after intravesical instillation, and no measurable urine exposure of total SN-38 and total antibody was observed at any time after the bladder emptying following 60-min intravesical instillation (data not shown). The no adverse effect level dose [NOAEL] in NHPs was determined to be at least 10 mg/mL when administered for two once-weekly events at 1 h each. The dose concentration at the NOAEL corresponds to a 2.5-fold exposure margin when compared with the predicted human efficacious dose of 4 mg/mL

Example 1.5 Safety margin

[0074] To explore dosing of SG by intravesical administration, a mathematical PK model was enacted for determining the human efficacious dose. This model included considerations of SG trough concentrations (Ctrough) maintained above target potency (IC95) value following 1 h intravesical instillation, and also accounted for interspecies differences in urine flow, SN-38 stability in urine, and free SN-38 in the formulation at baseline. The safety margin was calculated as a ratio of NHP dose and predicted human efficacious dose. The PK modeling predicted (FIG. 7) a human efficacious dose of 4 mg/mL (200 mg in 50 mL)

[0075] In summary, the In vitro data presented in this Example 1 (FIGs. 1-4) demonstrated high expression of Trop-2 (targeted by SG anti-TROP2 antibody) and TOPI (targeted by SG payload SN-38) in bladder cancer, as well as selectivity of SG mediated cytotoxicity for Trop-2-expressing tumor cells over normal bladder epithelial cells. As such, the in vitro data already indicated SG’s potential for reduced toxicity to normal, healthy tissue., This finding supported the subsequent in vivo testing of intravesical administration of SG. The resulting in vivo data presented here further demonstrated the tolerability of SG in non-human primates (NHP) when administered by intravesical instillation. A predicted human efficacious intravesical dose of 4 mg/mL was determined. By comparison, the approved dose for systemic injection is 10 mg/mL (Maximum Feasible Dose, MFD), supporting a safety margin of 2.5 (FIGs. 5-7). EXAMPLE 2. Efficacy analysis of SG in treating NMIBC

Materials

[0076] Three human bladder cancer cell lines sensitive to SN-38 and expressing endogenous TROP-2 were selected: TROP -2^high RT112, TROP -2^mtemedia,y J83 and TROP -2^low UM-UC-3 (Fig. 8A). A GFP/Luciferase transduced UM-UC-3 cell line (GFP/luc UM-UC3) was engineered by lentiviral transduction to express high level of TROP-2 and TROP-2^hlgh GFP/Luc+ UM-UC-3 cells were implanted subcutaneously in 5 nude mice and tested 25 days later for TROP-2 expression.

[0077] Cells were stained with anti-TROP2 antibody (hRS7 1/200) detected with AF-647 Goat anti-human IgG Fc (1/500). Live cells were selected with Sytox-blue staining. Staining was measured by flow cytometry analysis. FIG. 8A illustrates TROP-2 endogenous expression: high (RT112, green), intermediate (J82, orange), and low (UM-UM-3, blue); FIG. 8B illustrates engineered TROP-2 expression. Top panel: single cell suspension of 5 tumors derived from 5 mice implanted subcutaneously with TROP-2 transduced GFP/luc UM-UC3 cells. Tumors were harvested 25 days after implantation. Lower panels: mock-transduced (blue) vs. TROP-2 transduced UM-UC-3 cell line (orange). Values reflect geometric mean fluorescence intensity (gMFI) of TROP-2 expression. Higher values indicate higher surface TROP-2 expression. FIG. 8B shows that TROP-2 expression remains stable in vivo.

Example 2.1 In vitro analysis

Example 2.1.1 Impact on cell viability after 2 hours of SG exposure

[0078] The impact on cell viability after 2 hours of SG exposure was studied. Endogenous TROP-2 expressor cell lines were first validated for sensitivity to continuous exposure to SN-38. Cell viability after continuous exposure to SN-38 was measured with a colorimetric assay for assessing cell metabolic activity. NAD(P)H-dependent cellular oxidoreductase enzymes reflect the number of viable cells present and reduce the tetrazolium dye MTT [3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide] to its insoluble formazan, which has a purple color. The half maximal effective concentrations (EC 50) after continuous exposure to SN-38 of the 3 tested cell lines were the nanomolar range, demonstrating sensitivity to SN-38 (Table 6). Table 6. EC50 (pM) after continuous exposure to SN-38

[0079] Next, cell viability was measured after one 2 hours pulse/chase exposure to SG or control ADC (non-binding antibody linked to the same payload and linker than SG, at the same drug to antibody ratio, DAR), followed by 96 hours of incubation in flesh medium. Table 2 shows that EC50 in the nanomolar range could be obtained after 2 hours pulse-chase exposure, and that cells with high or intermediary expression level of TROP-2 responded better to SG than to control ADC.

Table 7. EC50 (pM) after 2-h exposure to SG or control ADC

Example 2.1.2 Induction of DNA damage after SG exposure in TROP-2 expressing cells

[0080] Table 8 show’s induction of DNA damage after 30 min of SG exposure in TROP-2 expressing cells. Table 8 show's that after 30-min pulsed-exposure to ADC follow'ed by 24 hours of incubation in fresh medium, SG mediated more DNA damage than control ADC in cells with high or intermediary expression level of TROP-2.

Table 8. DNA Damage After Pulse-chase exposure to SG

Example 2.1.3 Analyzing internalization of SG using live cell fluorescent imaging

[0081] Live cell fluorescent imaging demonstrated SG rapid and specific internalization in TROP-2 expressing cells (FIGs. 9A-9D).

[0082] To visualize and analyze the kinetics of SG internalization in live cells, SG (top panel) and a control ADC (bottom panel) were labeled with a pH sensitive dye to detect endocytosis via Incucyte (FIG. 9A). Fluorescent signal from labeled SG under acidic compartment such as endosomes and lysosomes is an indication for ADC internalization in UM-UC3-TROP-2 transduced. FIG. 9B shows that SG internalized within 1 hour and accumulated abundantly in UM-UC3-TROP-2 transduced cells compared to its control ADC. FIG. 9C shows that SG internalized much faster in TROP-2 transduced UM-UC3 than in all other tested cell lines; FIG. 9D shows that compared to other cells expressing endogenous TROP-2, TROP-2high RT112 internalized more SG compared to TROP-2int J82 and TROP-21ow UM-UM3 at 12-hr time point. SG internalized much faster in Trop-2 transduced UM-UC3 than in all other tested cell lines.

[0083] The studies showed rapid internalization of SG with colocalization with TROP-2 and lysosomes.

Example 2.1.4 Confocal analysis of internalization of SG

[0084] RT112 cells were fixed after pulse-chase exposure to 5 pg/mL SG ( 1 h at 4°C), followed by 3 washes and 1 h incubation at 37°C, and then SG, hTrop-2 and lysosomal associated membrane protein-1 (LAMP1) were visualized by confocal microscopy (FIGs. 10A and 10B). For confocal analysis, SG binding to nonpermeabilized cells was detected with an anti-human IgG (human immunoglobulin G) secondary antibody. FIG. 10A demonstrated the co-localization of SG with TROP-2 -positive vesicles and, to a lesser extent, with lysosomes based on LAMP1 staining (FIG. 10B)

[0085] UMUC3 hTrop-2+ cells were fixed after pulse-chase exposure to 5 pg/mL SG (1 h at 4°C), followed by 3 washes and 1 h incubation at 37°C, and then SG was visualized by confocal microscopy. For confocal analysis, SG binding to nonpermeabilized cells was detected with an anti-human IgG (human immunoglobulin G) secondary' antibody. The 3-D rendering (FIG. 10C) permitted visualization of the internalized SG as vesicles positive for human IgG.

[0086] Trodelvy (5 pg/ml) was applied for 1 hour at 4°C and was detected at the cell surface upon fixation in 4% paraformaldehyde followed by staining with secondary antibody. Additional incubation for 1 hour at 37 °C after 3 washouts, led to decline in Trodelvy signal in RT112 and UM-UC-3-Trop2 cells suggesting internalization of Trodelvy. FIG. 10D shows quantification of immunofluorescence emitted by SG-bound secondary' antibody against human IgG, before and after 1 hour incubation at 37°C.

[0087] Confocal analysis revealed SG internalization in both endogenous hTrop-2 expressors and hTrop-2 transduced cell lines, following binding to hTrop-2 -positive cells (RT112 and UM- UC3-Trop-2+). The colocalization of SG-positive lysosomes with hTrop-2 supports hTrop-2- dependent internalization of SG via endocytosis. SG cell surface detection after binding rapidly decreased in all four tested cell lines (RT112, 5637, and hTrop-2-transduced UM-UC3 cells and GFP/Luc+ UM-UC3) after 1 h incubation at 37°C; SG binding decrease varied nonsignificantly from 29.3 % ± 14.57 (hTrop2+UM-UC3) to 45.47% ± 5.33 (RT112) (results from 3 independent experiments).

[0088] In vitro data demonstrated SG cytotoxicity and internalization after short exposure of NMIBC cells to SG (less than 2 hours), modelling intravesical instillation with bladder voiding within 2 hours.

Example 2.1 Ex vivo analysis

Example 2.1.1 Stability of SG at low pH and in NHP and human urine [0089] The initial solution of SG was prepared in 20 mL of 0.9% sodium chloride (saline) for each vial that results in a concentration of 62.5 pM (10 mg/mL). The solution was further diluted with saline, or pH adjusted human or monkey urine. After 2 h of incubation, the free SN-38 (percentage of total) increased in both saline and pH adjusted urine. About 10% of free SN-38 was detected in the buffer containing 90% urine at pH 5 and pH 8, about 2-fold higher than that in the saline or 50% urine.

[0090] The studies showed stability of SG at low pH and in NHP and human urine. Incubation of SG in human or monkey urine under numerous physiological conditions for up to 2 h showed high stability and minimal liberation of the payload, SN-38, demonstrating the potential for low toxicity to normal bladder tissues when exposed via intravesical instillation (FIG. 11).

[0091] Ex-vivo data demonstrated SG stability in mine.

Example 2.2 In vivo analysis

Example 2.2.1 Effects of SG on tumor growth and survival of mice bearing human TROP-2 expressing UM-UC3 tumor cells

[0092] To control the in vivo sensitivity to SG of GFP/Luc+ TROP2+ UM-UC3 cells, 7-9 week-old female C.B-17 scid mice (purchased from Envigo) were implanted subcutaneously into the right hind flank with 1 million cells per mouse. When the mean tumor volume reached 150 mm³ (11 days after tumor injection) mice were randomized by tumor volume into 2 groups of 10 mice (FIG. 13A) The control group was dosed IP with lOmL/kg PBS, and the treatment group was dosed IP with 250 pg per mouse per dose of SG on days 11, 15 and 18 for 3 total doses. Tumor measurements and body weights were collected twice weekly. Mice were removed from study when their tumor volumes reached 2000 nun³. Tumor volumes were measured by caliper and calculated with the modified ellipsoidal formula: V = 16 (Length x Width2). 23 days after subcutaneous implantation of GFP/ Luc+ hTrop-2+ UMUC3 tumor cells (after 3 SG injections), it was confirmed that these tumors were sensitive to SG as evidenced by a significant 61% tumorgrowth inhibition (P<0.0001 SG vs. PBS control; 2-way ANOVA). Tumor growth was significantly impaired by SG treatment.

[0093] To mimic clinical intravesical instillations in NMIBC patients, an orthotopic model system for TROP-2 expressing NMIBC was developed with GFP/Luc+ TROP2+ UM-UC3 cells instilled in the bladder of 7-9 week-old female C.B-17 scid mice (3 million cells per mouse) after Trypsin pretreatment to improve tumor implantation. Three days after tumor cell injection, mice were randomized by in vivo imaging in 5 groups of 15 mice (FIG. 13B). Intravesical instillations were performed on days 4, 11, 18 and 25, for 4 total doses. The control groups were dosed with 10 mL/kg PBS, or with control ADC (same linker and payload as SG, but non-tumor targeting antibody) at 150 pg (FIG. 13B). The treatment groups were dosed with 150 pg of SG per mouse per dose (FIG. 13B). In vivo imaging tumor measurements were collected twice weekly for 60 days and body weights twice weekly for 90 days (FIG. 13C). Mice that were selected for randomization but that did not present any visible bladder tumors 20 days after tumor injection were removed from the study (0 to 3 mice/group).

[0094] Tumor burden was estimated by quantifying the total number of photons emitted per second (p/s) in the bladder area using bioluminescence Imaging (BLI). Mice with discrete bioluminescence signal ranging from 5xl0⁵ to 2x10' p/s 2 days after tumor cell injections in the bladder were selected for model development (FIGs. 12A-12C).

[0095] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

1. A method of treating bladder cancer comprising intravesical administration of an effective amount of an anti-Trop-2 antibody-drug conjugate (anti-Trop-2 ADC) to a subject having a bladder cancer.

2. The method of claim 1, wherein the anti-Trop-2 ADC comprises a topoisomerase I inhibitor.

3. The method of claim 2. wherein the topoisomerase I inhibitor is SN38 or Dxd.

4. The method of claim 2, wherein the topoisomerase I inhibitor is SN38.

5. The method of any one of claims 1 to 4, wherein the anti-Trop-2 ADC has a structural formula of mAb-CL2A-SN-38, with a structure represented by:

(described, e.g., in U.S. Patent No. 7,999,083).

6. The method of any one of claims 1 to 5, wherein the anti-Trop-2 ADC comprises sacituzumab (hRS7; described, e.g., in W02003074566, Figs 3 and 4).

7. The method of claim 1, wherein the anti-Trop-2 ADC is selected from sacituzumab govitecan, datopotamab deruxtecan (DS-1062), ESG-401, SKB-264, DAC-02 and BAT-8003.

8. The method of claim 1, wherein the anti-Trop-2 ADC is sacituzumab govitecan.

9. The method of any one of claims 1 to 8, wherein the bladder cancer is non-muscle invasive bladder cancer (NMIBC).

10. The method of claim 9, wherein the NMIBC is classified as Tis (carcinoma in site, CIS: flat, high-grade, non-papillary carcinomas confined to the urothelium), Ta (confmement to the epithelium or mucosa), or T1 (invasion of the subepithelial connective tissue or lamina propria).

11. The method of any one of claims 1 to 10, wherein the method further comprises co-administering an additional therapeutic agent or therapeutic modality'.

12. The method of claim 11, wherein the additional therapeutic agent or therapeutic modality comprises one, two, three, or four additional therapeutic agents and/or therapeutic modalities.

13. The method of claim I I or 12, wherein the additional therapeutic agent or therapeutic modality is administered via intravesical route.

14. The method of claim 11 or 12, wherein the additional therapeutic agent or therapeutic modality is administered intravenously.

15. The method of any one of claims 1 to 14, wherein the subject is treatment naive.

16. The method of any one of claims 1 to 14, wherein the bladder cancer has progressed following at least one prior anti-cancer therapy.

17. The method of any one of claims 1-14 and 16, wherein the subject has previously been treated with BCG and/or interferon.

18. The method of claim 17, wherein the subject failed to respond to BCG and/or interferon therapy or wherein the subject achieved a disease-free status following BCG and/or interferon therapy but disease subsequently recurred.

19. The method of any one of claims 1-18, wherein the bladder cancer is resistant or refractive to at least one anti-cancer therapy.

20. The method of any one of claims 1 to 19, wherein the bladder is emptied via a catheter prior to administration of the anti-Trop-2 ADC.

2 1. The method of any one of claim 1 to 20, wherein the anti-Trop-2 ADC is held in the bladder for about 2 hours.

22. The method of any one of claim 1 to 20, wherein the anti-Trop-2 ADC is held in the bladder for about 1 hour to about 3 hours.

23. The method of any one of claim 1 to 20, wherein the anti-Trop-2 ADC is held in the bladder for at least about 30 minutes.

24. The method of any one of claims 21-23, wherein the subject is positioned upright, prone, supine, and in the left and right lateral decubitus positions during the time that the anti-Trop-2 ADC is in the bladder.

25. The method of any one of claims 1 to 24, wherein the subject is human.

26. The method of any one of claims 1 to 25, wherein the anti-Trop-2 ADC is administered to the subject at a dose level of about 200 mg to about 500 mg per dose.

27. The method of any one of claims 1 to 25, wherein the anti-Trop-2 ADC is administered to the subject at a dose level of about 250 mg to about 500 mg per dose.

28. The method of any one of claims 1 to 25, wherein the anti-Trop-2 ADC is administered to the subject at a dose level of about 300 mg to about 500 mg per dose.

29. The method of any one of claims 1 to 25, wherein the anti-Trop-2 ADC is administered to the subject at a dose level of about 350 mg to 500 mg per dose.

30. The method of any one of claims 1 to 25, wherein the anti-Trop-2 ADC is administered to the subject at a dose level of about 400 mg to about 500 mg per dose.

31. The method of any one of claims 1 to 25, wherein the anti-Trop-2 ADC is administered to the subject at a dose level of about 450 mg to about 500 per dose.

32. The method of any one of claims 1 to 25, wherein the anti-Trop-2 ADC is administered to the subject at a dose level of about 200 mg per dose.

33. The method of any one of claims 1 to 25, wherein the anti-Trop-2 ADC is administered to the subject at a dose level of about 250 mg per dose.

34. The method of any one of claims 1 to 25, wherein the anti-Trop-2 ADC is administered to the subject at a dose level of about 300 mg per dose.

35. The method of any one of claims 1 to 25, wherein the anti-Trop-2 ADC is administered to the subject at a dose level of about 350 mg per dose.

36. The method of any one of claims 1 to 25, wherein the anti-Trop-2 ADC is administered to the subject at a dose level of about 400 mg per dose.

37. The method of any one of claims 1 to 25, wherein the anti-Trop-2 ADC is administered to the subject at a dose level of about 450 mg per dose.

38. The method of any one of claims 1 to 25, wherein the anti-Trop-2 ADC is administered to the subject at a dose level of about 500 mg per dose.